MARKER ASSISTED INTROGRESSION OF Sub1 LOCUS IN RICE · 2019-09-10 · marker assisted introgression of sub1 locus in rice by suchismita raha, b. sc. (agri.), i. d. no. 07-607-026

MARKER ASSISTED INTROGRESSION OF Sub1 LOCUS IN RICE

By

SUCHISMITA RAHA, B. Sc. (Agri.),

I. D. No. 07-607-026

DEPARTMENT OF PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY

CENTRE FOR PLANT MOLECULAR BIOLOGY

TAMIL NADU AGRICULTURAL UNIVERSITY

COIMBATORE – 641 003

2009


Thesis submitted in partial fulfillment of the requirements for the award of the degree of

MASTER OF SCIENCE IN BIOTECHNOLOGY to the

Tamil Nadu Agricultural University, Coimbatore – 3.

By

SUCHISMITA RAHA, B. Sc. (Agri.),

I. D. No. 07-607-026

DEPARTMENT OF PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY

CENTRE FOR PLANT MOLECULAR BIOLOGY

TAMIL NADU AGRICULTURAL UNIVERSITY

COIMBATORE – 641 003

2009

ACKNOWLEDGEMENTACKNOWLEDGEMENTACKNOWLEDGEMENTACKNOWLEDGEMENT

With a deep sense of gratitude, I express my heartfelt thanks to my chairman

Dr. N. SenthilDr. N. SenthilDr. N. SenthilDr. N. Senthil, , , , Associate Professor, Department of Plant Molecular Biology and Biotechnology, Centre

for Plant Molecular Biology and Biotechnology, for his learned counsel, unstinted attention and

scintillating support throughout the investigation.

I humbly express my indebtedness and deep sense of indelible gratitude from the core of my

heart to Dr.Dr.Dr.Dr. M.M.M.M. Raveendran,Raveendran,Raveendran,Raveendran, Associate Professor, Department of Plant Molecular Biology and

Biotechnology, Centre for Plant Molecular Biology and Biotechnology, for his valuable guidance,

incessant inspiration and wholehearted help and personal care throughout the course of this study and in

bringing out this thesis.

I record my sincere gratitude to members of the advisory committee,

Dr. C.Dr. C.Dr. C.Dr. C. VijayalakshmiVijayalakshmiVijayalakshmiVijayalakshmi, Professor, Department of Physiology and Dr. Dr. Dr. Dr. R. PushpamR. PushpamR. PushpamR. Pushpam, Assistant Professor,

Department of Rice, CPBG, for their valuable suggestions and guidance throughout the course of my

research.

I take immense pleasure to express my thanks to DrDrDrDr. S. Vellaikuma. S. Vellaikuma. S. Vellaikuma. S. Vellaikumarrrr, , , , Assistant Professor, Dr. Dr. Dr. Dr.

D. Vijayalakshmi,D. Vijayalakshmi,D. Vijayalakshmi,D. Vijayalakshmi, Assistant Professor, CPMB, for their untiring attention and timely help at each stage

of the research work.

I extent deep hearted thanks to Dr. P. BalasubramanianDr. P. BalasubramanianDr. P. BalasubramanianDr. P. Balasubramanian, Director, CPMB, and

Dr. V. KrishnasamyDr. V. KrishnasamyDr. V. KrishnasamyDr. V. Krishnasamy, Professor and Head, DPMB&B, CPMB, for providing constant encouragement

and facilities rendered to complete the course successfully.

I hold in high regard the efforts of all my all my all my all my teachersteachersteachersteachers for enriching my overall knowledge and help

rendered throughout the course of study.

I profoundly thank Dr. RajendranDr. RajendranDr. RajendranDr. Rajendran, Department of pathology, TNAU, for his timely help at

one stage or the other during the course of the research work.

I acknowledge Department of Biotechnology Department of Biotechnology Department of Biotechnology Department of Biotechnology for extending financial support for my

postgraduate programme.

I remember with gratitude, for kind co-operation and help of every staffstaffstaffstaff and other members of other members of other members of other members of

CPMBCPMBCPMBCPMB....

My heart is joyous to express the feelings with thanks to my labmates and friends, , , , Sruthi, Sruthi, Sruthi, Sruthi,

Cayal, Sanju, Ganesh, Kishor, Archana, Poorni, Trivima, Shweta, Ashok, Wadekar, Anu, Selva, Arul, Cayal, Sanju, Ganesh, Kishor, Archana, Poorni, Trivima, Shweta, Ashok, Wadekar, Anu, Selva, Arul, Cayal, Sanju, Ganesh, Kishor, Archana, Poorni, Trivima, Shweta, Ashok, Wadekar, Anu, Selva, Arul, Cayal, Sanju, Ganesh, Kishor, Archana, Poorni, Trivima, Shweta, Ashok, Wadekar, Anu, Selva, Arul,

Arvind, Ravi, Surender, Subru, Divya, Amudhan, Debayan, Beslin, Nirmal Arvind, Ravi, Surender, Subru, Divya, Amudhan, Debayan, Beslin, Nirmal Arvind, Ravi, Surender, Subru, Divya, Amudhan, Debayan, Beslin, Nirmal Arvind, Ravi, Surender, Subru, Divya, Amudhan, Debayan, Beslin, Nirmal and dearest junior friends,

Ramesh, Deepak, Arun, Malai, Rajguru, Kashmiri, Ranjini, Rajlakshmi, Tura, Karthik Ramesh, Deepak, Arun, Malai, Rajguru, Kashmiri, Ranjini, Rajlakshmi, Tura, Karthik Ramesh, Deepak, Arun, Malai, Rajguru, Kashmiri, Ranjini, Rajlakshmi, Tura, Karthik Ramesh, Deepak, Arun, Malai, Rajguru, Kashmiri, Ranjini, Rajlakshmi, Tura, Karthik and senior

research fellows Sowmya, Kalpana, Abirami, GowriSowmya, Kalpana, Abirami, GowriSowmya, Kalpana, Abirami, GowriSowmya, Kalpana, Abirami, Gowri and Dr. Dr. Dr. Dr. Mathiyazhagan Mathiyazhagan Mathiyazhagan Mathiyazhagan for their utmost

cooperation and help during the period of research.

My special thanks are due to my friends for their co-operation all through my journey till date,

Rashmi,Rashmi,Rashmi,Rashmi, Sonali,Sonali,Sonali,Sonali, Anu, Sumiya, Visha, Vimala, Ranjith, Rameshwar, Subhra, Ankita, Kamal, Mamta Anu, Sumiya, Visha, Vimala, Ranjith, Rameshwar, Subhra, Ankita, Kamal, Mamta Anu, Sumiya, Visha, Vimala, Ranjith, Rameshwar, Subhra, Ankita, Kamal, Mamta Anu, Sumiya, Visha, Vimala, Ranjith, Rameshwar, Subhra, Ankita, Kamal, Mamta

and all others all others all others all others for making these years ever memorable.

I wish to express my heartfelt thanks to my senior Mr. Mr. Mr. Mr. K. K. K. K. AshokAshokAshokAshok, , , , Phd Scholar for his whole

hearted and timely help in the progress of my research.

I also extend my sincere thanks to my seniors SudhaSudhaSudhaSudha, Phd Scholar; Shobhana, Shobhana, Shobhana, Shobhana, Phd Scholar; ; ; ;

Suresh, Suresh, Suresh, Suresh, PG scholar for generously helping me in every possible ways to complete my research successfully.

I am grateful to my beloved brothers and sisters, Subhasri, Subhajit, KrishSubhasri, Subhajit, KrishSubhasri, Subhajit, KrishSubhasri, Subhajit, Krishnedunedunedunedu, Sagnik, , Sagnik, , Sagnik, , Sagnik,

AbirAbirAbirAbirhotrahotrahotrahotra, Risha, Arindam , Risha, Arindam , Risha, Arindam , Risha, Arindam and Sujoy Sujoy Sujoy Sujoy who always stood by me with their boundless affection.

At this time of thesis submission, I remember my late mother Mrs. Chitrangada Mrs. Chitrangada Mrs. Chitrangada Mrs. Chitrangada who always

cared for me a lot and made me to learn more till the knowledge goes on.

I dedicate this thesis to my everloving parents Mr. Bhabatosh and Mrs. Sukla Mr. Bhabatosh and Mrs. Sukla Mr. Bhabatosh and Mrs. Sukla Mr. Bhabatosh and Mrs. Sukla and my uncles

and aunts, Ms. Bharati, Ms. Rudrani, Ms. Bharati, Ms. Rudrani, Ms. Bharati, Ms. Rudrani, Ms. Bharati, Ms. Rudrani, Dr.Dr.Dr.Dr. KKKKripan, Dr. Dhiman, Dr. Sangram, Mr. Raghubir, ripan, Dr. Dhiman, Dr. Sangram, Mr. Raghubir, ripan, Dr. Dhiman, Dr. Sangram, Mr. Raghubir, ripan, Dr. Dhiman, Dr. Sangram, Mr. Raghubir,

Mrs. Alo, Mrs. Bina, Mrs. Anuradha, Mrs. Sangeeta Mrs. Alo, Mrs. Bina, Mrs. Anuradha, Mrs. Sangeeta Mrs. Alo, Mrs. Bina, Mrs. Anuradha, Mrs. Sangeeta Mrs. Alo, Mrs. Bina, Mrs. Anuradha, Mrs. Sangeeta who showered their blessings in all my endeavors.

I humbly bow my head in front of my lordmy lordmy lordmy lord, who gave me everything to pursue this work into completion.

(Suchismita R(Suchismita R(Suchismita R(Suchismita Raha)aha)aha)aha)

ABSTRACT


By

SUCHISMITA RAHA

Degree : Master of Science in Biotechnology

Chairman : Dr. N.SENTHIL Associate Professor (Biotechnology),

Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore – 641 003. 2009

The present study was aimed at i) understanding the genetic variation for submergence

tolerance in rice and ii) understanding biochemical basis of improved submergence tolerance

exhibited by FR13A. Attempts were also made towards marker assisted introgression of Sub1 locus

controlling submergence tolerance in FR13A into a mega variety of TN namely, CO 43. Screening

for submergence tolerance revealed the superiority of FR13A over CO 43 for its ability to withstand

14 days submergence. FR13A was found to exhibit greater degree of recovery ability after 14 days

of submergence than CO 43. FR13A was found to accumulate significantly higher levels of total

carbohydrates than the susceptible CO 43. True F1 hybrids between CO 43 and FR13A were

selected through SSR genotyping using the SSR marker RM421 on chromosome 5. The quantitative

traits viz., days to flowering, plant height, number of tillers/hill, number of panicles/plant, panicle

length, number of grains per panicle, 100 grain weight and grain yield per plant were found to show

continuous variation within the population. Out of 232 SSR markers, 76 showed polymorphism

between FR13A and CO 43 which can be used in foreground selection, recombinant selection and

background selection. F2 plants harboring the Sub1 locus from FR13A were identified by genotyping

the population using RM219 which is tightly linked to Sub1 locus. Foreground selection revealed that

61 F2 plants were found carrying CO 43 allele of RM219, 125 F2 plants carrying both the alleles

(heterozygotes) and 64 F2s carrying FR13A allele of RM219. Phenotyping of selected F2 lines

confirmed the effect of Sub1 locus on tolerance against submergence and recovery after de-

submergence.

CONTENTS

CHAPTER NO.

TITLE

PAGE NO.

I

INTRODUCTION 1

II

REVIEW OF LITERATURE 3

III

MATERIALS AND METHODS 28

IV EXPERIMENTAL RESULTS 37

V

DISCUSSION 58

VI

SUMMARY 62

REFERENCES

APPENDX

LIST OF TABLES

Table No.

Title Page No.

1. Total carbohydrate contents (mg / 100mg Of leaf) in the control and submerged plants of CO 43 and FR13A

38

2. Morphological traits showed variation in F2 individuals of CO 43 and FR13A

44

3. Number of SSR primers (chromosomes wise) surveyed for assessing the polymorphism between the parents

51

LIST OF FIGURE

Figure No. Title Page

No.

1A Effect of submergence on the total carbohydrate levels in the tolerant FR13A

42

1B Effect of submergence on the total carbohydrate levels in the susceptible CO 43

42

2 Frequency distribution pattern for days to flowering in the F2 population 46

3 Frequency distribution pattern for plant height in the F2 population 46

4 Frequency distribution pattern for number of tillers among 256 F2 individuals

47

5 distribution for number of grains/panicle among the 256 F2 individuals 47

6 Frequency distribution for grain yield among the 256 F2 individuals 49

7A SSR primers used for surveying polymorphism between the parents FR13A and CO 43 from chromosome 1-6

52

7B SSR primers used for surveying polymorphism between the parents FR13A and CO 43 from chromosome 7-8 and from chromosome 10-12

53

8 List of SSR primers on chromosome 9 surveyed for polymorphism between the parents CO 43 and FR13A.

54

LIST OF PLATES

Plate No. Title Page

No.

1 Effects of submergence (13 days) on rice genotypes CO 43 and FR13A 39

2 Recovery of rice genotypes CO 43 and FR13A after 13 days of submergence 40

3 Agarose gel electrophoresis showing SSR fringerprinting of F1 hybrids along with the parents ( CO 43 and FR13A)

43

4 Grain type variations between parents and F2 population 48

5 Agarose gel electrophoresis illustrating the survey of polymorphism between CO 43 and FR13A using SSR markers located on chromosome 9

55

6 Genotyping of CO 43, FR13A and F2 individuals with SSR marker RM219 by PCR amplification and agarose gel electrophoresis

56

7 Response of progenies of selected F2 plants against 14 days submergence 57

INTRODUCTION

Rice, Oryza sativa L. (2n = 24) belonging to the family Graminae and subfamily Oryzoideae

is the staple food for half of the world’s population. With a compact genome, the cultivated rice

species Oryza sativa represents a model for other cereals as well as other monocot plants

(Shimamoto and Kyozuka, 2002). In India, the area under rice cultivation is 44.5 m ha with an annual

production of 96.69 million tonnes and an average productivity of 1.9 t ha–1 (http://indiabudget.nic.in).

About 32.4% of India’s total rice area, i.e., 15 m ha is under rainfed lowlands. Rainfed lowlands

constitute highly fragile ecosystems, always prone to flash-floods (submergence). Among the 42

biotic and abiotic stresses affecting rice production, submergence has been identified as the third

most important constraint for higher rice productivity causes total yield loss (Sarkar et al., 2006).

Scientists have estimated that 4 million tonnes of rice is being lost every year because of flooding

(IRRI, 2008).

Flood is the most damaging among the serious problems of agriculture. According to an

estimate of National Bureau of Soil Survey and Land Use Planning nearly 3.3 M ha of land is

affected by flood of varying degree. In Tamil Nadu, Cauvery river delta (rice bowl of TN) is facing

serious problems due to flash flooding during the monsoon period. About 3 lakhs ha of paddy area is

being affected severely every year due to submergence/flooding. The flooded area, severity of

flooding and the scale of damage are alarmingly increasing over the years. Moreover, under

changing climatic scenarios, crops will be exposed more frequently to episodes of drought, high

temperature and flood.

Even though rice is being cultivated under flooded and irrigated condition, most of the rice

varieties are susceptible to flooding if the plants are submerged under water for more than seven

days (Adkins et al., 1990). Hence, developing submergence/flooding tolerant rice genotypes will be

useful in reducing yield loss in rice in these areas. Submergence tolerance is a metabolic adaptation

in response to an anaerobiosis that enables cells to maintain their integrity to survive in hypoxia

without any major damage. High starch levels prior to submergence favoured tolerance by utilizing

non-structural carbohydrate to supply the required energy for growth and maintenance metabolism

(Jackson and Ram, 2003)

As with other major abiotic stresses, breeding and selecting successful submergence

tolerant rice cultivars have not yet met with notable commercial success till some years ago.

Germplasm survey revealed the existence of limited amount of genetic variation for submergence

tolerance. Intensive efforts at IRRI, Philippines resulted in the identification of a flood tolerant rice

line called “FR13A” which showed tolerance upto 14 days of flooding. Exploitation of this genetic

material in various breeding programs and mapping studies led to the understanding of genetic and

molecular basis of improved submergence tolerance in this rice genotype. Submergence tolerance in

FR13A is controlled by a putative Ethylene responsive Factor (Xu et al., 2006) located in the region

Sub1 on chromosome 9 (Xu and Mackill, 1996).

At IRRI, introgression of Sub-1 locus into a high yielding submergence susceptible Indian

variety "Swarna" was successfully carried out through marker assisted whole genome selection. The

improved Swarna called "Swarna Sub-1" showed improved level of tolerance to submergence than

the original Swarna and it possessed all the other desirable attributes of Swarna

(Neeraja et al., 2007). This report clearly showed the possibility of improving submergence tolerance

in rice through marker assisted introgression of Sub1 locus. The strategy of marker assisted

introgression of target locus through foreground and background selection improves the efficiency of

selection. Marker-assisted foreground selection would be especially effective for the transfer of

recessive genes since their classical transfer requires additional recurrent

selfing generations, a procedure that is prohibitively slow for most commercial breeders

(Welz and Geiger, 2000).

Based on these facts, the present study was undertaken with the following objectives:

1. Understanding genetic variation for submergence tolerance between CO 43, a popular long

duration rice variety of Tamil Nadu and FR13A

2. Understanding biochemical basis of improved submergence tolerance exhibited by FR13A

3. Marker assisted introgression of Sub1 locus controlling submergence into CO 43, a popular

variety of Tamil Nadu.

REVIEW OF LITERATURE

Rice is emerging as a model cereal for molecular biological studies. The main reasons for

this is complete genome has been sequenced. As it is the staple food for more than one-third of the

world’s population and is grown under a wide range of agroclimatic conditions in which it is subjected

to diverse biotic and abiotic stresses. Various abiotic stresses limit rice production in rainfed environments,

which comprise about 45% of the global rice area (Sripongpangkul et al., 2000). The rainfed lowland rice

ecosystem is affected by not only water deficit but also excess water. Even where rice response to

stress is superior to other crops, however, many rice-growing environments demand still greater

tolerance than is found in most improved germplasm (Lafitte et al., 2004).

2.1 Rice and submergence

Rice crop in lowland areas is invariably subjected to flooding stress continuously for various

periods. Nearly half of the ecosystem is prone to submergence damages caused by flash flooding.

Although the rice plant is well adapted to aquatic environments, it is unable to survive if it is

completely submerged in water for an extended period (Ito et al.,1999). Flooding is widespread in

southeast Asia, Bangladesh, and northeast India and about 15 million ha comes under potential

flash flood (short duration flood) in rainfed lowland rice areas and 5 million ha of deepwater rice

(Khush,1984). Flooding is a serious constraint to rice plant growth and survival in rainfed lowland

and deepwater areas because it results in partial or complete submergence of the plant. Flooding

imposes a severe selection pressure on plants principally because excess water in their

surroundings can deprive them of certain basic needs, notably of oxygen and of carbon dioxide and

light for photosynthesis. It is one of the major abiotic influences on species’ distribution and

agricultural productivity world-wide (Jackson et al., 2009).

In rice, young rice seedlings after transplanting are particularly vulnerable to submergence

Stress (Joho et al., 2008). The reproductive stage is the most sensitive to complete submergence,

followed by the seedling and the maximum tillering stages (Reddy and Mittra, 1985). The stage most

susceptible to partial submergence of at least 50% of plant height was the reductive division stage of

the pollen mother cells followed by the heading stage, the spikelet differentiation stage and all part of

the reproductive stage (Matsushima, 1962). Flooding during the seedling stage, increasing the water

depth inhibited the production of basal tillers and reduced tiller number, thereby decreasing eventual

grain yield (Lockard, 1958). The reduction in yield has been attributed to a decrease in the proportion

of ripened grains due to fertilization failure. Death in rice plants occurs when complete submergence

lasts longer than 1-2 weeks (Palada and Vergara, 1972).

Submerged rice plants experience two drastic environmental changes: the change from

aerobic to anaerobic conditions during submergence, and the subsequent change from anaerobic to

aerobic conditions when the floodwater recedes. Rice has well adapted to submergence-prone

environments by two strategies: (i) submergence tolerance to flash floods where a rapid increase in

water level causes partial to complete submergence for up to two weeks. (ii) shoot elongation by

types adapted to deepwater areas (>100cm) where water stagnates for several months and where

survival depends on shoot remaining in contact with the air. For deep water and floating rice, plants

have been selected to overcome lodging when the floodwater recedes by further elongation and

bending of the upper portions stems which keeps the panicle upright and off the soil surface (Catling

et al., 1988). How far submergence tolerant varieties have been developed (Mackill et al., 1993), but

have not been widely adopted. . A few tolerant landraces namely, FR13A, FR43B, Goda Heenati,

Kurkaruppan and Thavalu were identified that can withstand complete submergence for 10–14 days

(Xu and Mackill, 1996). Greater effort are needed to identify the traits required to improve genetic

adaptability of rice plants to those conditions, it is necessary to properly characterize the floodwater

environment and to closely investigate the physiological processes behind the plant response to the

changes (Ito et al., 1999).

2.2 Environmental characterisation of floodwater

Plant survival in submergence is greatly affected not only by depth of floodwater but also by

its physico-chemical characteristics (O2 and CO2 concentration, pH, turbidity, etc.). The adverse

effects on growth and metabolism are likely due to limited gas diffusion (Setter et al., 1988) and light

penetration (Palada and Vergara, 1972).

2.2.1 Gas diffusion

Limited gas diffusion is the most important factor during flooding (Setter et al., 1995). Since

gas diffusion is in 104-fold slower in solution than in air (Armstrong, 1979). Reduced movement of

gases to and away from submerged plant surfaces alters the concentration of O2, CO2 and ethylene

inside the plants. The depletion of O2 is a major feature of the flooded field which creates a condition

of low O2 (hypoxia) or no O2 at all (anoxia) around the plant tissues such as seeds or root apices and

stele. Though floodwater O2 concentration during flash floods is generally high, floodwater may

become anoxic in some environments, especially during the night when the O2 produced in the

daytime had been consumed for respiration. O2 concentration in stagnant air-saturated water of 0.25

mol m-3) was considered a reasonable threshold value required for respiration in germinating rice

seeds, coleoptiles, and embryos (Taylor, 1942).

2.2.2 Light

Light is another important factor to consider during submergence. When floodwater is turbid,

solar radiation under water becomes very low and limits the capacity of plants to photosynthesize.

Palada and Vergara (1972), observed a decrease in survival of rice seedlings after complete

submergence in turbid water because of lower light transmission (40% of that in air). There was a

reduction in solar radiation to <1% that in air at only 40 cm depth in one flood-affected location in

eastern India (Setter et al., 1995). Flood turbidity reduces light transmission and deposits silt on the

submerged plant. Low irradiance in surface water is occuring to surface algal colony and turbidity.

2.2.3 Temperature

Temperature is a further factor affecting the survival of plants during submergence. High

temperature (30ºC) accelerates plant mortality, where as low temperature (20ºC) improves survival.

High temperature decreases O2 and CO2 solubility in floodwater and accelerates anaerobic

respiration leading to faster starvation and faster death of the plant (Ram et al., 2002). Das et al.,

(2009), hypothesize that warmer water increases seedling mortality, possibly through increased

carbohydrate depletion during submergence and that turbid water will enhance plant mortality by

effects similar to those caused by natural shading, the common consequence of cloudiness during

the wet season. This could be caused by reduction in light penetration, the subsequent chlorophyll

degradation and reduced under-water photosynthesis.

2.2.4 Effect of nitrogen and phosphorous supply

Submergence strongly affects protein content, while nitrogen and phosphorous availability

and assimilation can influence submergence response. Protein reserves rapidly depletes due to

submergence through hydrolysis to amino acids and other soluble nitrogen-containing compounds

(Yamada, 1959). Palada and Vergara (1972) found that the increase in the percentage of nitrogen

content that normally occurs between 10 and 20 days after germination (from 3.1 to 4.3%) to be

abolished by submergence even reversed if the water is turbid. Mazaredo and Vergara (1982)

supported that the shoots of tolerant lines viz., FR13A were found to be richer in nitrate, containing

70 µg per plant shoot than susceptible one containing 20 µg per plant shoot. The effects of N

treatment during submergence increases chlorophyllase activity. Chlorophyllase activity increase in

the presence of ethylene, suggesting presence of higher leaf nitrogen in nitrogen treated seedling,

which enhances leaf senescence and greater chlorosis during submergence (Ella et al., 2003).

Ramakrishnayya et al., (1999) reported that applying phosphate to the plant at the time of

submergence reduce rice plant survival by 35%. The adverse effects of high phosphorous

concentration in flood water were mainly attributed to a promotion of algal growth resulting

competition between algae and submerged plant for CO2 and light.

2.3 Mechanisms of submergence tolerance

2.3.1 Morphological adaptation

2.3.1.1 Arenchyma

The presence of gas –filled spaces, known as aerenchyma, in roots of numerous plant species is

considered to be an important anatomical adaptation for survival under flooded conditions (Justin

and Armstrong, 1987). Although O2 transport through aerenchyma is most significant when the shoot

is above water, this pathway may be used to transport some of the O2 produced in the underwater

photosynthesis when suffcient light penetrates into the above canopy through water. The formation

of aerenchyma during flooding occurs not only in the roots but also in the leaves (Vartapetian and

Jackson, 1997). Aerenchyma allows rapid gas movement from shoots to roots and promote root

growth and survival in O2-deficient conditions (Ap Rees and Wilson, 1984; Armstrong, 1979). Such

gas-filled channels would supply O2 for root respiration and release O2 into the rhizosphere for

oxygenation.

2.3.1.2 Shoot elongation

Most rice cultivars elongate their shoot during total submergence. In small seedlings, this

response is restricted to emerging leaves. This is one of the escape strategies for adaptation to

submergence that promotes a return of part of the foliage to the air (Kende et al., 1998). The

mechanisms of plant adaptation to excessive flooding depend on the water regime. In deepwater

areas with >100 cm water depth for 2-3 months, cultivars with sufficient capacity for internode

elongation maintain their foliage above the water surfaces to sustain leaf photosynthesis and oxygen

transport, leading to better survival. Most rice cultivars show shoot elongation in response to

submergence, which enables rice plants to resume aerobic metabolism and photosynthesis fixation

of CO2 by raising their shoots above the shoot surface (Ram et al., 2002). Reduced elongation of

plants occurs under flash flood conditions which is necessary for survival because elongating plants

would tend to lodge as soon as the water level recedes (Jackson and Ram, 2003). The negative

relationship between flash-flood tolerance and shoot elongation during submergence was confirmed

using 903 cultivars from the International Rice Research Institute (IRRI) gene bank collection (Setter

and Laureles, 1996). Elongation is maximum at 14 days old seedlings followed by 21 and 30 days.

Survival is least in young seedlings and improved with seedling age. Leaf elongation during

submergence by the application of GA3, paclobutrazol which is known as an inhibitor of kaurene oxidation in

the gibbrellin biosynthesis pathway (Rademacher, 1992) and cycocel (CCC) have further confirmed the

hypothesis that minimum under water elongation is associated with increased survival. Increased in

the number of survival after complete submergence in submergence tolerant rice genotypes and the

addition of gibberellin reversed the effect. Like rice, Rumex has a range of genotypes with various

tolerance levels and adaptation to submergence: Rumex acetosa, intolerant of submergence,

ethylene-insensitive such that the petioles do not elongate either during submergence or exposure to

ethylene (Blom et al., 1990). Kawano et al., (2009) investigated that shoot elongation during

submergence uses energy and seems to consume carbohydrate in the leaves developed before the

submergence under water, where photosynthesis is limited. All cultivars are achieved by means of

extension growth by developing leaf sheaths during submergence. Submerged Saligbeli and Ballawe

cultivars did not show significant correlations between whole-plant dry matter weight and shoot

elongation during submergence indicating no photosynthetic gain as the plants extended upwards.

2.3.2 Hormonal regulation

2.3.2.1 Ethylene –a key regulator of submergence responses in rice

Ethylene is a major regulator of submergence where it acts as a major regulator of

submergence responses in rice (Oryza sativa). This gaseous phytohormone rapidly accumulates in

tissues of submerged plants due to physical entrapment and active biosynthesis during the stress,

triggering a range of acclimation responses including shoot elongation, adventitious root formation

and carbohydrate metabolism. In addition, ethylene coordinates the balance of gibberellic acid (GA)

and abscisic acid (ABA) contents, which facilitates GA-promoted elongation of shoots during

submergence. Besides cell elongation, the interaction of ethylene with GA and ABA also regulates

adventitious root formation, but these developmental processes are modulated by distinct regulatory

networks of the three hormones (Benning and Kende, 1992; Vriezen et al., 2003). Rapid stem

elongation is mediated by ethylene, where a genomic clone (OS-ACS5) encoding 1-

aminocyclopropane-1-carboxylic acid (ACC) synthase, which catalyzes a regulatory step in ethylene

biosynthesis, has been isolated from cv IR36, a lowland rice variety. Expression was induced upon

short- and long-term submergence in cv IR36 and in cv Plai Ngam, a Thai deepwater rice variety

(Straeten et al., 2001). In addition to ethylene, the phytohormones, gibberellic acid (GA) and abscisic

acid (ABA) are key signaling components in the orchestration of shoot elongation during

submergence in rice. Application of GA promotes cell division and cell elongation in internode

sections, as observed in response to ethylene. GA biosynthesis inhibitors, tetcyclacis (TCY) and

paclobutrazol (PB), restrict the elongation of shoots during submergence (Fukao and Serres, 2008).

In case of adventitious root formation consists of three developmental steps, death of the epidermal

cells which cover adventitious root initials, penetration of the roots from the epidermis, and initiation

of elongation growth. Ethephon treatment triggered all the developmental processes of adventitious

root development in nodes of deepwater rice even under aerobic conditions (Steffens and Sauter,

2005).

2.3.2.2 Expression analysis of ABA 8´-hydroxylase genes under submergence

The reduction in the ABA levels was caused by the activation of the ABA 8´-hydroxylation

pathway. The mRNA levels of three CYP707A genes from Nipponbare, designated as OsABA8ox1, -

2 and -3, by quantitative reverse transcription–PCR. The mRNA levels of OsABA8ox2 and OsABA8ox3 did

not increase, but instead decreased gradually (Krochko et al., 1998, Kushiro et al., 2004).

2.3.2.3 Expression of ABA biosynthetic genes under submergence

The expression of four genes involved in the biosynthesis of ABA. The mRNA levels of

OsZEP and OsNCED3 began to decrease 1 h after submergence, and those of OsNCED1 and

OsNCED2 began to decrease 2 h after submergence. Expression of all four genes tended to

decrease gradually, although the expression of OsZEP and OsNCED2 showed slight increases

8–12 h after submergence (Saika et al., 2007).

During stress, submergence-stimulated decrease in ABA content was Sub1A-independent,

whereas GA-mediated underwater elongation was significantly restricted by Sub1A. Transgenics that

ectopically express Sub1A displayed classical GA-insensitive phenotypes, leading to the hypothesis

that Sub1A limits the response to GA. Notably Sub1A increased the accumulation of the GA

signaling repressors Slender Rice-1 (SLR1) and SLR1 Like-1 (SLRL1) and concomitantly diminished

GA-inducible gene expression under submerge conditions. In the Sub1A overexpression line, SLR1

protein levels declined under prolonged submergence but were accompanied by an increase in

accumulation of SLRL1, which lacks the DELLA domain (Fukao and Serres, 2008).

2.4 Carbohydrate reserves sustained sugar supply and energy metabolism

Submergence tolerance is related to high carbohydrate supply during submergence.

Carbohydrate metabolism during submergence seems to be an important factor in flash-flood

tolerance and this strategy is characterized by slow expansion growth that is presumed to conserve

energy (Singh et al., 2001). The role carbohydrate plays in submergence tolerance is presumably

through energy supply needed for maintenance processes. The impact of respirable reserves on the

extent of submergence tolerance shows variation in carbohydrate levels. Yamada (1959) reported

the rice plant exhausts a rapid loss of starch and total carbohydrate during submergence in leaves,

leaf sheaths and roots occurs during submergence stress. Pre –submergence stored carbohydrate

are reported to be associated with enhanced survival under flooded conditions, possibly by supplying

energy for maintenance through anaerobic respiration (Das et al., 2005). Boamfa et al., (2003) reported that

in double haploid population and the parents, FR13A and CT6241 showed poor survival in the morning and

better survival during submergence in the evening when plant carbohydrate concentrations were high. The

high starch levels prior to submergence favored tolerance. As starch is the limiting factor for survival in

submerged plants. The ability to store carbohydrates in underground organs before the wet season is one of

the strategies favoring survival during flooding (Crawford, 1992), which even enable the forest trees to

survive under water of Amazonian floodplain forests (Parolin, 2009). Since anaerobic metabolism is

costly in terms of carbohydrate consumption as compared with normal aerobic respiration.

The genetic diversity in carbohydrate concentrations of plants prior to submergence and its

implication on plant survival has been emphasizes (Mallik et al., 1995). Old seedlings tend to have

large carbohydrate reserves and therefore good survival during submergence (Adkins et al., 1990).

Culms of submergence –tolerant plants contained starch even after being submerged whereas

intolerant rice cultivars were exhausted during the same period (Mallik et al., 1995). Ricard et al.,

(1991) reported that the induction of sucrose synthase in anoxic rice seedlings is a response to increased

demand for sugars at the onset of fermentative metabolism. Due to increase in the flux of carbohydrates

through the glycolytic pathway would thus enable rice seedlings to produce more energy for longer periods

and thus increase survival under anoxia. The protective effects of glucose during anoxia have been

observed in excised tissue of rice seedlings (Valdez, 1995), in roots of 4-5 days old, intact wheat seedlings

(Waters et al., 1991), and in germinating rice seeds (Ella and Setter, 1999)

It has been reported that transcripts of rice α-amylases accumulate in the seed embryo and

aleurone during germination even under anoxia (Hwang et al., 1999). In addition, α-amylase protein levels

and activity were shown to be induced by anoxia in rice seedlings (Guglielminetti et al., 1995). Semi-

quantitative RT-PCR detection of the transcripts of three α-amylase genes, Rice Amylase-3C (RAmy3C),

RAmy3D, and RAmy3E, revealed that their up-regulation was controlled by the Sub1 locus (Ismail et al.,

2009).

2.5 Anaerobic protein

Submergence or anoxia –tolerant and intolerant species may differ in the number and the

level of production of anaerobic proteins due to repression of most aerobic protein synthesis, during

response of plant tissues to O2 depletion. Most of the anaerobic proteins, however, are enzymes

involved in carbohydrate metabolism and alcoholic fermentation. Six of the inducible proteins have

been identified as cytosolic enzymes; alcohol dehydrognase, aldolase, glucose phosphate

isomerase, sucrose synthase, pyruvate decarboxylase, and glyceraldehydes phosphate

dehydrogenase in many crops including rice (Walker et al., 1987). In rice, Reggiani et al., (1990)

observed that repression was greater in membrane proteins than in soluble proteins. In maize, a set

of 20 anaerobic polypeptides is selectively expressed in primary roots (Sachs et al., 1980). A similar

pattern of altered gene expression was observed in barley (Hoffman et al., 1986), in cottonwood

(Kimmerer, 1987), in tomato (Tanksley and Jones, 1981), in pea (Llewellyn et al., 1987), and in soybean

(Tihanyi et al., 1989). Vartapetian and Poljakova (1994) demonstrated the inhibition of ANPs

(anaerobic proteins) synthesis in rice coleoptiles and a consequent decline in anoxia tolerance when

treated with cycloheximide, a protein synthesis inhibitor.

2.6 Alcoholic fermentation

Anaerobic response of plant tissues is the adaptive metabolic mechanism of increasing rate

of alcoholic fermentation (AF) which involves alcohol dehydrogenase (ADH) and pyruvate

decarboxylase (PDC) as the two key enzymes. Submergence can shift aerobic respiration to the less

efficient anaerobic fermentation pathway as the main source of energy production. Acetaldehyde is

one of the intermediate of alcoholic fermentation, which can be oxidized by aldehyde dehydrogenase

(ALDH) and found to be low in plants having higher activities ALDH with concomitant increase in

submergence tolerance (Sarkar et al., 2006). The beneficial effect of alcoholic fermentation in

growth and survival of rice under anoxia due to several points of view: (i) enzymes of alcoholic

fermentation often increase (Drew et al., 1994), (ii) hypoxic pretreatment increased anoxia tolerance

in submerged rice seedlings (Ellis and Setter, 1999), (iii) higher sugar supply improves survival.

Schwartz (1969), observed that in maize, Arabidopsis ADH-null mutants, and rice ADH-reduced

mutants showed lower tolerance to anaerobic conditions.

2.7 Post-submergence injury

The re-entry of air after de-submergence introduces higher O2 concentration relative to the

very low concentration under water. Injury of the submerged plant generally develops after de-

submergence (Gutteridge and Halliwell, 1990; Crawford, 1992) and is possibly caused by active O2

species (Hunter et al., 1983; Crawford, 1992). O2 is one possible source of active O2 species. When

O2 gets reduced, one electron leaks out from the electron transfer system, converting it into

superoxide anion (O-2). Superoxide anion, in return, produces more active O2 species; hydrogen

peroxide (H2O2) and hydroxyl radical (OH-).

The active O2 species are cytotoxic because these are highly reactive. It oxidizes

unsaturated fatty acids of the lipid layers in cellular membrane or in intercellular organelles, known

as lipid peroxidation. Lipid peroxidation is at its peak soon after de-submergence. The level of lipid

peroxidation in anoxia-intolerant Iris germanica increased by 157-fold relative to the non-submerged

control, where in case anoxia-tolerant I. pseudacorus increased by only 1.2-fold (Hunter et al., 1983).

Lipid peroxidation and associated harmful effects of anoxia and submergence can be reduced by

substances like α-tocopherol and carotenoids. The level α-tocopherol was three times higher in

submerged rice seedlings than that of aerobically grown controls and remained higher for 24 h after

transfer of seedlings to air (Ushimaru et al., 1994).

2.8 Genetics of submergence

The expression of submergence tolerance is known to be environmentally dependent and

genetically complex (Suprihatno and Coffman, 1981; Setter et al., 1997). Genetics studies suggested

both simple and quantitative inheritance for submergence tolerance. Using population derived from a

cross between an indica submergence –tolerant line (‘IR40931-26’) and a susceptible japonica line

(‘P1543851’), a mojor QTL was fine mapped on chromosome 9, designated as Sub1. The locus

showed 70% of phenotypic variation in submergence tolerance. A few cultivars, such as the O.

sativa ssp. indica cultivar FR13A, are highly tolerant and survive up to two weeks of complete

submergence owing to a major quantitative trait locus Submergence 1 (Sub1) near the centromere

of chromosome 9. Cluster of three genes at Sub1 locus, encoding putative ethylene response factors

(ERFs)/ethylene-responsive element binding proteins/Apetala2-like proteins (Xu et al., 2006; Perata

and Voesenek, 2007). All three Sub1 region genes fall in the B-2 subclass of ERF proteins, which

contains a single 58- to 59-residue ERF domain. Two of these genes, Sub1B and Sub1C, are

invariably present in the Sub1 region of all rice accessions analysed. In contrast, the presence of

Sub1A is variable. A survey identified two alleles within those indica varieties that possess this gene:

a tolerance-specific allele named Sub1A-1 and an intolerance-specific allele named Sub1A-2. Over

expression of Sub1A-1 in a submergence-intolerant O. sativa ssp. japonica conferred enhanced

tolerance to the plants, down regulation of Sub1C and up regulation of Alcohol dehydrogenase 1

(Adh1), indicating that Sub1A-1 is a primary determinant of submergence tolerance.

The molecular mechanism behind the deepwater rice responses through the identification of

the genes viz., SNORKEL1 (SK1) and SNORKEL2 (SK2), which trigger deepwater response by

encoding ethylene response factors involved in ethylene signalling. The products of SNORKEL1 and

SNORKEL2 that trigger remarkable internode elongation via gibberellin. The deepwater rice C9285

possesses SK1 and SK2, although both genes are absent from the non-deepwater rice T65. SK1

and SK2 possess a putative nuclear localization signal and a single APETALA2/ethylene response

factor (AP2/ERF) domain. The ERF domains in the SK genes showed a high similarity to those of

Arabidopsis thaliana (At) ERF1, Oryza sativa (Os) ERF1 and SUB1A-1. The SK genes were significantly

expressed under deepwater conditions, whereas these expressions were low under dry conditions in

C9285. Compared to transgenic plants developed which overproduced SK genes driven by the OsAct1

promoter in T65 background showed SK1 gene drives elongation one to three internodes and SK2-

overproducers elongated one to seven internodes, even under dry conditions (Hattori et al., 2009).

2.9 Phylogenetic analysis of Sub1 genes

Orthologues of the Sub1 genes were isolated from O. rufipogon and O. nivara by use of

oligonucleotide primers corresponding to the most highly conserved regions of the Sub1 genes of

domesticated rice, in search of orthologues of Sub1 locus in the closest relatives of O. sativa to

provide insight into the origin of gene and allelic variation. For direct comparison of Sub1

genes/alleles, the nucleotide and amino acid sequences were subjected to pairwise and multiple

alignment analyses using EMBOSS (Labarga et al., 2007) and ClustalW2 (Larkin et al., 2007). The

Sub1 orthologues of O. nivara and O. rufipogon used for the analysis are OnSub1B-1 (EU429442),

OnSub1B-2 (EU429443), OnSub1C-1 (EU429445), OrSub1B-1 (EU429444) and OrSub1C-1

(EU429446). Phylogenetic analyses were done by truncated nucleotide sequences were aligned with

ERF2 under ERF gene family (Subgroup VII). The evolutionary relatedness of Sub1 genes in O.

sativa, O. nivara and O. rufipogon, a neighbor-joining method of phylogenetic analyses was used.

The three Sub1 genes, Sub1A, Sub1B and Sub1C, were separated into three distinct clades with

significant bootstrap value supporting the phylogeny. The Sub1A gene, which is present only in

some indica varieties, was more related to Sub1B than Sub1C. The Sub1B alleles of O. sativa were

resolved into two subgroups, which corresponded to the Sub1 locus haplotype. The Sub1B alleles of

O. nivara and O. rufipogon co-clustered with the alleles of rice accessions that lack Sub1A, which is

in accordance with the absence of the Sub1A gene in the wild-rice germplasms examined whereas

the Sub1C alleles were resolved only in two subgroups (Fukao et al., 2008)

2.10 Submergence-induced gene OsCTP in rice

The PCR based suppression subtractive hybridization (SSH) method to identify

submergence-induced genes from the submergence-tolerant rice cultivar, FR13A. These genes

putatively encode four proteins including a cation transport protein (OsCTP), monogalactosyl–

diacylglycerol synthase, a cold-induced protein and glutathione synthetase. OsCTP was generated

by rapid amplification of cDNA ends that encodes a putative protein of 137 amino acids. OsCTP

expression is enhanced by submergence as well as stress induced by abscisic acid, salt or drought.

OsCTP might encode a novel cation transport protein similar to Escherichia coli ChaC and may be

associated with a general defensive response to various environmental stresses (Qi et al., 2005).

2.11 QTLs affecting flood tolerance in rice

The Sub1 locus maps to a region of chromosome 9 near to centromere is not very dense in

RFLP marker, RFLP marker R1164 from Genome Programme of Japan has been measured to be

1cM from the gene (Xu et al., 2000). Quantitative trait locus (QTL) mapping determines the number,

genome location and effect of QTLs associated with responsive traits to submergence stress. Major

QTL determinig traits associated with submergence tolerance was mapped in vicinity on rice

chromosome 9 with and small–effect QTLs on rice chromosomes 2, 5, 7, 10, 11 (Kamolsukyunyong

et al., 2001). An AFLP map constitute of 202 AFLP marker with a map length of 1756cM, detected

QTL associated with submergence tolerance on chromosome 6,7,11 and 12 (Nandi et al., 1997).

QTLs affecting elongation ability was identified in IR74/Jalmagna RI populations was Qph at

chromosome 1, 2, 3, 4, 6, 7. QTL for initial plant height was mapped in chromosome 1,3,10 with

60.3% total phenotypic variation, was sd-1 (Sripongpangkul et al., 2000).

Four putative QTLs for submergence tolerance during germinating in rice were detected in

the backcross population of IR64/KHAIYAN. Each QTL on chromosome 1 (qAG-1), 2 (qAG-2), 11

(qAG-11) and 12 (qAG-12), with a LOD value range from 3.66 to 5.71 and phenotypic variance

ranged from 12 to 29.24% (Angaji, 2008).

The internodes of deepwater rice can elongate in response to rises in water level. Inouye

(1983) proposed using position of the lowest elongated internode (LEI) and total internode

elongation (TIL), the number of elongated internodes (NEI) to evaluate total internodel elongation.

These three parameters, Hattori et al., (2008) detected five QTLs viz., qTILI on chromosome 1,

qLEI3 on chromosome 3 and qTIL12 , qNEI12 and qLEI12 on chromosome 12.

Deepwater rice (floating rice) can survive under flooded conditions because of their floating

ability. The position of the lowest elongated internode (LEI) and the rate of internode elongation

(RIE) were used to measure floating ability. QTL qLEI3 on chromosome 3 and qLEI12 on

chromosome 12 were detected for LEI and qRIEI1 on chromosome 1 and qRIEI12 on chromosome

12 were detected for RIE (Kawano et al., 2008).

2.12 Microsatellites

The genomes of higher organisms contain tree types of multiple copies of simple repetitive

DNA sequences (satellite DNAs, minisatellites, and microsatellites) arranged in arrays of vastly

differing size (Armour et al., 1999; Hancock, 1999). Microsatellites (Litt and Luty, 1989), also known

as simple sequence repeats or SSRs (Tautz et al., 1986), short tandem repeats (STRs) or simple

sequence length polymorphisms or SSLPs (McDonald and Potts, 1997), are the smallest class of

simple repetitive DNA sequences. Microsatellites are born from regions in which variants of simple

repetitive DNA sequence motifs are already over represented. SSR allelic differences are, therefore,

the results of variable numbers of repeat units within the microsatellite structure. The repeated

sequence is often simple, consisting of two, three or four nucleotides (di-, tri-, and tetranucleotide

repeats, respectively). One common example of a microsatellite is a dinucleotide repeat (CA)n,

where n refers to the total number of repeats that ranges between 10 and 100. These markers often

present high levels of inter- and intra-specific polymorphism, particularly when tandem repeats

number is ten or greater (Queller et al., 1993).

2.12.1 Application of microsatellite markers

SSR markers have been used as powerful tools in the assessment of genetic variation and

in the elucidation of genetic relationships within and among species. It has been reported that the

genetic diversity and DNA fingerprinting of 15 elite rice genotypes using 30 SSR primers on

chromosome numbers 7-12 revealed that all the primers showed distinct polymorphism among the

cultivars studied indicating the robust nature of microsatellites in revealing polymorphism

(Chakravarthi and Naravaneni, 2006). SSR are economically employed in hybrid rice breeding

programs. These markers have been used to define heterotic groups in rice (Xiao et al., 1996), to

study the genetics of heterosis (Hua et al., 2000), transgressive variation (Xiao et al., 1998), hybrid

fertility (Zhang et al.,1997), to transfer the traits via marker-assisted selection (He et al., 2000), to

define introgressions in wide hybridization programs (Brar et al., 2000), to construct ordered sets of

substitution lines (Lorieux et al., 2000) and for the study of microsynteny in the chloroplast genomes

of Oryza and eight other Graminae species (Ishii and McCouch, 2000). Microsatellite in rice called

rice microsatellite, which is co-dominant nature and highly reproducible and easy to optimized

(Semagn et al., 2006).

2.13. Molecular breeding for rice improvement

Earlier reported by Mohanty and Khush (1985), explained the diallele analysis of submergence

tolerance in rice. It indicated Tolerance was dominant over non-tolerance and the average

dominance was within the range of incomplete dominance. Dominant alleles were more

concentrated in submergence tolerant. Mishra et al., (1996) revealed that submergence tolerance in

tolerant rice cultivar is governed by one dominant gene. Genetic improvement of submergence

tolerant lines for increasing the grain yield can be obtained by modified breeding and selection

strategy (Cooper et al., 1999). The development of DNA (or molecular) markers has irreversibly

changed the disciplines of plant genetics and plant breeding. It has been used effectively to identify

resistance genes, and MAS has been applied for integrating different resistance genes into rice

cultivars lacking the desired traits (Jena and mackill, 2008). While there are several applications of

DNA markers in breeding, the most promising for cultivar development is called marker assisted

selection (MAS). The fundamental advantages of MAS compared to conventional phenotypic

selection are:

• Simpler compared to phenotypic screening

• Selection may be carried out at seedling stage

• Single plants may be selected with high reliability.

DNA markers that are linked to them are accomplished via QTL mapping experiments. QTL

mapping represents the foundation of the development of markers for MAS (Mackill et al., 1999;

Collard et al., 2005, 2008).

2.13.1 Marker assisted selection for submergence tolerance rice

MAS is relatively more efficient than selection by phenotype alone (Lande and Thompson,

1990). The success of MAS depends on location of the markers with respect to genes of interest.

The relationships between the markers and respective genes could be distinguished; the molecular

marker is located within the gene of interest, which is the most favourable situation for MAS and in

this case, it could be ideally referred to as gene-assisted selection (Babu et al., 2004).

Microsatellite markers are highly suitable for MAS in rice (Mackill et al., 2006) compared to

RFLP and RAPD markers. The microsatellite marker RM219 and the codominant PCR-based marker

RM464A (derived from a microsatellite marker, RM464) was selected for submergence tolerance.

The two markers RM219 and RM464A were found to be linked to Sub1 by 3.4 and 0.7 cM,

respectively further tested in 55 diverse indica and japonica rice cultivars and breeding lines (Xu et

al., 2004). Amplification products of five microsatellite markers, RM285, RM316, RM444, RM464,

and RM219 (Chen et al., 1997; Temnykh et al., 2001) linked to Sub1, were compared between the

two parents (DX202-9 and M202).

2.14 Marker assisted backcross approach for submergence tolerance rice

The basis of a marker-assisted backcrossing (MAB) strategy is to transfer a specific allele at

the target locus from a donor line to a recipient line while selecting against donor introgressions

across the rest of the genome. The use of molecular markers, which permit the genetic dissection of

the progeny at each generation, increases the speed of the selection process, thus increasing

genetic gain per unit time (Tanksley et al., 1989). MAB has previously been used in rice breeding to

incorporate the bacterial blight resistance gene Xa21 (Chen et al., 2000, 2001), waxy gene (Zhou et

al., 2003). It has been shown an effective means of utilizing QTLs with large effects like Sub1 in rice

breeding programs (Toojinda et al., 2003, 2005). It is reported that the molecular markers that were

tightly linked with Sub1, flanking Sub1, and unlinked to Sub1 were used to apply foreground,

recombinant, and background selection, respectively. The selected markers (two to four markers on

a chromosome of 100 cM) provide adequate coverage of the genome in backcross programs (Servin

and Hospital, 2002). In backcrosses between a submergence tolerant donor and the widely grown

recurrent parent Swarna. Generation of Swarna–Sub1 was produced by crossing the Indian mega-

variety Swarna to the FR13A and subsequent backcross to Swarna. Introgression of Sub1 did not

have any negative impact under control conditions with respect to phenology, yield, and grain

quality; however, Sub1 lines showed substantially higher yields after submergence. Flowering and

maturity is earlier, and had better grain filling (Singh et al., 2009). MAB scheme has been

investigated by computer stimulations and marker data points reduced (MDPs) by determining

minimum population sizes required for recombinant selection and appropriate population sizes,

ratios and selection strategies for background selection (Visscher et al., 1996). Rather than rice

Jiang et al., (2000) reported that the transmission genetics of advanced- generation backcross

progenies of a cross between two recently diverged allotetraploid cotton species, Gossypium

hirsutum L. and G. barbadense L. through introgression of RFLP markers.

2.14.1 Foreground selection

The selection of the Sub1 locus (foreground) was done by the reported rice microsatellite

(RM) markers RM219 and RM464A, which were found to be linked to Sub1 by 3.4 and 0.7 cM were

used (Xu et al. 2004), and RM316 was also used for foreground selection, distance of 1.5 cM from

RM464A according to the published map (Temnykh et al., 2001).

2.14.2 Recombinant selection

Based on the fine mapping of the Sub1 locus and sequence information (Xu et al. 2006),

four Bacterial Artificial Chromosome (BAC) clones (AC090056, AP005705, AP005907 and

AP006758) of japonica Nipponbare (IRGSP 2005) corresponding to the Sub1-linked marker

(RM464A) was identified. Motifs of SSRs in the BAC clones were identified using the SSRIT or

Simple Sequence Repeat identification Tool (Temnykh et al., 2001). Recombinant selection was

done by flanking markers, flanked about 5 Mb regions on each side of the Sub1 locus. Microsatellite

markers were identified from the reported 20 BACs flanking the Sub1 locus

(IRGSP 2005).

2.14.3 Background selection

The availability of closely linked markers and/or flanking markers for the target locus, the

size of the population, the number of backcrosses, the position and number of markers develop

background selection (Frisch and Melchinger, 2005). Microsatellite markers unlinked to Sub1

covering all the chromosomes including the Sub1 carrier chromosome 9, that were polymorphic

between the two parents, were used for background selection to recover the recipient genome.

Based on the polymorphism information, initially evenly spaced microsatellite markers were selected

per chromosome. At least three polymorphic microsatellite markers per chromosome were used,

which revealed fixed (homozygous) alleles at non-target loci at one generation (Neeraja et al., 2007).

2.14.4 Sub1-specific markers

In the early applications of MAB for developing submergence-tolerant varieties the

diagnostic marker used was Gns2, a cleaved amplified polymorphic sequence (CAPS) marker that

was used in combination with several markers flanking Sub1 (Neeraja et al., 2007). Additional allele

specific and intragenic markers were developed to measure the introgressed region of Sub1 locus

more precisely. A specific single nucleotide polymorphism (SNP) within the Sub1 coding region that

causes an amino acid substitution (intolerant: CCC=proline; tolerant: TGC=serine) were targeted for

marker design. A dominant sequence tag site marker was developed by designing a PCR primer

with SNP at 3´ end named AEX marker and two CAPS marker designed in the promoter region of

Sub1A, named IYTI and IYT3 (Septiningsih et al., 2009). Sequencing of Sub1C revealed seven

allelic groups and a unique phosphorylation site are found for the tolerant lines FR13A, Kurkaruppan,

Goda Heenati, IR40931 and IR40981 (Xu et al.,, 2006). An insertion–deletion (indel) marker for

Sub1C named SUB1C173, was designed and used in addition to the Sub1A markers.

MATERIALS AND METHODS

The present study was undertaken with the aim of i) understanding the genetic variation and

physiological basis of submergence tolerance in rice and ii) marker assisted introgression of Sub1

locus controlling submergence tolerance into CO 43, a popular rice variety of Tamil Nadu. All the

field experiments were conducted at Paddy Breeding Station, Tamil Nadu Agricultural University

(TNAU), Coimbatore. All the biochemical and genotyping experiments were conducted at

Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology,

TNAU, Coimbatore-03 during 2007-2009. The materials used and methods adopted in this study are

described below.

3.1. Understanding genetic variation for submergence tolerance in rice

3.1.1. Genotypes used

Two rice genotypes namely FR13A (Flood Resistant 13A) and CO 43 were selected for

various physiological studies. FR13A is a photoperiod-sensitive and highly submergence tolerant

rice genotype but possessing undesirable agronomic traits viz., low yield, awns and poor cooking

quality (Siangliw et al., 2003). CO 43 is a long duration rice variety released from Paddy Breeding

Station, TNAU, Coimbatore and it is popularly grown in irrigated areas of Tamil Nadu. It is derived

from a cross between Dasal X IR20 and known for its high level of salinity tolerance.

Seeds of FR13A were obtained from Central Rice Research Institute, Cuttack and seeds of

CO 43 were obtained from Paddy Breeding Station, Tamil Nadu Agricultural University (TNAU).

Evaluation of rice genotypes for submergence tolerance was performed under green house

conditions. Rice plants were grown under normal conditions with six pots for each variety. After 21

days, a set of three pots for each variety was submerged in 1.5 m height plastic tanks filled with

water. Plants were monitored at every 3 days interval (3rd, 7th, 10th, 14th days after submergence and

10 days after de-submergence) and leaf samples were collected for carbohydrate estimation. After

14 days of submergence, pots were taken-out from the tanks and evaluated for their level of

tolerance. Recovery ability of genotypes was assessed after 10 days of de-submergence.

3.2 Biochemical basis of submergence tolerance in rice

3.2.1 Estimation of total carbohydrates in rice shoots

Leaf tissues were collected from the control, submerged (0, 3rd, 7th, 10th, 14 days after

submergence) and de-submerged plants of CO 43 and FR13A were dried at 70ºC for 48 hrs and

ground in a mortar and pestle. Total carbohydrate contents of the above samples were estimated as

described by Fales, (1951).

3.2.2 Requirements

• Dried ground leaf samples

• 2.5N HCl

• Anthrone reagent (C14H10O; M.W.- 194.24)

o Anthrone reagent: 200 mg of Anthrone was dissolved in 100 ml of ice cold 95%

H2SO4 (It should be freshly prepared before use)

• Standard glucose:

o Stock solution- 100 mg dissolved in 100 ml of distilled water (concentration mg/ml)

o Working standard – 10 ml of stock solution diluted to 100 ml with distilled water.

(Stored under refrigeration after adding a few drops of toluene)

3.2.3 Carbohydrate estimation

• About 100 mg of ground leaf samples were taken into boiling test tubes (50ml)

• The hydrolysis was done by keeping the tubes in water bath for 3 hours with 5ml of 2.5 N

HCl and cool down to room temperature.

• The hydrolysed samples were neutralised with solid sodium carbonate until the

effervescence ceases. And the volume was made upto 100 ml using distilled water.

• 10 ml was taken from the sample solution and centrifuged at 5000 rpm for 10 minutes.

• The supernatant was collected in a falcon tube and 0.1 ml and 0.2 ml aliquots were taken for

analysis.

• Standards were prepared by taking working standard at different concentrations- 0 ml as

blank, 0.2 ml, 0.4 ml. 0.6 ml, 0.8 ml and 1 ml in test tubes.

• Volume was made upto 1 ml using distilled water and 4 ml Anthrone reagent was added in

each tube (The contents of all the tubes were cooled on ice before adding ice –cold

Anthrone reagent).

• All the test tubes were heated for eight minutes in boiling water bath.

• Test tubes were rapidly cooled to room temperature and the absorbance of standards and

samples were measured using a spectrophotometer at 630 nm

3.2.4 Calculation

mg of glucose Amount of carbohydrates present in 100 mg of leaf sample = X 100 ml Volume of test sample

3. 3 Introgression of Sub1 locus controlling submergence tolerance from FR13A into CO 43

3.3.1 Generation and evaluation of F1 hybrids between CO 43 and FR13A

Crosses were made between CO 43 and FR13A during Rabi’2007 by keeping CO 43 as a

female parent and FR13A as a male parent. Obtained seeds were raised during summer 2007-08

and F1 hybrids were evaluated in the field. True F1 hybrids were selected based on morphological

markers. To confirm the hybridity, SSR genotyping was done using the genomic DNA isolated from

F1s and parents and true hybrids were tagged.

3.3.2 Isolation of genomic DNA

DNA was extracted from fresh leaf tissue for all the F1 individuals and their parents using the

modified CTAB protocol as described by Ausubel et al., (1994). The quality of DNA was checked by

agarose gel electrophoresis and quantified by Nanodrop Spectrophotometer.

3.3.2.1 Requirements

a) Leaf samples (leaf samples were collected from 30 days old seedlings and stored at –80°

C till use.

b) Cetyl Trimethyl Ammonium Bromide (CTAB) Extraction buffer (100 ml):

1. CTAB 2% (w/v)

2. Tris HCl (pH 8.0) 100 mM

3. Sodium chloride 1.4 M

4. EDTA 20 mM

(Tris, sodium chloride and EDTA were autoclaved and 2% CTAB was added after

autoclaving and preheated before using the buffer)

a) Tris EDTA (TE) Buffer

Tris HCl (pH 8.0) 10 mM

EDTA (pH 8.0) 1 mM

(This was dissolved and made up to 100 ml, autoclaved and stored at 4°C)

b) Ice-cold Isopropanol

c) Chloroform: Isoamylalcohol 24:1 (v/v)

d) Sodium acetate (3.0 M, pH 5.2) (pH adjusted using glacial acetic acid)

e) Ethanol (70% and 100%)

f) RNase A - 10 mg/ml

(RNase A was dissolved in TE buffer and boiled for 15 minutes at 100°C to destroy

DNase and stored at -20°C).

3.3.2.2 Protocol

• About 200 mg of leaf samples were cut into small bits with the help of sterile scissors and

transferred to sterile mortar.

• The leaf tissues were ground in liquid nitrogen and extracted with 600 µl of CTAB buffer and

incubated for 30 minutes at 65°C in water bath with occasional mixing.

• The tubes were removed from the water bath and equal volume of chloroform: Isoamyl

alcohol mixture (24:1 v/v) was added and mixed by inversion for 15 minutes.

• It was centrifuged at 10,000 rpm for 20 minutes at room temperature.

• The clear aqueous phase was transferred to a new sterile eppendorf tube.

• Equal volume of ice cold isopropanol was added and mixed gently by inversion and then

kept in the freezer until DNA was precipitated out.

• Using blunt end tips, the precipitated DNA was spooled out into an eppendorf tube.

• The spooled DNA was air dried after removing the supernatant by brief spin.

• 100 µl of TE was added to dissolve the DNA and then 10 µl of RNase was added and

incubated at 37°C for 35 minutes.

• 500 µl of Chloroform: Isoamylalcohol mixture was added and centrifuged for 10 minutes.

• Aqueous phase was transferred to another eppendorf without disturbing the inner phase.

• 2.5 volume of absolute alcohol and 1/10 volume of sodium acetate were added and kept for

overnight incubation.

• Then it was centrifuged and the supernatant was discarded. To this 500 µl each of 70% and

100% ethanol was used subsequently to wash the DNA by centrifugation.

• The alcohol was discarded and DNA was completely air-dried.

• Then the DNA pellet was dissolved in 100 µl of TE and stored at -30°C.

3.4 Assessing the quality of DNA by agarose gel electrophoresis

3.4.1 Chemicals used

a) Loading Dye

Glycerol 50% (v/v)

Bromophenol blue 0.5% (w/v)

b) 10X TAE (Tris Acetate EDTA buffer)

Tris Base 48.4 g

Acetic acid 11.42 ml

0.5MEDTA 20 ml

(Dissolved in 800 ml of sterile water and made up to 1000 ml)

3.4.2 Protocol

• The Pyrex gel casting plate open ends were sealed with cello tape and the comb was placed

properly in casting plate kept on a perfectly horizontal platform.

• 0.8 % (0.8 g/100 ml) agarose was added to 1X TAE, boiled until the agarose dissolved

completely and then allowed to cool. Ethidium bromide (DNA intercalating agent) was added

when temperature reached 55-600 C as a staining agent.

• Then it was poured into the gel mould and allowed to solidify.

• The comb and the cello tape were removed carefully after solidification of the agarose.

• The casted gel was placed in the electrophoresis unit with wells towards the cathode and

submerged with 1X TAE to a depth of about 1cm.

3.4.3 Loading the DNA samples

• 2 µl of DNA sample dissolved in TE was pipetted onto a parafilm and mixed well with 4 µl of

6X loading dye by pipetting up and down several times.

• The gel was run at 8 V/cm for 1 hour

• Post staining was done by keeping the gel in Ethidium bromide (DNA intercalating agent)

staining agent and bands were visualized and documented using a gel documentation

system (Model Alpha Imager 1200, Alpha Innotech Corp., USA).

3.5 Quantification of DNA

DNA was quantified by using Nanodrop (Nanodrop Spectrophotometer ND-1000). 1 µl of

genomic DNA was loaded for quantification. 1µl of TE buffer was used as blank. The absorbance for

all samples was measured at 260 nm as double stranded DNA has maximal absorbance at 260 nm.

If the quantified DNA in Nanodrop shows ‘x’ ng/µl, then dilution is done ‘y’ times (where, ‘y’ = ‘x’/50).

Based on the quantification data; DNA dilutions were made in 1X TE buffer to a final concentration of

50ng/µl and stored in -20°C for further use.

3.6 SSR genotyping of parents and F1 hybrids

Microsatellite (SSR) markers showing polymorphism between the parents CO 43 and

FR13A were used for identifying true F1 hybrids. SSR genotyping includes the following steps:

• PCR amplification of genomic DNA was done using forward and reverse microsatellite primers

• Resolution of polymorphism through agarose gel electrophoresis

• Staining and developing the gel

• Analysis of banding pattern

3.6.1 PCR amplification

The cocktail for PCR amplification was prepared as follows:

A) Reaction mixture (15 µµµµl)

Stock Aliquot Final concentration

a) DNA 50 ng/µl 2.00 µl 66.7ng

b) dNTPs (2.5 mM) 0.50 µl 75.0mM

c) Forward primer (10 µM) 1.00 µl 1.5µM

d) Reverse primer (10 µM) 1.00 µl 1.5 µM

e) Assay buffer (10 X) 1.50 µl 1 X

f) Taq DNA polymerase (3 units/µl) 0.20 µl 0.04 units

g) Sterile distilled H20 8.80 µl

Total 15µl

(dNTPs, assay buffer and Taq DNA polymerase used were obtained from Bangalore

Genei Ltd., India and primers used were obtained from Research Genetics Inc., USA).

B) The reaction mixture was given a momentary spin for through mixing of the cocktail

components. Then 0.20 ml PCR tubes were loaded in a thermal cycler.

C) The reaction in thermal cycler (PTC-100TM, MJ Research Inc, Massachusetts, USA and

BIO-RAD, DNA Engine®, Peltier Thermal Cycler) was programmed as follows:

a) Profile 1: 95˚C for 5 minutes Initial denaturation

b) Profile 2: 94˚C for 1 minute Denaturation

c) Profile 3: 56-61˚C for 1 minute Annealing

d) Profile 4: 72˚C for 1 minute Extension

e) Profile 5: 72˚C for 5 minutes Final extension

f) Profile 6: 4˚C for hold Hold the samples

Profiles 2, 3 and 4 were programmed to run for 36 cycles.

After PCR amplification, the products were resolved by agarose gel electrophoresis and banding

pattern was scored after EtBr staining. True hybrids were identified based on the presence of

SSR alleles of both the parents.

3.7 Evaluation of F2 generation

Seeds were collected from a single F1 hybrid plant and F2 generation (256 plants) was

raised along with the parents in the field during Rabi’2008. Observations on days to 50 %

flowering, plant height, panicle length, number of tillers per plant, number of grains per panicle,

100 grain weight and grain yield were recorded from the F2 population and their parents.

3.7.1 Days to flowering/ heading

The number of days from sowing to panicle emergence was counted.

3.7.2 Plant height

Plant height was measured from the ground level to the tip of the primary panicle and

expressed in centimeters.

3.7.3 Number of tillers per plant

In each plant, number of tillers at the time of harvest was counted and recorded

3.7.4 Panicle length

The length of the primary panicle was measured from the base to the tip and recorded in

centimeter.

3.7.5 Number of grains per panicle

A number of seeds in the first 2-3 panicles were counted and recorded as number of

grains per panicle.

3.7.6 100 Grain weight

Total of 100 filled grains were counted from each plant and weighed in the electronic

balance and expressed in grams/ plant.

3.7.7 Grain yield

The weight of dried and cleaned grains from each plant was recorded and expressed in

gram/plant.

3.8 Marker assisted selection

3.8.1 Parental polymorphism survey

A total of 232 SSR markers (Table 3; Fig 7) were selected for surveying the

polymorphism between the parents viz, CO 43 and FR13A to facilitate foreground selection

(selection at target Sub1 locus) and background selection. Out of 232 SSR markers, 50 markers

located on chromosome 9 were chosen for genotyping the parents to facilitate precise selection

of recombinants at the Sub1 locus among the segregants.


In order to select the F2 segregants harboring the Sub1 locus, a SSR marker namely

RM219 linked to the Sub1 locus was used for genotyping the F2 segregants. Leaf samples were

collected from the 30 days old seedlings of F2 population and used for genomic DNA extraction.

Isolated genomic DNA of parents and 256 F2 plants was assessed for its quality and

concentration and the DNA samples were diluted based on Nanodrop quantification.

Primers of RM219 SSR marker were used for PCR amplification in the DNA isolated

from parents and 256 F2 plants. PCR products were separated by 3% agarose gel

electrophoresis and scored for genotyping. F2 segregants possessing, i) CO 43 allele of RM219,

ii) FR13A allele of RM219 and iii) heterozygotes were identified.

3.9 Phenotyping of selected F2 lines for their response against submergence

With a view to verify the effect of introgression of Sub1 locus in terms of tolerance

against submergence, progenies of 5 F2 plants possessing CO 43 allele of RM219, progenies of

5 F2 plants possessing heterozygote alleles of RM219 and progenies of 5 F2 plants possessing

FR13A allele were screened for their tolerance against submergence. About

25 plants of each progeny were grown in pots for 25 days and submerged inside water for

14 days. Water level was maintained well above the tip of the plant and de-submerged after

14 days. Plants were evaluated for their survival during submergence and recovery ability.

EXPERIMENTAL RESULTS

The present study was undertaken with a view of i) Understanding genetic variation for

submergence tolerance between CO 43 and FR13A; ii) Understanding biochemical basis of

improved submergence tolerance exhibited by FR13A and iii) Marker assisted introgression of Sub1

locus controlling submergence into CO 43, a popular variety of Tamil Nadu. The results obtained are

presented as below:

4.1 Understanding genetic variation for submergence tolerance

Two rice varieties namely CO 43 (long duration, submergence susceptible rice variety popularly

grown in Tamil Nadu) and FR13A (a submergence tolerant rice variety grown in eastern India) were

grown in pots for 20 days and submerged inside water for 13 days. The results showed that CO 43

was not able to withstand submergence and its leaves and stems showed rotting symptoms (Plate

1). FR13A plants were not affected much after 13 days of submergence and leaves remained green.

After de-submergence, FR13A plants were able to recover very rapidly and regained the greenness

of leaves. Whereas CO 43 plants were not able to recover after de-submergence (Plate 2).

4.2 Understanding the biochemical basis of submergence tolerance in FR13A

Submergence tolerance is a metabolic adaptation in response to anaerobiosis that enables

cells to maintain their integrity so that the plant survives hypoxia without major damages. High

carbohydrate status after submergence, which is the consequence of its level before submergence

and extent of turnover and consumption during submergence, is the key factor that determines the

ability of plants to withstand submergence stress. In the present study, physiological responses of

rice genotypes viz., FR13A and CO 43 with respect to the total carbohydrate level before and

submergence was estimated.

Plants were completely submerged and water depth was maintained 75cm above the plant

for 14 days. After de-submergence, the plants were allowed to recover for 10 days. Total

carbohydrate content (mg/100 mg of leaf tissue) was estimated. Before submergence, the

submergence tolerant FR13A was found to contain almost double the quantity of total carbohydrate

than CO 43 (Table 1).

Table 1. Total carbohydrate content (mg /100 mg of leaf) in the control and submerged

plants of CO 43 and FR13A.

Variety/Cultivar Seedling stage during submergence

Total carbohydrate content ( mg / 100mg of leaf)

Control Submerge treated

FR13A

0th day ( 20th day) 20.25 ±0.24 20.25 ±0.24

3rd day ( 23rd day) 24.50 ±2.50 19.00±0.49

7th day (27th day) 29.25 ±2.75 17.50 ±0.49

10th day (30th day) 33.50 ±4.51 11.75 ±0.75

14th day (34th day) 38.25 ±5.76 09.00 ± 1.5

10 days after de-sub - 14.50 ±1.00

C043

0th day ( 20th day) 12.75 ±2.75 12.75 ± 2.75

3rd day ( 23rd day) 12.75 ±4.26 9.25 ±0.75

7th day (27th day) 16.75 ±3.00 8.5 ± 0.70

10th day (30th day) 18.50 ±2.23 8.5 ±0.70

14th day (34th day) 20.25 ±3.50 7.25 ±0.25

10 days after de-sub - 7.02±0.02

Each value is a mean of two independent replications.

The total carbohydrate content in the control plants showed an increasing trend along with

duration. In the submerged plants, the total carbohydrate content was found to be consumed very

rapidly which resulted in the reduced level of total carbohydrates. The consumption rate of total

carbohydrates in the tolerant FR13A was found to be lower than the susceptible CO 43 (Table 1).

After de-submergence, the tolerant FR13A plants were able to synthesize and accumulate

carbohydrates (Fig 1A) whereas the susceptible CO 43 plants were found to be dead and thereby no

accumulation of carbohydrates (Fig 1B).

4.3 Introgression of Sub1 locus from FR13A into CO 43 through marker assisted selection

Most of the cultivated rice varieties in Tamil Nadu Viz., White Ponni, ADT 36, ADT 39, CO

43 and CO 48 are susceptible to flooding or submergence. Among these varieties, CO 43 is a high

yielding, long duration popular variety in Tamil Nadu known for its salinity tolerance worldwide. This

variety is cultivated widely in the Cauvery delta areas where flash flooding is a common problem. In

the present study, efforts were made to improve the submergence tolerance of CO 43 by

introgressing Sub1 locus from the tolerant FR13A through marker assisted breeding.

4.3.1 Evaluation of F1 generation and identification of true hybrids

Crosses were made between these two rice varieties by keeping the submergence tolerant

line FR13A as a donor parent and CO 43 as a recipient parent. Obtained F1s were evaluated in the

field and true F1 hybrids were identified by using both morphological markers and molecular markers.

A SSR marker namely RM421 (Chromosome 5; 101.5 cM) which was found to be polymorphic

between CO43 and FR13A was used for genotyping the hybrids and parents for selecting true

hybrids (Plate 3). The true F1s possessing both male and female alleles of RM421 were selected,

selfed and forwarded to F2 generation.

4.3.2 Evaluation of F2 generation

The F2 population (256 plants) from a single F1 plant was raised in the field along with the

parents. All the F2 plants were evaluated for the morphological traits namely, days to flowering, plant

height, number of tillers/hill, number of panicles/plant, panicle length, number of grains per panicle,

100 grain weight and grain yield per plant. All the data recorded in the F2 individuals were subjected

to the basic statistical analysis viz., mean, range and standard deviation (Table 2).

Figure 1A. Effect of submergence on the total carbohydrate levels in the tolerant FR13A

Figure 1B. Effect of submergence on the total carbohydrate levels in the susceptible CO 43

0

37

10

14

0

3 7 1014 18

0

5

10

15

20

25

30

0 5 10 15 20

No.of days

Total ca

rbohyd

rate (m

g/100

mg o

f leaf) Control

Submerged

03

710

14

0 3 7

1014

18

0

5

10

15

20

25

30

35

40

45

0 5 10 15 20

No.of days

Total ca

rbohyd

rate (m

g/100

mg o

f leaf) Control

Submerged

Table 2. Morphological traits showed variation in F2 individuals of CO 43 and FR13A

SL. No. Traits

Parents F2 Individuals

FR13A CO 43 Mean Range Standard Deviation

1 Days to flowering 114 109 99 75-130 11.59

2 Plant height 76.9 74 66.26 25-100 17.76

3 Number of tillers 12.2 14.4 11.60 4-29 5.24

4 Number of panicles 7.5 11.6 9.43 1-25 4.89

5 Panicle length 22.7 23.9 18.29 8-30 4.66

6 Number of grains /panicle

103.5 298.4 72.4 2-204 38.52

7 100 grain weight 2.34 1.85 2.01 0.8-3.6 0.56

8 Grain yield 11.7 27.5 10.6 0.12-53.6 13.30

The morphological traits viz., days to flowering, plant height, number of tillers, panicle length,

number of seeds per panicle, 100 grain weight and grain yield showed continuous variation among

the F2 individuals.

4.3.2.1 Days to flowering

Days to flowering ranged between 75 – 130 days in the F2 population with a mean value of

99 days. The character showed continuous variation in the population with some transgressive

segregants (Figure 2).

4.3.2.2 Plant height (cm)

Plant height ranged between 25-100 cm among the F2 individuals. The mean of plant height

recorded was 66.26 cm with a standard deviation of 17.76. A maximum of 77 plants were found to

possess the plant height between 59 – 69 cm (Figure 3).

4.3.2.3 Number of tillers

The average number of tillers per plant among the F2 population was 11.60 and it ranged

from 0 - 29. The class interval 10 -12.9 was found to possess maximum number of 80 plants (Figure

4).

4.3.2.4 Number of panicles and panicle length

The F2 population recorded an average of 9.43 panicles per plant and the number of

panicles/plant ranged between 0 and 25. Length of the panicle ranged from 8 - 30 cm among the F2

individuals. The recorded mean was 18.29 cm.

4.3.2.5 Number of grains per panicle and 100 grain weight

Total number of grains per panicle ranged from 0 -204 among the F2 population. The

recorded mean was 72.4 grains per panicle with a standard deviation of 38.52 (Figure 5).

An average of 100 grain weight among the F2 individuals was 2.01 grams. It ranged from

0.8 - 3.6 gms (Plate 4).

4.3.2.6 Grain yield

Regarding grain yield/plant, the F2 population recorded the mean value of 9.59 gms per

plant with the range between 0 - 53.6 grams (Figure 6).

Frequency distribution pattern for morphological traits evaluated in the 256 F2 population of

CO 43 and FR13A

Figure 2. Frequency distribution pattern for days to flowering in the F2 population

Figure 3. Fequency distribution pattern for plant height among 256 F2 individuals

82

23 24

77

17

33

11

0

10

20

30

40

50

60

70

80

90

0-10 25-35 37-47 48-58 59-69 70-80 82-92 >93

Plant height in cm

No. of in

divid

uals

FR 13A (76.9 cm)

CO 43 ( 74 cm)

4

3540

21

56

44

3023

3

0

10

20

30

40

50

60

74-79.6 80-85.6 86-91.6 92-97.6 98-103.6 104-109.6

110-115.6

116-121.6

>127

No. of days

No. of F2 in

divid

uals FR 13A

CO 43

52

80

50

60

70

80

90

individ

uals

FR 13A

CO 43

Figure 4. Frequency distribution pattern for number of tillers among 256 F2 individuals

Figure 5. Frequency distribution for number of grains/panicle among the 256 F2 individuals

23

36

49

62

42

2114

62

0

10

20

30

40

50

60

70

0-20.4 22-42.4 43-63.4 64-84.4 85-105.4 106-126.4

129-149.4

150-170.4

>190

No. of grains/panicle

No. of F2

indiv

idual

s FR 13A

CO 43

Figure 6. Frequency distribution for grain yield among the 256 F2 individuals

98

76

39

22

7 8 4 10

20

40

60

80

100

120

0-5.36 5.5-10.86 10.97-16.33

17.2-22.56 23.6-28.96 29.2-34.56 35.1-40.46 >41

Grain yield in gram

No. of F2 in

divid

uals

FR 13A

CO 43

4.4 Marker Assisted Selection

4.4.1 Surveying parental polymorphism using SSR markers

To select SSR markers for foreground selection and background selection in the segregating

progenies, 232 microsatellite (SSR) markers covering all 12 chromosomes in the rice genome were

used for genotyping the parents FR13A and CO 43. Out of 232 SSR primers, 76 primers showed

polymorphism between two parents which accounts for 32.7 percentage (Table 3; Figure 7). The

number of SSR markers produced polymorphism between two parents on all the twelve

chromosomes of rice are listed in (Appendix 1 to 4).

The maximum polymorphism was observed (38.4%) on chromosome 5 with 5 polymorphic

SSR markers out of 13 SSR markers surveyed. The lowest level of polymorphism percentage

observed was in chromosome 6 (22.3). The maximum number of SSR markers (50), were surveyed

on chromosome 9 which recorded polymorphism of 36.0 % (Figure 8).


Rice microsatellite markers RM444 (3.2cM), RM285 (1.8cM), RM464A (0.7cM), RM316

(1.5cM) and RM219 (3.4cM) which were found to be closely linked to Sub1 locus on chromosome 9

were surveyed for parental polymorphism. Out of these five primers only RM219 showed

polymorphism between CO 43 and FR13A (Plate 5) and it was used for foreground selection.

In order to select F2 plants harboring the Sub1 locus from FR13A, genomic DNA was

isolated from all the 256 F2 individuals and they were genotyped using the SSR marker RM219

(Plate 6). Genotyping of F2s and parents using RM219 revealed that 61 F2s were found to possess

CO43 allele, 125 F2 plants were found to possess both the alleles (heterozygotes) and 64 plants

were found to possess FR13A allele. This fit well with the expected ratio of 1:2:1.

4. 5 Phenotyping of selected F2 lines for their response against submergence

With a view to verify the effect of introgression of Sub1 locus in terms of tolerance against

submergence, progenies of 5 F2 plants possessing CO 43 allele of RM219, progenies of

5 F2 plants possessing heterozygote alleles of RM219 and progenies of 5 F2 plants possessing FR

13A allele were screened for their tolerance against submergence. About 25 plants of each progeny

were grown in pots for 25 days and submerged inside water for 14 days. Water level was maintained

well above the tip of the plant and de-submerged after 14 days.

Table 3. Number of SSR primers (chromosome wise) surveyed for assessing the polymorphism

between the parents

Chromosome No. No. of RM primer pairs surveyed

No. of primer pairs produced

polymorphism

Percentage of polymorphism

1 20 7 35.0

2 14 5 35.7

3 20 7 35.0

4 15 5 33.4

5 13 5 38.4

6 18 4 22.3

7 17 5 29.4

8 22 6 27.3

9 50 18 36.0

10 16 5 31.3

11 12 4 33.4

12 15 5 33.4

Total 232 76 32.7

The results revealed that the progenies of F2 plants possessing FR13A allele and

heterozygote allele were found to exhibit greater degree of tolerance against submergence when

compared to the progenies of F2 plants possessing CO 43 allele (Plate 7). Lines with FR13A allele

and heterozygotes were able to maintain greenness of leaves and recovered rapidly after de-

submergence. Whereas the lines with CO 43 allele showed high degree of rotting symptoms during

14 days submergence and were not able to recover after de-submergence.

DISCUSSION

Rice (Oryza sativa L.) is the well-known holder of two important titles: the most important

food crop in the world and a model crop for genomic studies among the cereal species. Rice is the

staple food in many developing countries in Asia, Africa, and Latin America. Rice production is

severely hampered by many abiotic stresses viz., drought, salinity, submergence/flooding, high

temperature etc., when compared to other cereal crops. The projected increase in global population

to 9 billion by 2050 and predicted increase in water scarcity, decrease in arable land, the constant

battle against new emerging pathogens and pests and possible adverse effects from climate change

will present great challenges for rice breeders and agricultural scientists (Collard et al., 2008). In this

context, increasing rice production with no additional lands available for cultivation depends on

increasing the rice production under marginal cultivation i.e., by developing rice varieties suitable for

drought, salinity and submergence prone areas.

Flooding is one of the most important environmental stresses worldwide. Flash floods or

short-term submergence regularly affect around 15 million hectares of rice (Oryza sativa L.) growing

areas in South and Southeast Asia. Even more favorable irrigated areas experience flooding

problems during the monsoon season. In India, area under rice cultivation is 44.5 m ha -1 (data of

2000–01) with an annual production of 85.5 million tonnes and an average productivity of 1.9 t ha–1.

Rice is grown in a wide range of ecologies ranging from irrigated to uplands, rainfed lowland, deep

water and tidal wetlands. Out of 44.5 m ha, 3 m ha is submerged or flood-prone, where plants are

completely submerged for 1–2 weeks or so, resulting in partial or even complete crop failure.

Submergence tolerant varieties have been developed (Mackill et al., 1993), but have not

been widely adopted. One reason is that these tolerant varieties lack many of the desirable traits of

the widely grown varieties, referred to as “mega varieties” that are popular in major rice-growing

areas of Asia, because of their high yield and grain quality. Hence the acceptable strategy would be

to improve the existing “mega varieties” for their tolerance against submergence. But this approach

needs availability of suitable donor and precise identification of genomic regions controlling

submergence tolerance which would enable us to introgress the trait very precisely into any desired

background through marker-assisted breeding. The availability of the large-effect QTL Sub1 for

submergence tolerance, availability of genomic resources in rice genome, a theoretical frame-work

for MAB and the existence of intolerant varieties that are widely accepted by farmers provided an

opportunity to develop cultivars that would be suitable for larger areas of submergence prone rice

(Mackill et al., 2006).

A major QTL (Sub1) explaining about 70% of phenotypic variation in submergence tolerance

has been identified and fine mapped on chromosome 9 in the submergence tolerant cultivar FR13A

(Xu et al., 2000). Even though a single gene namely Sub1A (ethylene response factor) controlling

tolerance against submergence has been identified, the transfer of this gene through conventional

breeding combined with MAS (marker assisted breeding) is still the most effective way to develop

submergence tolerant cultivars (Xu et al., 2006). The basis of a marker-assisted backcrossing (MAB)

strategy is to transfer a specific allele at the target locus from a donor line to a recipient line while

selecting against donor introgressions across the rest of the genome. The use of molecular markers

is that it permits the genetic dissection of the progeny at each generation and thus increases the

speed of the selection process. The main advantages of MAB are: (1) efficient foreground selection

for the target locus, (2) efficient background selection for the recurrent parent genome, (3)

minimization of linkage drag surrounding the locus being introgressed and (4) rapid breeding of new

genotypes with favorable traits. The effectiveness of MAB depends on the availability of closely

linked markers and/or flanking markers for the target locus, the size of the population, the number of

backcrosses and the position and number of markers for background selection (Frisch and

Melchinger, 2005). MAB has previously been used in rice breeding to incorporate the bacterial blight

resistance gene Xa21 (Chen et al., 2000, 2001), waxy gene (Zhou et al., 2003) and submergence

tolerance (Neeraja et al., 2007) into elite varieties.

Based on the above facts, the present study was undertaken with the objectives: (1) to

develop a submergence-tolerant version of the widely grown cultivar in Tamil Nadu namely CO 43.

The level of genetic variation between CO 43 and FR13A for submergence tolerance was assessed

under green house conditions. The relation between the levels of total carbohydrate reserves and

submergence tolerance was investigated and finally attempts were made to implement MAS for

improving submergence tolerance of local variety CO 43.

The cultivar FR13A was found to be superior to CO 43 in terms of submergence tolerance.

The tolerant FR13A was able to retain greenness of leaves even after 14 days of submergence

whereas the susceptible CO 43 was found to be severely affected by submergence which resulted in

rotting and decay of leaves (Figure 1). Complete submergence hastens degradation of chlorophyll

content in susceptible rice cultivars compared to tolerant ones (Ella et al., 2003) which can also be

used as an indicator of submergence tolerance.

Even though rice is predominantly grown under flooded conditions, most of the existing rice

cultivars are seriously damaged if they are completely submerged for more than three days (Sarkar

et al., 2006). Germplasm survey revealed the existence of only limited amount of genetic variation

for submergence tolerance among rice cultivars. A few tolerant landraces namely, FR13A, FR43B,

Goda Heenati, Kurkaruppan and Thavalu were identified that can withstand complete submergence

for 10–14 days (Xu and Mackill, 1996). Tolerant breeding lines with improved agronomic

characteristics have now been developed which are equivalent to the irrigated checks (Mohanty et

al., 2000). Recent efforts have resulted in identification of some new rice landraces viz., Atiranga,

Khoda, Khadara, Kusuma and Kalaputia possessing reasonably higher levels of tolerance to

submergence but with better agronomic traits than the landraces identified before (Sarkar et al.,

2006). However the mechanisms controlling submergence tolerance in these land races have not yet

been understood.

In the present study, the tolerant FR13A was found to recover very rapidly after de-

submergence whereas the popular CO 43 was failed completely to recover after de-submergence

(Figure 2). Quick regeneration following submergence is a desirable trait under frequent or

prolonged flooding, as it can ensure early recovery and production of sufficient biomass for optimum

productivity. The old leaves die after flooding, particularly when floodwater is turbid or when flooding

is prolonged. Initiation of new leaves and their subsequent growth requires availability of non-

structural carbohydrates (Sarkar et al., 2006).

In the present study it was observed that the tolerant FR13A was found to accumulate

significantly higher levels of total carbohydrates (20.25 mg/100 mg) in the shoots than the

susceptible CO 43 (12.75 mg/100 mg) at same stage of development. Maintenance of high levels of

stored carbohydrates in the seedlings prior to submergence coupled with minimum shoot elongation

and retention of chlorophyll are all desirable traits for submergence tolerance (Sarkar et al., 2006).

Cultivars that maintained more than 6% of their initial non-structural carbohydrate at the time of re-

aeration were found to be capable of developing new leaves rather quickly (Das et al., 2005). In the

present study also it was observed that the tolerant FR13A recorded slow consumption of reserves

and it was able to resume the accumulation of carbohydrate reserves very rapidly after de-

submergence. Hence, it is assumed that high carbohydrate status after submergence, which is the

consequence of its level before submergence and extent of turnover and consumption during

submergence, is the key factor that determines the ability of plants to withstand submergence stress.

Current understanding of the physiological and biochemical basis of submergence tolerance

has progressed well in recent years, making it possible to design efficient phenotyping protocols and

has laid the infrastructure for further genetic and molecular studies, to discover genes underlying

component traits associated with tolerance. This will subsequently speed up the breeding process if

individual genes can be combined in favourable phenotypes through marker-assisted selection as

the example shown with Sub1 locus (Neeraja et al., 2007). In this study, attempts were made to

develop a submergence tolerant version of CO 43, a ruling “mega variety” variety of Tamil Nadu

through marker assisted introgression of Sub1 locus from the tolerant FR13A. MAB strategy has

been shown to be an effective means of utilizing QTLs with large effects like Sub1 in rice breeding

programs (Toojinda et al., 2005; Neeraja et al., 2007). Molecular markers such as SSRs have been

efficiently utilized in many crop improvement programs viz., hybrid identification, testing seed genetic

purity and linkage mapping. In this study also, the true F1 hybrids between CO 43 and FR13A were

identified and confirmed by using the method of SSR genotyping.

Evaluation of 256 F2 plants under field conditions revealed the presence of continuous

variation for the targeted quantitative traits viz., days to flowering, plant height, panicle size, number

of grains per panicle, grain weight etc., This indicated the suitability of population for selection

process from the early stage itself.

In this study, the Sub1 locus was monitored by markers shown to be closely linked with the

gene. In order to select the F2 segregants harboring the Sub1 locus, a SSR marker namely RM219

linked to the Sub1 locus was used for genotyping the F2 segregants. Survey on foreground lines

indicated that 61 F2s were found carrying CO 43 allele, 125 F2 plants carrying both the alleles

(heterozygotes) and 64 F2s carrying FR13A allele assuring the expected ration of 1:2:1. The effect of

introgression of Sub1 locus in terms of tolerance against submergence, progenies of 5 F2 plants

possessing CO 43 allele of RM219, progenies of 5 F2 plants possessing heterozygote alleles of

RM219 and progenies of 5 F2 plants possessing FR13A allele were screened for their tolerance

against submergence. The progenies of F2 plants possessing FR13A allele and heterozygote allele

showed higher degree of tolerance level than progenies of F2 plants possessing CO 43 allele.

Recovery level after de-submergence were more in lines with FR13A allele and heterozygote, where

all the plants carrying lines with CO 43 allele were dead, assuring the phenotypic association of

submergence tolerance with Sub1 locus.

In order to retain the positive attributes of CO 43, it is planned to employ SSR markers for

background selection which will lead to great acceleration of recipient genome recovery in the

present study. In order to identify SSR markers for background selection, about 232 microsatellite

(SSR) markers covering all 12 chromosomes in the rice genome were used for genotyping the

parents FR13A and CO 43. Out of 232 SSR primers, 76 primers (minimum of 4-5 markers per

chromosome) showed polymorphism between two parents which accounts for 32.7 % (Table 3). This

is in accordance with the conclusion made by Servin and Hospital, (2002) and Neeraja et al., (2007)

to provide adequate coverage of the genome in backcross programs.

In summary, efforts are in progress to enhance the submergence tolerance level of mega

rice variety of Tamil Nadu, CO 43 by using a marker assisted backcrossing approach. In future,

individual F3 plants grown by single seed descent method will again be genotyped using Sub1 linked

marker and plants harboring Sub1 locus from FR13A will be identified and subjected to further

recombinant selection, background selection and used for back crossing with CO 43. Preliminary

results of this study indicated the potential and superiority of MAS over conventional approaches to

improve submergence tolerance in rice.

SUMMARY

The present study was aimed at i) understanding the genetic variation for submergence

tolerance in rice and ii) understanding biochemical basis of improved submergence tolerance

exhibited by FR13A. Finally attempts were made towards marker assisted introgression of Sub1

locus controlling submergence tolerance in FR13A into a mega variety of TN namely, CO 43. The

results obtained in this study are summarized as below.

1) Screening for submergence tolerance revealed that the mega variety CO 43 was not able to

withstand submergence (14 days) where as FR13A exhibited greater degree of tolerance

against submergence.

2) The leaves and shoots of CO 43 showed rotting symptoms during submergence which may

be due to degradation of chlorophyll. In contrast, FR13A leaves were remained green.

3) Assessment of recovery ability after 14 days submergence revealed the better recovery of

FR13A. FR13A plants were able to recover very rapidly and regained the greenness of

leaves. In contrast, CO 43 plants could not recover after de-submergence due to

degradation of chlorophyll and found dead.

4) FR13A was found to accumulate significantly higher levels of total carbohydrates (20.25

mg/100 mg) in the shoots than the susceptible CO 43 (12.75 mg/100 mg) at same stage of

development

5) True F1 hybrids between CO 43 and FR13A were selected through SSR genotyping using

the SSR marker RM421 on chromosome 5. .

6) All the 256 F2 plants were evaluated for the morphological traits namely, days to flowering,

plant height, number of tillers/hill, number of panicles/plant, panicle length, number of grains

per panicle, 100 grain weight and grain yield per plant. All these traits were found to show

continuous variation within the population.

7) With a view of identifying SSR markers for selection, 232 SSR primers were surveyed

between the two parents. Out of this 232 markers, 76 showed polymorphism which can be

used in foreground selection, recombinant selection and background selection.

8) F2 plants harboring the Sub1 locus from FR13A were identified by genotyping the population

using RM219 which is tightly linked to Sub1 locus.

9) Foreground selection revealed that 61 F2 plants were found carrying CO 43 allele of RM219,

125 F2 plants carrying both the alleles (heterozygotes) and 64 F2s carrying

FR 13A allele of RM219.

10) Phenotyping of selected F2 lines confirmed the effect of Sub1 locus on tolerance against

submergence and recovery after de-submergence.

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De

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of s

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info

rmat

ion,

repe

at ty

pe, a

nnea

ling

tem

pera

ture

and

pro

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size

are

ava

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http

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w.gr

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g/m

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Documents

MARKER ASSISTED INTROGRESSION OF Sub1 LOCUS IN RICE · 2019-09-10 · marker assisted introgression of sub1 locus in rice by suchismita raha, b. sc. (agri.), i. d. no. 07-607-026