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Application of
Molecular Markers to
Rice Breeding in
Australia
Molecular markers for the sd-1 and fgrgenes
A report for the Rural Industries Research
and Development Corporation
by Stephen Garland &Robert Henry
May 2001
RIRDC Publication No 01/38
RIRDC Project No. USC-2A
© 2001 Rural Industries Research and Development Corporation.
All rights reserved.
ISBN 0 642 58260 2
ISSN 1440-6845
Application of molecular markers to rice breeding in Australia
Publication No. 01/38
Project No. USC-2A
The views expressed and the conclusions reached in this publication are those of the author
and not necessarily those of persons consulted. RIRDC shall not be responsible in any way
whatsoever to any person who relies in whole or in part on the contents of this report.
This publication is copyright. However, RIRDC encourages wide dissemination of its
research, providing the Corporation is clearly acknowledged. For any other enquiries
concerning reproduction, contact the Publications Manager on phone 02 6272 3186.
Researcher Contact Details
Professor Robert Henry
Centre for Plant Conservation Genetics
Southern Cross University PO Box 157
Lismore NSW 2480
Phone: 02 66203010
Fax: 02 66222080
Email: [email protected]
RIRDC Contact Details
Rural Industries Research and Development Corporation
Level 1, AMA House
42 Macquarie Street
BARTON ACT 2600
PO Box 4776
KINGSTON ACT 2604
Phone: 02 6272 4539
Fax: 02 6272 5877
Email: [email protected].
Website: http://www.rirdc.gov.au
Published in May 2001
Printed on environmentally friendly paper by Canprint
ii
Foreword
The major objectives of the project were to identify, adapt and evaluate molecular markers for routine
use in the Australian rice-breeding program. The major component of the work involved the
development of markers for the semi-dwarfing gene sd-1 and the major fragrance gene fgr.
This publication provides a description of the development and assessment of four useful markers for
sd-1 and three useful markers for fgr. The utilisation of these markers within the Australian rice-
breeding program is discussed.
This project was funded from industry revenue which is matched by funds provided by the Federal
Government and is an addition to RIRDC’s diverse range of over 600 research publications. It forms
part of our Rice R&D program, which aims to improve the profitability and sustainability of the
Australian rice industry.
Most of our publications are available for viewing, downloading or purchasing online through our
website:
• downloads at www.rirdc.gov.au/reports/Index.htm
• purchases at www.rirdc.gov.au/eshop
Peter Core
Managing Director
Rural Industries Research and Development Corporation
iii
Acknowledgments
Thanks to all the staff from Yanco Agricultural Institute and Southern Cross University who
provided invaluable assistance. Thanks to Dr Susan McCouch and the Cornell Research
Foundation Inc, New York, their contribution was critical to the success of this project.
iv
Contents
Foreword ................................................................................................................................ii
Acknowledgments ................................................................................................................. iii
Executive Summary............................................................................................................... v
1. Introduction .................................................................................................................... 1
1.1 Semi-dwarfism ............................................................................................................ 1
1.2 Fragrance ................................................................................................................... 1
2. Objectives ...................................................................................................................... 3
3. Methodology................................................................................................................... 3
3.1 Mapping Population .................................................................................................... 3
3.2 DNA extraction............................................................................................................ 4
3.3 Supply and investigation of the RFLP probes............................................................. 4
3.4 Evaluation of marker utility.......................................................................................... 5
4. Results ........................................................................................................................... 6
4.1 The development of markers for sd-1 ......................................................................... 6
Number of alleles............................................................................................................. 10
4.2 The development of markers for fgr.......................................................................... 10
5. Discussion.................................................................................................................... 13
5.1 Markers for sd-1........................................................................................................ 13
5.2 Markers for fgr .......................................................................................................... 14
6. Implications .................................................................................................................. 15
7. Recommendations ....................................................................................................... 17
8. References................................................................................................................... 19
v
Executive Summary
The major objectives of the project were to identify, adapt and evaluate molecular markers for routine
use in the Australian rice-breeding program. The major component of the work involved the
development of markers for the semi-dwarfing gene sd-1 and the major fragrance gene fgr.
The semi-dwarfing gene (sd-1) is responsible for producing sturdy and moderately short plants with
higher harvest index compared to similar tall plants. sd-1 is the source of semi-dwarfism in the
Australian rice varieties. The genetics of fragrance in rice is more complicated and involves the
production of basmati or jasmine style aroma. Several chemicals are involved with aroma, however a
single constituent (2-acetyl-1-pyrroline) is believed to be a major component and is controlled by a
single gene.
The application of marker assisted selection for these genes, has potential to greatly improve the
efficiency of the Australian rice-breeding program. Markers have the capacity to assist in the selection
of traits that are expensive or laborious to assess. Molecular markers can be evaluated from a single
seedling leaf or seed samples, allowing selection to occur before the trait is expressed. The semi-dwarf
character, for example, could be detected before maturity or even as a heterozygote thus eliminating the
need for progeny testing in a backcrossing program. Grain quality characters such as aroma are very
difficult to assess accurately. Markers used in preliminary screening would reduce labour costs and
assist in selection.
Fragments of DNA linked to sd-1 and fgr were assessed for differences to develop markers for these
genes. The techniques utilised, ensured that the markers would be easily produced and identified.
Four useful markers have been produced for sd-1. Three useful markers have also been produced or
identified for fgr. The markers for fgr and sd-1 will be useful in the Australian rice-breeding program
and in rice breeding programs in general. This project has demonstrated that molecular markers are
suitable for variety identification and marker assisted selection within the Australian Industry.
It will be necessary to develop more markers and to assess those markers in a wider selection of rice
lines. The presence of a database containing the alleleic characterisations of general markers or those
markers linked to genes or quantitative traits, would allow for the selection of a suite of markers which
are polymorphic between Australian varieties, breeding lines and breeding samples. The markers could
be selected for use in germplasm identification, genetic mapping, and marker assisted selection
programs.
The challenge is to maximise the potential benefits of markers through the appropriate integration of
marker assisted selection into the breeding program. It may be necessary to restructure the breeding
program to obtain maximum benefits.
This project was the subject of a PhD project for Stephen Garland providing important training in rice
molecular biology.
1
1. Introduction
1.1 Semi-dwarfism
The agronomic benefits of shorter rice plants can include increased lodging resistance and increased
harvest index (reviewed by Mackill and Rutger 1979), however most dwarfing genes are associated with
unsuitable agronomic characteristics (reviewed by Cho et al., 1994, Maluszynski et al., 1986, Mackill
and Rutger, 1979).
One semi-dwarfing gene, sd-1 (d-47), has been used predominantly in the development of highly
productive rice cultivars. sd-1 increases lodging resistance, harvest index, responsiveness to nitrogen
fertilisation and improves general plant type (reviewed by Cho et al., 1994). The semi-dwarfing gene sd-
1 is incompletely recessive (Murai et al., 1995), and as a homozygote reduces total culm height by
about 25% (Rutger et al., 1986). The absolute reduction of internode length decreases from the first to
the sixth internode (Murai et al., 1995). There is currently no hard evidence to implicate sd-1 with the
biosynthesis of, or response to, any particular phytohormone involved with stem elongation.
Most of the Australian semi-dwarf cultivars have M7 (Carnahan et al., 1978) in their pedigree. M7 is a
cultivar that possesses the sd-1 locus. sd-1 is therefore the likely candidate dwarfing gene predominating
in the Australian industry.
sd-1 is partially recessive and the identification of the correct genotype is not completely accurate based
on phenotype (height class). The degree of expression is dependent on environmental conditions such as
fertiliser levels (Murai et al., 1995a) and problems with genotyping would be exasperated under
variable field conditions. A further complicating factor, that would also tend to widen genotype height
classes and increase class overlap in breeding populations, is the presence of numerous QTL loci
associated with height (see Li et al., 1995, Lu et al., 1996, Zhuang et al., 1997, Yan et al., 1998). The
correct identification of heterozygotes, that could be produced after a tall recurrent parent is
backcrossed to a semi-dwarf individual, would not be definite under controlled conditions.
Given the benefits of incorporating sd-1 into new varieties, the utilisation of molecular markers for the
identification of the genotype for the sd-1 locus would increase the efficiency of a breeding program.
Heterozygotes developed from a backcrossing exercise could be identified from small samples of
seedling leaves, alleviating the need for maturation of plants and genotyping based on the identification
of segregation in the progeny.
1.2 Fragrance
Several chemical constituents are important to the aroma of cooked rice (Grosch and Schieberle, 1997).
However, 2-acetyl-1-pyrroline (AP) is regarded as the most important component of aroma in the
basmati and jasmine style fragrant rices (Lorieux et al., 1996). AP is found in all parts of the rice plant,
except for the roots (Lorieux et al., 1996) and is also found, at concentrations up to 100 times lower, in
non-fragrant varieties (reviewed by Grosch and Schieberle, 1997).
Lorieux et al. (1996) reviewed the genetics of aromatic fragrance and concluded that a single recessive
gene was responsible for the production of fragrant rice plants.
2
Fragrance can be detected by tasting the associated flavour in individual seeds or assessing the aroma of
leaf tissue or grains after either heating in water or reacting with solutions of KOH or I2-KI (reviewed
by Tragoonrung et al., 1996).
Tasting individual grains has been the preferred method for quality selection of aromatic rice varieties
within the Australian breeding program (Reinke et al., 1991). However, there are problems with the
sensory detection of fragrance. There is considerable variation between analysts in their ability to detect
fragrance or the associated flavour. Some individuals have difficulty in detecting the aroma or taste. A
rapid decline of an individuals ability to distinguish between fragrant and non-fragrant samples with
each analysis performed is also experienced. An analyst's ability declines as the senses become saturated
or actual physical damage occurs. Abrasions to the tongue, causing bleeding, often result from chewing
numerous seeds. Caustic substances such as KOH would also cause damage to the nasal passages.
Sensory methods are therefore not suitable for processing large numbers of samples.
The chemical detection of AP is also possible but is time consuming and requires large samples (eg.
hundreds of grams, Widjaja et al., 1996, Lorieux et al., 1996). The development of a PCR based
molecular marker for the major component of fragrance (AP), for use within breeding programs, would
have many advantages over sensory or chemical detection methods. Many more plants could be
processed and sample sizes of 0.1g or less could be analysed.
Plants from a breeding program for fragrant lines, in the early stages of cultivar development, could be
assessed before maturity from small amounts of leaf tissue to reduce the number of seed samples tested
by sensory methods. Non-fragrant or heterozygous individuals would be avoided for the sensory or
chemical assessment of aroma or flavour quality or tests for other grain quality characteristics. As the
recessive gene could be detected in a heterozygous state, the marker would be useful for the
identification of offspring possessing fgr after backcrossing with homozygous non-fragrant plants.
3
2. Objectives
Several authors have reported polymorphisms within rice RFLP probes and sequence-tagged-sites
(Williams et al., 1991, Fukuoka et al., 1994, Ghareyazie et al., 1995, Xu et al., 1998a). Based on this
success and the many benefits associated with a PCR based marker, the objectives of this study were to
convert RFLP probes and other loci genetically mapped near sd-1 and fgr into PCR based markers.
Polymorphic sites could then be identified by sequencing homologous regions in parent cultivars of a
mapping population or identified as size differences between the PCR products. The polymorphic
markers could then be assessed for linkage to semi-dwarfism and fragrance and ultimately used to
produce PCR based markers for assessment and utilisation within the Australian rice breeding program.
The Australian cultivar Doongara, has an unknown source of semi-dwarfism, as no short stature plants
are present in its pedigree (Bluebelle / Calrose // Jojutla, Ko et al., 1994). A secondary aim of this study
was to determine if the semi-dwarfism in Doongara was also due to sd-1.
3. Methodology
Cho et al. (1994) identified the close linkage of the RFLP markers RG220 and RG109 (co-segregated),
to sd-1 (0.8 cM) on chromosome 1. Maeda et al. (1997) also identified linkage between these two
markers and sd-1. RG220 and RG109 were found to be at a genetic distance of 0.3 and 0.9 cM. The
single recessive fragrance gene (fgr) has been linked to the RFLP clone RG28 on chromosome 8, at a
genetic distance of 4.5cM (Ahn et al., 1992). Lorieux et al. (1996) confirmed the close linkage between
RG28 and fgr (5.8cM).
We screened for polymorphism in PCR amplified regions homologous to RFLP clones linked to sd-1
and fgr to develop PCR based markers. In addition, microsatellite markers previously mapped in the
vicinity of fgr were assessed for polymorphism information content to provide alternative markers for
fragrance.
3.1 Mapping Population
A population of 215 F2 seedlings, derived from a cross between Kyeema (tall, fragrant, long-grain,
Australian cultivar) and Doongara (semi-dwarf, non-fragrant, long grain, Australian cultivar) and 100
seedlings of each parent cultivar were transplanted to the field at 30cm intervals at Yanco Agricultural
Institute, NSW Agriculture, Yanco, in November 1997. Leaf material was collected in January 1998
and frozen for DNA extraction at a later date. Height measurements for phenotype determination were
taken in early March 1998 and were determined for the tallest panicle as the distance from the lower
node of the third internode to the top of the panicle. Height genotypes were determined for 50 F2 plants
by assessing height and height segregation for 8 F3 individuals from each F2 plant. The F3 plants were
grown in 20cm diameter pots from November 1998 to February 1999 at Southern Cross University.
The observed segregation ratios between height genotypes, for the 50 genotyped F2 individuals, were
tested by χ2 analysis against the expected ratio for a single gene (1 : 2 : 1).
124 F2 plants (including the 50 F2 genotyped for height) were classified as fragrant, segregating or non-
fragrant by tasting dehulled F3 seed (ground between front teeth before tasting). If five F
3 seeds were
tasted and all were fragrant, the F2 individual was considered fragrant, otherwise at least 12 seeds were
tasted to separate segregating from non-fragrant F2 individuals. The observed segregation ratio of non-
4
fragrant:segregating:fragrant, was tested by χ2 analysis against the expected ratio for a single gene (1 :
2 : 1).
3.2 DNA extraction
DNA was extracted from approximately 0.6 g of frozen leaf tissue as generally described by Weining
and Henry (1995) for 50 F2 individuals genotyped for height and parent cultivars (Kyeema and
Doongara). The following modifications were included; 2 ml of 2.5% sarkosyl extraction buffer and 2
ml of phenol/chloroform/isoamyl alcohol (25:24:1) were used; DNA was precipitated with 1.3 volumes
of isopropanol in addition to 0.13 volumes of 3M sodium acetate (pH 4.8) and then washed twice with
70% ethanol. The DNA was resuspended in 200 µl of Tris-EDTA (TE) buffer. DNA preparations were
diluted with TE buffer to a final concentration of approximately 50 to 100 ng per µl.
3.3 Supply and investigation of the RFLP probes
The probes RG109, RG220 and RG28 (rice-etiolated-leaf genomic library, PstI restricted, pUC9
vector, cultivar IR36) were supplied by Dr. Susan McCouch and the Cornell Research Foundation Inc,
New York. The clones were bi-directionally sequenced using commercial pUC DNA sequencing primers
and the ABI PrismTM, BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit. Gel separation and
electropherogram production were performed by the Australian Genome Research Facility, University
of Queensland.
Probe-specific PCR primers were designed with the aid of MACVECTORTM 6.0, Sequence Analysis
Software, Oxford Molecular Group Inc. Primers were synthesised by Pacific Oligos Pty Ltd, SCU,
Lismore, NSW, Australia. PCR reactions using template DNA from Kyeema and Doongara were
carried out on a Perkin Elmer, Gene Amp PCR System 9700. The reaction volume was 20µl containing
1X Boehringer Mannheim PCR Buffer (1.5 mM MgCl2), 50 to 100 ng of genomic DNA and 200µM
dNTPs. The PCR reaction mix also included 150 nM of each primer, 2 mM total MgCl2, and 0.5 units
of Taq DNA Polymerase (Boehringer Mannheim). The temperature cycling conditions were 1 minute at
94° C; followed by 35 cycles of 94° C for 30 seconds, 54° C for 30 seconds and 72° C for 1 minute;
with a final hold at 72° C for 5 minutes.
PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN) and sequenced as
discussed above. Sequence polymorphism between Kyeema and Doongara, the parents of the mapping
population were identified. Primers flanking the polymorphism, if required, were designed and
synthesised as previously indicated, except the forward primer was 5´end labeled with the fluorescent
phosphoramidite dye, HEX or FAM (Perkin-Elmer). Sizing of the PCR products were performed on an
ABI Prism 310 Genetic Analyser with the following run and analysis conditions (Module GS STR
POP4 C, 20 minute run time, size standard GENESCAN 500 - TAMRA, Local Southern Sizing
method, Capillary-length-to-detector 30 cm). The sizing of DNA fragments was relative and not
necessarily an absolute measure of size.
Sequence tagged sites from the genetic map of Harushima et al. (1998) were also selected for
assessment. These STS were developed from RFLP probes produced from the cultivar Nipponbare and
included C86, C1439, G1133, L543, L819, R2625, S2523, S2596, S13312, and S13471. These STS
were expected to be near sd-1, based on the relative position of integrated markers from the genetic
maps of Harushima et al. (1998) and Causse et al. (1994) (the latter map includes sd-1) as presented in
the genetic map of Maeda et al. (1997). The STS and clone RZ538 (EcoR1, rice etiolated leaf cDNA
library, variety IR36, Causse et al., 1994) was also selected for assessment as it had been genetically
5
mapped close to sd-1 and end sequence was available. DNA sequences for the STS were obtained from
the Ricegenes database.
Fifty F2 individuals from the mapping population were genotyped for the polymorphic markers. The
genetic distance between fgr or sd-1 and the linked markers were determined as the percentage of
recombinant chromosomes (cM).
3.4 Evaluation of marker utilityTo assess the usefulness of the PCR based markers within the Australian rice industry, the marker
alleles were characterised for 24 rice samples. The rice samples analysed included 16 Australian
breeding lines or Australian commercial cultivars. Half of the 24 samples were fragrant lines and also
included 13 tall and 11 semi-dwarf lines. Rice seed samples were supplied by Yanco Agricultural
Institute (YAI), New South Wales Agriculture, Yanco, Australia. (See Ko et al. (1994) for some of the
Australian pedigrees, others are available through the YAI if required). Five plants for each variety
were grown in a glasshouse at Southern Cross University, Lismore, NSW, Australia. Approximately 1
to 3 cm of mature leaf was sampled from each of the five plants for each cultivar. The leaf material
from the five plants for each cultivar was bulked producing a total weight of about 0.3 g. The DNA was
then extracted as described above.
Microsatellite markers RM42 and RM223 (Chen et al., 1997) were selected in addition to the markers
developed from the RFLP clone RG28 for assessment of polymorphism and allele characterisation.
These markers are linked to fgr and will provide alternate markers for this gene. RG28 and RG1 are
linked at a map distance ranging from 10 cM (Causse et al., 1994) and 12 cM (Lorieux et al., 1996) to
25.5 cM (Cho et al., 1998). RG28 and RG1 flank the genome region that contains fgr. RG28 was not
mapped by Chen et al. (1997) and Xu et al. (1998b). However, the location of RG28 and fgr, in relation
to RM42 and RM223, can be estimated based on the relative position of RG1 and other nearby
markers, in these genetic maps, and in the genetic maps of Cho et al. (1998), Lu et al. (1996) and
Causse et al. (1994).
RG28 probably lies between RM42 and RM223. RM42 may therefore be a few cM further from fgr
than RG28 and RM223 is possibly closer. In most cases these microsatellite markers are likely to be
within 10 cM of fgr and therefore useful for gene tagging within a breeding program.
Primer sequences for RM42 and RM223 are available from Chen et al. (1997). Primers were
synthesised by Pacific Oligos Pty Ltd, SCU, Lismore, NSW, Australia. PCR reactions were carried out
on a Perkin Elmer, Gene Amp PCR System 9600 or 9700. The reaction volume was 20µl containing 1X
Boehringer Mannheim PCR Buffer (1.5 mM MgCl2), approximately 50 ng of genomic DNA, 200µM
dNTPs and 200 nM [R110] dUTP (Perkin Elmer). The PCR reaction mix also included 100 nM of each
primer, 3mM total MgCl2, and 0.5 units of Taq DNA Polymerase (Boehringer Mannheim). The
temperature cycling conditions for the microsatellite markers were 1 minute at 94° C followed by 30
cycles of 94° C for 15 seconds, 54° C for 15 seconds and 72° C for 1 minute. Sizing of microsatellite
alleles was performed on an ABI Prism 310 Genetic Analyser
Polymorphism Information Content values (PIC) were calculated for each marker using Nei’s Gene
Diversity measure Hs (Nei 1973)
n
Hs = 1 - Σ xk
2
k = 1
Where xk is the frequency of the kth allele.
6
4. Results4.1 The development of markers for sd-1
The observed segregation ratios between height genotypes, for 50 F2 individuals, are presented in Table
1. The results are consistent with a single gene controlling semi-dwarf stature χ2 (0.05, df=2), p=0.5)].
Table 1 Observed segregation ratio between height genotypes for 50 F2 individuals from the mapping
population (Kyeema/Doongara) and the expected segregation ratio for a single gene. χ2 (0.05, df=2)
analysis indicated no significant difference between observed and expected ratios, p=0.5.
Homozygous
Semi-dwarf
Heterozygote Homozygous
Tall
Significnce
Observed
frequency11 23 16
not significant
Expected
frequency12.5 25 12.5
Four DNA polymorphisms were identified in homologous regions of RFLP clones and STS, between
Kyeema and Doongara, that could be detected as a size difference or the loss or gain of restriction site.
The markers, primer details and alleleic detection methods are supplied in Table 2. The PCR conditions
for the sequence tagged sites (STS) listed in Table 2 were described in the methods section as indicated
for the conversion of the RFLP and STS to PCR based markers. The PCR conditions for SCU-Rice-
SSR-2 were the same as for the RM prefixed microsatellite markers without the addition of flourescent
dUTPs.
Table 2. Name and primer details for the PCR based markers for sd-1. The RFLP clones used to
develop the PCR based markers are included in the marker name. The exception is the microsatellite
marker SCU-Rice-SSR-2 that was developed from RG109.
Marker Name Primer pairDetection
Method
SCU-Rice-SSR-2
5'-AGCTACTATCAGACAACAGAAAACG-
3' Size Separation
(Source RG109) 5'-ATACGCCAAGATTTCCTAAAC-3'
SCU-Rice-STS-13312 5'-GGAACTGCTCTGCTACTAACCCTG-3' Size Separation
5'-CGTCGGGAAACGAATCCAAC-3'
SCU-Rice-STS-S13471 5'- GCACCACATACTCACTTCTGCTTG-3' Restriction
5'-GCAGGTGTAGCTCTTGTCACAGTG-3' Digest (Pst I)
SCU-Rice-STS-
RG22O 5'-ATTGACCTGTGGATTGCTGTCTG-3' Restriction
7
5'-TGGCGTCTGCTGCTCATTTG-3' Digest (Hae III)
Alleles for SCU-Rice-STS-S13312 are presented if Figure 1. Alleles for SCU-Rice-SSR-2 are
presented in Figure 2. Alleles for SCU-Rice-STS-S13471 and SCU-Rice-STS-RG220 after agarose gel
electrophoresis and ethidium bromide staining of restriction-enzyme digested, PCR-products, are
presented in Figures 3 and 4.
Figure 1 Capillary electrophoresis output, demonstrating alleles for the marker SCU-Rice-STS-13312,
for the varieties of Kyeema and Doongara and one heterozygote F2 individual from the mapping
population (Kyeema / Doongara). Alleles differ by 1 bp. Horizontal scale in bp. Size is relative and not
an accurate measure. Expected allele sizes based on sequence information were 206 and 207 bp.
Doongara allele - 305
bp
Heterozygote
Kyeema allele - 297
bp
209 bp allele - Kyeema
Heterozygote
208 bp allele-Doongara
8
Figure 2 Capillary electrophoresis output, demonstrating alleles for the marker SCU-Rice-SSR-2, for
the varieties of Kyeema and Doongara and one heterozygote F2 individual from the mapping population
(Kyeema / Doongara). Alleles differ by 8 bp. Horizontal scale in bp. Size is relative and not an accurate
measure. Expected allele sizes based on sequence information were 303 and 311 bp.
Figure 3 Hae III digestion of PCR products of marker SCU-Rice-STS-RG220 after agarose gel
electrophoresis and ethidium bromide staining. Lanes 1 to 3 are individuals homozygous for the allele
found in Doongara. Note the presence of 2 bands approximately positioned at the expected size of 312
and 365 bp. A band is also faintly visible at the expected size of 110 bp. Lanes 7 to 9 are individuals
homozygous for the allele found in Kyeema or Amaroo. Note the presence of 1 distinct band
approximately positioned at the expected size of 677 bp. The product at 110 bp was very faint but could
be detected on the original gel. Lanes 4 to 6 are heterozygous individuals from the mapping population
(Kyeema / Doongara).
Figure 4 PstI digested PCR products of the marker SCU-Rice-STS-S13471 after agarose gel
electrophoresis and ethidium bromide staining. Lanes 1 and 4 are individuals homozygous for the allele
1 2 3 4 5
100 bp ladder
..500
300
..200
9
found in Kyeema. Note the presence of 2 bands approximately positioned at the expected size of 100
and 185 bp. Lanes 5 to 7 are individuals homozygous for the allele found in Doongara. Note the
presence of 1 distinct band approximately positioned at the expected size of 283 bp. Lanes 2 and 3 are
heterozygous individuals from the mapping population (Kyeema / Doongara).
No recombinants in 50 F2 individuals were found for sd-1, SCU-Rice-SSR-2 (RG109), and SCU-Rice-
STS-S13471. One recombination (map distance of 1 cM) was identified between sd-1 and both, SCU-
Rice-STS-RG220 and SCU-Rice-STS-S13312. Two recombination events were recorded between
SCU-Rice-STS-RG220 and SCU-Rice-STS-S13312 (2 cM).
Markers SCU-Rice-SSR-2, SCU-Rice-STS-RG220, SCU-Rice-STS-S13312 were assessed for levels
for polymorphism-information-content (PIC) across 24 rice samples. The marker SCU-Rice-STS-
S13471 was assessed for 5 cultivars as low levels of polymorphism and similar polymorphism patterns
suggested further screening would be unproductive.
Varietal screening and Polymorphism Information Content (PIC) values for markers SCU-Rice-STS-
RG220, SCU-Rice-STS-S13312, SCU-Rice-STS-S13471 and SCU-Rice-SSR-2 are presented in Table
3. SCU-Rice-SSR-2 was the most polymorphic marker with a PIC value of 0.50 and three alleles
identified.
All of the semi-dwarf varieties analysed were Australian breeding lines or cultivars. The tall varieties of
Kyeema, Pelde, Goolarah, Inga and YRF203 are Australian. The rest of the tall samples were more
distantly related foreign varieties, mostly indica subspecies. (see Garland et al., 1999, for an assessment
of genetic similarity between these varieties).
No polymorphisms were identified, for the markers linked to sd-1, between alleles possessed by the tall
Australian cultivars and most of the Australian semi-dwarf lines. Alleleic differences were only present
between both Doongara and YRF204, and the tall Australian varieties of Kyeema, Pelde, Goolarah,
Inga and YRF203. Most of the tall foreign varieties possessed distinct alleleic differences for the
markers SCU-Rice-STS-S13312 and/or SCU-Rice-SSR-2, to the semi-dwarf specimens. Exceptions
were for the comparison of Doongara and YRF204 with Khao Dawk Mali 105, and Della with all semi-
dwarf cultivars except for Doongara and YRF204. Low levels of polymorphism were detected for the
marker SCU-Rice-STS-RG220.
Table 3 Varietal screening and Polymorphism Information Content values for the markers SCU-Rice-
SSR-2, SCU-Rice-STS-RG220, SCU-Rice-STS-S13312, and SCU-Rice-STS-S13471. Alleles for
SCU-Rice-SSR-2 are in base pairs as determined by capillary electrophoresis. D = allele found in
Doongara. K = allele found in Kyeema. nd = no data.
Marker SCU-Rice-Semi-Dwarf SSR- 2 STS - RG220 STS -S13312 STS - S13471
Doongara 305 D D D
YRF204 305 D K D nd
Amaroo 297 K K K
Namaga 297 K K K
Millin 297 K K nd
Echuca 297 K K nd
Jarrah 297 K K nd
Illabong 297 K K nd
10
YRW4 297 K K nd
YRK4 297 K K nd
Langi 297 K K nd
TallKyeema 297 K K K
Pelde 297 K K K
Goolarah 297 K K nd
Inga 297 K K nd
YRF203 297 K K nd
Della 297 K K nd
Azucena 297 K D nd
Millagrossa 295 K D nd
Dumsiah 295 K D nd
Dumsorkh 295 K D nd
Basmati 370 295 K D nd
Moosa Taroom 110 295 K D D ndKhao Dawk Mali 105 305 D D nd
Number of alleles 3 2 2 2PIC value 0.5 0.2 0.4 -
4.2 The development of markers for fgr
Segregation ratios for the fragrance genotypes were found to be consistent with that of a single
fragrance gene (Table 4).
Table 4 Observed segregation ratio for 124 F2 individuals (Kyeema / Doongara) and the expected
segregation ratio for a single gene. χ2(0.05, df = 2) analysis indicated no significant difference between
observed and expected ratios, p = 0.12.
Fragrant Segregating Non fragrant Significan
Observed
number 28 55 41
not
significant
Expected
number 31 62 31
A small microsatellite mono T repeat in homologous fragments of RG28 was identifed that differed in
repeat number between Kyeema and Doongara. Doongara had 9 tandem T repeats, RG28 (Variety
IR36) had 8 and Kyeema 7. The primers SCU-Rice-SSR-1.F (HEX labelled) (5´-
GATCTCACTCCAAGTAAACTCTGAC-3´) and SCU-Rice-SSR-1.R
(5´-ACTGCCATTGCTTCTGTTCTC-3´) were designed to flank the microsatellite region producing an
expected product of 130 bp for Doongara (marker SCU-Rice-SSR-1). The primers amplified a single
product that was determined as 125 bp in Kyeema and as expected, 2 bp larger in Doongara. Size
determined by the 310 Genetic Analyser is not necessarily an absolute measure when using a general
size standard. PCR conditions for SCU-Rice-SSR-1 were the same as those for the RM prefixed
markers without the addition of fluorescent dUTPs. Alleles for SCU-Rice-SSR-1 are presented if Figure
5.
11
Figure 5 Capillary electrophoresis output, demonstrating alleles for the marker SCU-Rice-SSR-1 for
the varieties Kyeema, Dumsiah and Doongara. Horizontal scale in bp. The peak to the right of the
Kyeema allele is an artefact. Results are also presented for 2 homozygous and one heterozygote F2
individual from the mapping population (Kyeema / Doongara).
The microsatellite marker SCU-Rice-SSR-1 was closely linked to the fragrance gene in the mapping
population. Four recombinants were found in the 50 F2 plants (4cM). The four recombinants included 1
individual homozygous for fgr and heterozygous for SCU-Rice-SSR-1 and 3 homozygous non-fragrant
individuals that were heterozygous for SCU-Rice-SSR-1.
Alleles for the markers SCU-Rice-SSR-1, RM223, and RM42, for a range of fragrant and non-fragrant
rice samples are presented in Table 5. The three alleles found for SCU-Rice-SSR-1 in the fragrant
cultivars represented 7, 8 and 9 T repeats (PIC = 0.48). Two alleles were found in the non-fragrant
cultivars, the 9 bp allele predominated and only the breeding line YRK4 possessed the second allele of 8
bp. The markers RM223 (PIC = 0.66) and RM42 (PIC = 0.64) were more polymorphic. Forty-eight
percent of the possible 144 pair-wise comparisons between the fragrant and non-fragrant cultivars for
SCU-Rice-SSR-1 did not produce a distinguishing allelic difference. For this same assessment, 12.5%
for RM42, 33.3% for RM223 and 8% for all three markers combined were determined. The rice
samples that could not be comprehensively distinguished by allelic differences were Della from
Doongara, Amaroo, Echuca, Inga, Jarrah, Langi, Millin, Namaga, Pelde and YRW4; and YRF203 and
YRF204 from Doongara.
Kyeema - 7 T repeats
Dumsiah - 8 T repeats
F2 Homozygote - 7 T repeats
F2 Heterozygote
F2 Homozygote - 9 T repeats
Doongara - 9 T repeats
12
Table 5 Microsatellite alleles in bp (based on 310 Genetic Analyser results) for the markers SCU-Rice-SSR-1,
RM42 and RM223 for a range of fragrant and non-fragrant cultivars. (PIC–Polymorphism Information Content)
(*more alleles may be present)
Fragrant
CultivarsSCU-Rice-SSR-1 RM42 RM223
Kyeema 125 167 151
Goolarah 125 167 145
Khao Dawk
Mali 105125 167 145
Azucena 125 167 147
Millagrossa 126 160 169 145 153*
Dumsiah 126 160 147
Moosa
Tarom 110126 127 160 149
Dumsorkh 127 160 149
Basmati 370 127 160 149
YRF203 127 167 151
YRF204 127 167 151
Della 127 165 167 149 151
Non-
Fragrant
Cultivars
SCU-Rice-SSR-
1RM42 RM223
Doongara 127 167 151
Amaroo 127 165 149
Echuca 127 165 149
Illabong 127 165 147
Inga 127 165 149
Jarrah 127 165 149
Langi 127 165 149
Millin 127 165 147 149
Namaga 127 165 149
Pelde 127 165 149
YRW4 127 165 149
YRK4 126 165 140 149
Number of Alleles 3 4 6
PIC value 0.48 0.64 0.66
13
5. Discussion
5.1 Markers for sd-1
The Australian semi-dwarf cultivars have a pedigree that indicates the presence of the sd-1 gene. The
exception is Doongara, however this study demonstrates that the source of semi-dwarfism in Doongara
is also sd-1. Four useful PCR based markers, closely linked to sd-1, have been produced. The marker
SCU-Rice-SSR-2 has the greatest immediate utility. Alleleic detection for this marker simply involves
size separation and the marker was able to distinguish between a reasonable proportion of the tall and
semi-dwarf samples tested in this investigation.
A suitable polymorphic marker was detected for most of the pair-wise comparisons between the semi-
dwarf and tall varieties. Most of the semi-dwarf or tall varieties had comprehensively different alleles
for the markers SCU-Rice-SSR-2 or SCU-Rice-STS-S13312, for 9 to 12 varieties from the opposing
height category. However, the marker alleles from the tall varieties of Pelde, Goolarah, Della and
YRF203 were only different to two semi-dwarf cultivars.
The lack of polymorphisms between some of the tall and semi-dwarf cultivars is not unexpected due to
the narrow genetic base of the Australian industry (reviewed by Ko et al., 1994) and the high levels of
genetic similarity between Australian varieties (Garland et al., 1999). Conversely, because the
Australian cultivars are highly related, it could be expected that markers linked to sd-1 would be
different between the tall and semi-dwarf lines. Possible explanations for the lack of polymorphism
between some of the tall and semi-dwarf Australian lines include:
1) Recombination between the linked markers present in the tall parent lines and sd-1
during cultivar development;
2) Lack of polymorphism between the tall and semi-dwarf parent lines originally used to
produce the cultivars analysed; or
3) Through mutation.
The lack of alleleic differences between the genetically divergent cultivars of Doongara (semi-dwarf)
and Khao Dawk Mali 105 (tall), certainly supports the possibility that a lack of polymorphism existed
between parent lines. Recombination between loci within 1 cM is also quite possible given the number
of meiotic events that would occur over the several years needed for cultivar development. Selection for
certain traits, either naturally or as part of the breeding program, could also greatly increase the chance
of producing cultivars where recombinations have occurred between sd-1and linked loci. It is of interest
to note that agronomic traits and QTLs that would be under selective pressure have been identified in
the same genome region as sd-1. These traits include, shattering resistance (Oba and Kikuchi,1991);
number of panicles per plant, number of filled grains per panicle, total number of spikelets per panicle,
% spiklet fertility, 1000 grain weight, grain weight per plant (Zhuang et al., 1997); and heading date
(see Ricegenes consensus map).
In order to confirm the cause for the lack of universality of different alleles to distinguish between tall and semi-
dwarf cultivars, we would need to:
1) Know what alleles were present in the parent cultivars,
2) Observe nucleotide instability to demonstrate mutational causes, and
14
3) Know the associations between allele types and the degree of expression of the
quantitative characteristics, in order to assess the effect of selective pressures on
recombination in the vicinity of sd-1. Obviously more work is necessary to identify the
mechanism involved. Undertaking such an investigation would be of interest, especially
for the assessment of marker stability.
Further markers for sd-1 may be needed for some crosses between semi-dwarf and tall varieties. As
indicated by the success of this investigation, markers could be developed from the numerous other
clones and STS linked to sd-1. Clones genetically mapped between G370 and C86 (Harushima et al.,
1998) and the clones RZ739 and RZ161 from the Ricegenes consensus map
(http://genome.cornell.edu/rice/quickqueries/) could be included. These genetic maps are also suitable
resources for the development of further markers for fgr.
5.2 Markers for fgr
Given that SCU-Rice-SSR-1, RM223 and RM42 are suitable markers for all sources of fragrance (fgr)
and the large proportion of fragrant and non-fragrant lines distinguished, these markers will be highly
useful in the Australian rice breeding program and in rice breeding programs in general. Della was the
only fragrant variety tested with low levels of separation from the non-fragrant lines. This was due to
the identification of two common alleles for the markers RM223 and RM42. Markers for fgr in Della
individuals will be distinguishable from alleles in non-fragrant lines in some cases, depending on which
allele(s) for the marker is present for the individual specimen. The heterogeneity in the Della sample was
not unexpected as it is susceptible to out-crossing (Garland et al. 1999). As heterogeneity was detected
in several samples, it is recommended that further screening be performed for different accessions or
seed samples to make the allelic characterisation comprehensive. In addition, it would be wise to verify
polymorphisms between parents of a cross before a program of marker aided selection is undertaken.
To summarise, four useful PCR based, co-dominant markers, have been produced for sd-1, the semi-
dwarfing gene utilised in the Australian industry. Three useful PCR based co-dominant markers, have
also been produced or identified for fgr, the gene responsible for the major component of fragrance in
rice. The markers for fgr and sd-1 will be useful in the Australian rice-breeding program and in rice
breeding programs in general.
15
6. Implications
It is envisaged that the markers developed in this study will be used for accelerated backcrossing and for
selection during inbreeding. Marker assisted backcrossing can halve the number of backcrosses
necessary to incorporate a gene of interest into a preferred genetic background (reviewed by Dudley,
1993). Marker assisted backcrossing is especially attractive for recessive genes, like sd-1 and fgr. There
is no need to identify heterozygous individuals in the backcross generations by traditional methods. The
heterozygous individuals carry the gene of interest and are needed for the production of the next
backcross. The traditional genotyping procedure is time consuming and involves the detection of
segregation in progeny produced by selfing individuals from each backcross generation.
A marker assisted exercise to incorporate fgr or sd-1 into a desirable genetic background would involve
a cross between a superior cultivar and one possessing the gene of interest, followed by repeated
backcrossing of selected progeny to the superior cultivar. Progeny would be selected, before maturity
and without genotype assessment, that had the lowest genome composition from the varietal source of
fgr or sd-1. Genome composition would be assessed and progeny selected based on the presence of the
lowest proportion of alleles from the varietal source of fgr or sd-1, for markers unlinked or distantly
linked to the gene of interest, and the presence of alleles for the markers closely linked to those genes.
The number of backcrosses are greatly reduced when markers for the recurrent parent are incorporated
into a marker assisted backcrossing exercise (reviewed by Dudley, 1993).
Markers for other traits of interest could also be incorporated into such an exercise. The Australian rice
industry is currently trying to develop a basmati style variety suitable for Australian conditions.
Basmati style cultivars are fragrant, experience grain elongation with little increase in breadth after
cooking, have low to moderate amylose content (<25%), and a soft texture (Ahn et al., 1993). A topical
example of accelerated backcrossing can be provided using the development of a basmati style variety
and utilizing markers for sd-1, fgr, markers for other traits of interest and randomly distributed markers.
Marker assisted selection may provide the power necessary for the development of productive semi-
dwarf Basmati style cultivars given the low levels of success achieved by traditional breeding.
Difficulties experienced, include a lack of desirable recombinants and reversion to the recurrent parent
type in backcrossing exercises (reviewed by Ahn et al., 1993). This may reflect problems with
segregation distortion biased towards the genotype of the recurrent parent. Markers will be able to
identify the possibly lower than expected numbers of individuals possessing important genomic regions
from the basmati cultivar.
The initial cross, in our example of marker assisted backcrossing, could involve a highly productive
semi-dwarf Australian long grain cultivar, with grain quality characteristics of greatest similarity to
basmati rice, crossed with a traditional basmati cultivar. F2 progeny would be selected based on the
presence of marker alleles for sd-1, fgr, and the least proportion of basmati derived alleles for markers
in other genome regions. The selection of alleles for markers linked to other specific genes that could be
incorporated into this process, include 2 that are important to cooking quality. Developed microsatellite
markers for the Waxy gene, a gene that controls amylose content (Ayres et al., 1997, Tan et al., 1999),
should definitely be utilized. Markers linked to the genome region containing the starch branching
enzyme III (Harrington et al., 1997), a gene that controls the relative proportions of amylose and
amylopectin in rice grains, should also be incorporated into the exercise.
Progeny could also be selected based on the presence of alleles for QTL loci from the parent line
possessing the superior phenotype for the trait of interest. A major QTL for cooked-kernel elongation
16
has been identified on chromosome 8 (Ahn et al., 1993). It would probably be appropriate to select
progeny that contain alleles for markers in this genome region that originated from the basmati parent.
Markers for other QTL, such as those traits with numerous QTL of low effect (see review by Yano and
Sasaki, 1997) could also be considered. However, as indicated by Redona and Mackill (1996) alleles for
markers linked to QTLs that are associated with superior measurement for the traits, do not necessarily
come from the superior parent. The appropriate alleles for selection will be cross specific and possibly
environmentally influenced. These markers will need to be identified through empirical studies.
17
7. Recommendations
1) It is recommended that the markers for sd-1 be applied to marker assisted backcrossing. The marker
assisted conversion of tall varieties, for example Kyeema or Pelde, to semi-dwarfs is an example.
The markers for sd-1 are probably not of great benefit to the selection of semi-dwarfs during
inbreeding. Visual selection in the standard breeding program is probably sufficient.
2) Markers for fgr should be applied to selection in both inbreeding and backcrossing exercises due to
the time consuming and inaccurate nature of assessing fragrance.
3) Markers for both traits should be assessed on the parents of the initial cross to confirm marker
polymorphism, on the F1 plants to confirm the success of the cross and in subsequent generations.
Marker assessment of the F2 generation is particularly important during inbreeding to ensure that
recombination does not occur between the gene of interest and the marker and thus reduce the
accuracy of detection of the gene in later generations.
4) Heterogeneity was detected in several samples, for some of the markers linked to genes of interest
identified in this study, and it is recommended that further screening be performed. Alleleic
screening of different accessions or seed samples, that may be involved in marker assisted selection
programs, should be undertaken in order to make the allelic characterisation comprehensive. In
addition, it would be wise to verify polymorphism between parents of a cross before a program of
marker aided selection is undertaken.
5) There were some potential crosses identified that did not possess a suitable marker for either sd-1 or
fgr. Given the success of this investigation further screening of linked RFLP probes would probably
be the most efficient method for the development of further markers for these two genes.
6) A comprehensive fingerprinting exercise should now be undertaken to extend the work of Garland et
al., 1999). There are hundreds of microsatellite markers now available for rice. Alleles for 100 or
more microsatellite markers, distributed through the genome, should be determined for numerous
individuals, from several accessions, for each cultivar or rice sample of importance to the
Australian rice-breeding program. The presence of a database containing the alleleic
characterisations would allow for the selection of a suite of markers which are polymorphic between
Australian varieties, breeding lines and breeding samples, for use in germplasm identification,
genetic mapping, and marker assisted selection programs.
7) The challenge is to maximise the potential benefits of markers through the appropriate integration of
marker assisted selection into the breeding program. It may be necessary to restructure the breeding
program to obtain maximum benefits. For example, it will be necessary to screen F2 individuals
during inbreeding exercises. In general, individual plants will need to be identified. It may be
necessary to transplant by hand or reduce the density of planting to enable tagging of individuals or
groups of individuals for accurate re-sampling in the selection process.
8) In order to facilitate the integration of markers into the breeding program it may be appropriate to
create a new position of Molecular Plant Breeder. This position would work in close collaboration
with breeders and cereal chemistry. The initial appointment could involve a specific task. For
example, marker assisted development of a suitable Basmati style variety through accelerated
backcrossing. Suitable procedures would be developed and refined for possible extension of marker
assisted selection into the wider breeding program.
18
19
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