UNIVERSITY GRANTS COMMISSION BAHADUR SHAH ZAFAR … · KSSR-17. iv. Molecular diversity analysis: For deducing relationship among individual plants and/ or populations, each band

UNIVERSITY GRANTS COMMISSION BAHADUR SHAH ZAFAR MARG

NEW DELHI – 110 002

Final Report of the work done on the Major Research Project (Report to be submitted within 6 weeks after completion of each year)

1. Project report No. 1st /2nd /3rd :

/Final Final

2. UGC Reference No. : F. No. 41-533/2012(SR) dated 17.07.2012

3. Period of report from : 01.07.2012 to 30.06.2015

4. Title of research project : Molecular characterization and micropropagation of Salampanja (Dactylorhiza hatagirea) for rapid multiplication and conservation

5. a) Name of the Principal Investigator

b) Deptt.

c) University/College where work has progressed

: : :

Dr R K Kapila

Department of Agricultural Biotechnology

CSKHPKV, Palampur, HP, India 176 062.

6. Effective date of starting the

project : 01.07.2012

7. Grant approved and

expenditure incurred during the period of the report:

a. Total amount approved Rs.

: Rs. 11,00,800/-

b. Total expenditure Rs. : Rs 9,90,640/- c. Report of the work done : Section-wise given below

i) Brief objective of the project : 1. Survey and collection of ecotypes of Salampanja (Dactylorhiza hatagirea)

endemic to alpine regions of Himachal Pradesh and their conservation in ex-situ Field Gene Bank,

2. Morphological and molecular characterization of available genetic diversity among natural populations/ecotypes of Salampanja (Dactylorhiza hatagirea) from alpine regions of Himachal Pradesh-North Western Himalayas using morphological and DNA markers, and

3. Development of efficient in vitro protocols for rapid multiplication of quality planting material of Salampanja.

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ii) Work done so far and results achieved and publications, if any, resulting from the work (Give details of the papers and names of the journals in which it has been published or accepted for publication)

a.

Objective 1: Survey and collection of ecotypes of Salampanja (Dactylorhiza hatagirea) endemic to alpine regions of Himachal Pradesh and their conservation in ex-situ Field Gene Bank

Objective-wise work done and detailed results

a. Survey and collection of planting material (seeds/tubers) of Salampanja (Dactylorhiza hatagirea): An extensive survey of alpine regions of districts Kinnaur, Kullu and Lahaul & Spiti including places like Sangla, Chhitkul, Rangrik, Shakoli, Bathard ,Tosh, Mane Koma, Sichling, Shego and Giyu was conducted in the months of July/August/September 2012 & 2013 and a total population of 48 plants representing ten ecotypes were collected (Plates 1 & 2). The details of location, its geographical coordinates, altitudes and number of plants collected are tabulated below in Table 1. Table 1. Details of ten collections of Salampanja collected from alpine regions of Himachal Pradesh during September 2012 & 2013 Collection Plant

Nos. Location Longitude Altitude

(amsl) DHC-1 1-5 Sangla, Kinnaur (HP) 310

7825.114 N

02616 m

16.422 E DHC-2 6-9 Chhitkul, Kinnaur (HP)

310

7821.044 N

03393 m

25.284 E DHC-3 10-14 Rangrik, Spiti Valley

(HP) 320

7815.103 N

03270 m

02.218 E DHC-4 15-18 Shakoli, Lahaul Valley

(HP) 320

7641.694 N

02991m

40.581 E

DHC-5 19-23 Bathardh, Kullu (HP) 310

7751.416 N

02527 m

19.024 E DHC-6 24-27 Tosh, Kullu (HP) 310

7759.375 N

02910 m

28.149 E DHC-7 28-32 Mane Koma, Spiti

Valley (HP) 320

7801.609 N

03318 m

14.441 E DHC-8 33-37 Sichling, Spiti Valley

(HP) 320

7803.734 N

03275 m

13.061 E DHC-9 38-41 Shego, Spiti Valley (HP) 320

7810.609 N

03533 m

06.069 E DHC-10 42-48 Giyu, Spiti Valley (HP) 320

7804.520 N

03195 m

36.737 E

b. Establishing ex situ Field Gene Bank: All these ten collections are being maintained at Department of Agriculture Biotechnology, CSK HPKV Palampur as ex situ Field Gene Bank under protected conditions for their further use in tissue culture and molecular fingerprinting studies (See Plate 3). The respective collections have also been maintained at Research Centres of the University at Sangla (Kinnaur) and Kukumseri (Lahaul & Spiti) also for their further use as well as ex situ Field Gene Bank.

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Plate 1. Dactylorhiza hatazirea plants at maturity growing under natural conditions at

the sites of collection

Plate 2. Dactylorhiza hatazirea plants at maturity and uprooted tuber being collected

for maintaining ex situ Field Gene bank

Plate 3. Ex situ maintained plants of ten collections at Department of Agricultural Biotechnology CSKHPKV Palampur for further studies

Objective 2: Morphological and molecular characterization of available genetic diversity among natural populations/ecotypes of Salampanja (Dactylorhiza hatagirea) from alpine regions of Himachal Pradesh - North Western Himalayas using morphological and DNA markers

a. Morphological characterization: The data on 6 morphological traits of representative plant samples of all the 10 collections are presented in Table 2. Most of the collections exhibited minor differences for morphological traits which were available in the species for data recording. Besides plant height and flower length, minor differences were observed with respect to flower colour and number of florets/

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capsules per inflorescence. Due to negligible observed differences among the different ecotypes for morphological traits, these data were not used for further clustering and diversity analysis along with molecular data. Table 2. Morphological features/data of ten collections of Salampanja collected from alpine regions of Himachal Pradesh Collection Location Plant

height at initiation of flowering (cm)

Plant height at maturity(cm)

Flower Colour

Flower/Spike length (cm)

Mean number of florets/ capsules per spike

Leaf lamina width (mm)

DHC-1 Sangla, Kinnaur (HP)

18.4 28.6 Light purple

7.6 14.2 26.6

DHC-2 Chhitkul, Kinnaur (HP)


8.4 13.6 24.4

DHC-3 Rangrik, Spiti Valley (HP)

14.8 26.6 Purple 9.6 12.8 23.6

DHC-4 Shakoli, Lahaul Valley (HP)


7.4 10.4 22.4

DHC-5 Bathardh, Kullu (HP)

13.8 26.6 Purple 8.8 11.4 20.2

DHC-6 Tosh, Kullu (HP)


6.6 10.8 19.4

DHC-7 Mane Koma, Spiti Valley (HP)


5.4 10.2 18.6

DHC-8 Sichling, Spiti Valley (HP)


6.4 10.8 16.4

DHC-9 Shego, Spiti Valley (HP)


7.2 11.2 17.4

DHC-10 Giyu, Spiti Valley (HP)


7.4 11.6 17.8

b. Molecular Characterization: For molecular characterization, a set of 48 plants of ten different collections (Table 1) collected under this project was used. For molecular characterization, step-wise the following work was accomplished: i. DNA Isolation: DNA of all 48 plants of ten collections was extracted using CTAB method and its quality and quantity assessed by running on agarose gel (Plate 4).

Plate 4. Quality and quantity assessment of 25 samples of DNA isolated from Dactylorhiza hatagirea collections

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 M Lanes 1-25 are DNA samples

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ii. Designing of new Dactylorhiza SSR primers: For designing Dactylorhiza hatageriea specific primers, a total of 745 sequences were downloaded from NCBI website. These sequences belong to different species of Dactylorhiza viz., Dactylorhiza fuchsii x Dactylorhiza incarnate, Dactylorhiza viridis, Dactylorhiza majalis, Dactylorhiza maculate, Dactylorhiza foliosa, Dactylorhiza elata, Dactylorhiza sambucina, Dactylorhiza romana, Dactylorhiza occitanica, Dactylorhiza insularis, Dactylorhiza ochroleuca, Dactylorhiza iberica, Dactylorhiza praetermissa, Dactylorhiza aristata, Dactylorhiza angustata, Dactylorhiza sphagnicola etc. These sequences were downloaded from EST database of NCBI in FASTA format and masking was done using Repeat Masker software. Only those sequences were selected whose matching repeat were between 2 to 6 base pairs. In all, following 40 microsatellite primers were designed for Dactylorhiza hatagirea using Primer 3 software to achieve better molecular characterization of collected population using species specific SSR markers (Table 3).

Table 3. Sequences of a set of 40 new SSR primers developed from available sequences of related species on NCBI

S. No. Seq. No.

Primer name Sequence No of bases

KSSR1-F 5 Left primer GCCCGCGAACACTTTATTTA 20 KSSR1-R Reverse CTCCTCGCGAATGAAATGAT 20 KSSR2-F 15 forward GGTCCAGGGGGATAAGTTCT 20 KSSR2-R Reverse AGAAAGAACGCCAAAGACGA 20 KSSR3-F 29 forward GCCCGCGAACACTTTATTTA 20 KSSR3-R Reverse CTCCTCGCGAATGAAATGAT 20 KSSR4-F 33 forward CGCGAAGTCAAGATTGAAAA 20 KSSR4-R Reverse CCCGGCCAGTACTTAACCAG 20 KSSR5-F 41 forward GGAGATGCAGTTTGTCGTGA 20 KSSR5-R Reverse TTACCAAGTGCACACCAAGC 20 KSSR6-F 43 forward CATGACATGGATGAAGGAGTAGA 23 KSSR6-R Reverse GCCCAGGTGGAGAAACTCTT 20 KSSR7-F 188 forward AAACAAACATGCCCCAGTTA 20 KSSR7-R Reverse GAGCCGGACATGAGAGTTTC 20 KSSR8-F 189 forward GCCCGCGAACACTTTATTTA 20 KSSR8-R Reverse CTCCTCGCGAATGAAATGAT 20 KSSR9-F 190 forward GCCCGCGAACACTTTATTTA 20 KSSR9-R Reverse CTCCTCGCGAATGAAATGAT 20 KSSR10-F 379 forward TCCTCTGCAGTCTTGTTCCA 20 KSSR10-R Reverse AAAGCGCATGAGAAAGAACG 20 KSSR11-F 380 forward TCCTCTGCAGTCTTGTTCCA 20 KSSR11-R Reverse GAGAAAGAACGCCAAAGACG 20 KSSR12-F 381 forward CAGGGGGATAAGTTCTCGAC 20 KSSR12-R Reverse AGAAAGAACGCCAAAGACGA 20 KSSR13-F 384 forward GGTGTTCCTAACTGCCCACT 20 KSSR13-R Reverse GAGAAAGAACGCCAAAGACG 20 KSSR14-F 385 forward GGTGTTCCTAACTGCCCACT 20 KSSR14-R Reverse GAGAAAGAACGCCAAAGACG 20 KSSR15-F 386 forward GGTGTTCCTAACTGCCCACT 20 KSSR15-R Reverse GAGAAAGAACGCCAAAGACG 20 KSSR16-F 387 forward CCCGTGGGATTCTTTTTCAT 20 KSSR16-R Reverse AGAAAGAACGCCAAAGACGA 20 KSSR17-F 388 forward CCGTGGGATTCCTTTTTCAT 20 KSSR17-R Reverse GAGAAAGAACGCCAAAGACG 20 KSSR18-F 399 forward CGCGAAGTCAAGATTGAAAA 20 KSSR18-R Reverse GGGAAATGAACCTTTTGCAC 20 KSSR19-F 407 forward TGCCAAGTGTATTGTGGAGGT 21 KSSR19-R Reverse TTCTATCTTGGCCCAAATGC 20

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KSSR20-F 410 forward CGCCGACAAACTCTACATCG 20 KSSR20-R Reverse CGATCCTCATCCTGTTTTGC 20 KSSR21-F 411 forward CTGGAAGTAGGGGGAGCAAT 20 KSSR21-R Reverse CTCAATCATCCAAAGGGACAA 21 KSSR22-F 414 forward AAGGTACCACGCTTCGTCAG 20 KSSR22-R Reverse GACTGCAGGTAAGGGCTCAG 20 KSSR23-F 416 forward AGGGTGAAAAACCACACGAC 20 KSSR23-R Reverse CACCCTGGTCTCAAAAGGAA 20 KSSR24-F 417 forward CACTCTCCTCCTTGATTCAGC 21 KSSR24-R Reverse CACCCTGGTCTCAAAAGGAA 20 KSSR25-F 418 forward ACAAAGCAGAGGCAAGGATG 20 KSSR25-R Reverse CAATTCCATCCAAACGAACA 20 KSSR26-F 419 forward TCCCCCATCACAACATACAA 20 KSSR26-R Reverse GATCATTATCCCGAGCCAAG 20 KSSR27-F 420 forward GATCCGATGTTGGGTATTGG 20 KSSR27-R Reverse GGGCACCTTAGAAGGCAAAG 20 KSSR28-F 421 forward CGGTTAGATGAGTCCTTGCTG 21 KSSR28-R Reverse GCGCCAAGAAGTTCTCTCAT 20 KSSR29-F 462 forward CAACAGCATGTTCGTGTGC 19 KSSR29-R Reverse GCTCGCTTTCTGTCGGTATT 20 KSSR30-F 490 forward GCCCGCGAACACTTTATTTA 20 KSSR30-R Reverse CTCCTCGCGAATGAAATGAT 20 KSSR31-F 597 forward GCCAGCATTGAGAGTGATGA 20 KSSR31-R Reverse GACTGGACCTGGAGCAGAAA 20 KSSR32-F 618 forward CGATGGAAGCTGTTCTAACGA 21 KSSR32-R Reverse TGGGACTCTCTCTTTATTCTCGTC 24 KSSR33-F 619 forward CGATGGAAGCTGTTCTAACGA 21 KSSR33-R Reverse TGGGACTCTCTCTTTATTCTCGTC 24 KSSR34-F 620 forward GGAGATGCAGTTTGTCGTGA 20 KSSR34-R Reverse TGTGCACACCAAGCTTCC 18 KSSR35-F 621 forward TCAGCGGAGGAGAGGTAGAA 20 KSSR35-R Reverse TGGCCACTTGTAGTGAGCTG 20 KSSR36-F 628 forward AAACCTTTCGCTTCTGATCG 20 KSSR36-R Reverse GCGTCCTCGACGAATAAGAC 20 KSSR37-F 714 forward CATGCCCCAGTTATCCACTT 20 KSSR37-R Reverse GAGCCGGACATGAGAGTTTC 20 KSSR38-F 719 forward GCCCGCGAACACTTTATTTA 20 KSSR38-R Reverse CTCCTCGCGAATGAAATGAT 20 KSSR39-F 720 forward TAAACAAACATGCCCCAGTT 20 KSSR39-R Reverse CTCCTCGCGAATGAAATGAT 20 KSSR40-F 744 forward GGTGCGGTTATGCCAGTATT 20 KSSR40-R Reverse CACCTGCCCTTTTGATCACT 20

iii. Screening of primers: Forty newly developed SSR primers were screened for successful amplification and assessment of polymorphism on a subset of 10 randomly selected plants and the primers exhibiting successful amplification on the subset were selected for running PCR and final assessment of polymorphism at population level (Plate 5). Out of 40 newly developed primers, 28 exhibited successful amplification. Fifteen of those exhibiting polymorphism among populations were used for final molecular characterization of the population (Plates 6-9).

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Plate 5. Screening for amplification of products of newly developed SSR markers, KSSR-1 and KSSR-2 on a subset of 10 random plants

Plate 6. Polymorphism in 48 accessions of Dactylorhiza hatagirea using SSR marker KSSR-1


Plate 8. Polymorphism in 48 accessions of Dactylorhiza hatagirea using SSR marker, KSSR-16


iv. Molecular diversity analysis: For deducing relationship among individual plants

and/ or populations, each band of a specific molecular weight in the DNA profile of an

individual was treated as locus/marker. A binary data matrix with ‘1’ indicating the

presence and ‘0’ the absence of a particular band was generated for all 15 SSR

primers. The binary data matrix was prepared for each molecular marker separately

and they were clubbed as combined data for overall analysis. The binary data for all

markers were further analyzed with the help of GenAlex software (Peakall and

Smouse, 2006) for analysis of molecular variance (AMOVA), principle coordinate

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analysis (PCoA), Fst estimation, Nei’s genetic similarity, estimated gene flow (Nm),

Mantel test of geographic and genetic distance etc.

The molecular data generated using 15 SSR primers revealed considerable diversity

among 48 individuals of 10 ecotypes/collections. Fifteen primers generated a total of

38 bands/alleles with an average polymorphism of 54.93% ranging from 0.00 to

95.83% (Table 4).

Table 4. Descriptors of 15 SSR primer sets used to elucidate population structure of Salampanja and level of polymorphism explained by each primer

Primer name Primer Sequence (5’-3’) Repeat motif Ta

PIC (%) (°C)

KSSR-02 F-GGTCCAGGGGGATAAGTTCT R-AGAAAGAACGCCAAAGACGA

(TTC)4 53 57.03

KSSR-04 F-CGCGAAGTCAAGATTGAAAA R-CCCGGCCAGTACTTAACCAG (TA)6 50 37.50

KSSR-07 F-AAACAAACATGCCCCAGTTA R-GAGCCGGACATGAGAGTTTC (TA)6 51 53.01

KSSR-11 F-TCCTCTGCAGTCTTGTTCCA R-GAGAAAGAACGCCAAAGACG (TTC)4..(TTCCTC)3 53 30.91

KSSR-12 F-CAGGGGGATAAGTTCTCGAC R-AGAAAGAACGCCAAAGACGA (AGA)3 53 61.83

KSSR-15 F-GGTGTTCCTAACTGCCCACT R-GAGAAAGAACGCCAAAGACG (TTC)4 53 57.03

KSSR-18 F-CGCGAAGTCAAGATTGAAAA R-GGGAAATGAACCTTTTGCAC (TA)6 50 17.08

KSSR-20 F-CGCCGACAAACTCTACATCG R-CGATCCTCATCCTGTTTTGC (GAA)6 52 0.00

KSSR-21 F-CTGGAAGTAGGGGGAGCAAT R-CTCAATCATCCAAAGGGACAA (AGA)6 51 18.66

KSSR-22 F-AAGGTACCACGCTTCGTCAG R-GACTGCAGGTAAGGGCTCAG (TCT)8 56 53.50

KSSR-30 F-GCCCGCGAACACTTTATTTA R-CTCCTCGCGAATGAAATGAT (TA)8 54 34.96

KSSR-32 F-CGATGGAAGCTGTTCTAACGA R-TGGGACTCTCTCTTTATTCTCGTC (CAA)3 53 53.80

KSSR-35 F-TCAGCGGAGGAGAGGTAGAA R-TGGCCACTTGTAGTGAGCTG (GAA)6 56 30.34

KSSR-37 F-CATGCCCCAGTTATCCACTT R-GAGCCGGACATGAGAGTTTC (TA) 53 8

15.27

KSSR-39 F-TAAACAAACATGCCCCAGTT R-CTCCTCGCGAATGAAATGAT (TA) 50 15

35.22

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The binary data used for principal coordinates analysis (PCoA) distributed the samples

in two coordinates, coordinate one accounted for 36.78% and coordinate two for

22.51% of the total variation among populations (Fig 1). Distribution pattern of all the

samples from different populations revealed consistency with their geographical origin.

It shows that there is much lesser variation within population as compared to among

populations. The same was evident from the analysis of molecular variance (AMOVA),

which showed 27% (p = 0.01) variation within population as compare to 73% (p = 0.01)

among populations (Table 5).

Table 5. Analysis of Molecular Variance (AMOVA) of 48 samples of 10 populations of Salampanja

Df = degree of freedom, SS = sum of squares, MS = Mean square.

Fig 1. Principal Coordinate Analysis of genetic differences among 10 populations of Salampanja

-Values in parenthesis shows level of variation explained by the coordinate

1 2 3 4 5 6 7 8 9 10 11

12 13

14

15 16 17 18

19 20 21 22 23

24 25 26 27

28

29 30

31 32 33 34 35 36 37

38 39 40

41

42

43 44 45 46 47 48

Coor

d. 2

(22.

51%

)

Coord. 1 (36.78%)

Pop1

Pop2

Pop3

Pop4

Pop5

Pop6

Pop7

Pop8

Pop9

Pop10

Source df Sum of Squares

Mean Squared Deviation

Estimated Variance

% Total Variance

Prob

Among Pops. 9 181.213 20.135 3.904 73%

<0.01

Within Pops. 38 55.579 1.463 1.463 27%

<0.01

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The binary data was further used for calculation of genetic similarity index (Table 6)

which was used for generation of dendrogram to view the relatedness among

populations. Based on similarity index, populations have been grouped in four different

clusters at 60% of genetic similarity (Fig. 2). The populations of Sangla, Chitkul,

Shakoli and Tosh grouped in one cluster at 92% of genetic similarity, followed by

ManeKoma, Sichling, Giyu and Shego at 87% of genetic similarity. Bathard and

Rangrik populations out grouped indicating diversity and their separate origin.

Fig 2. Dendrogram of 10 populations of Salampanja representing clustering of populations in four major groups on the basis of Nei’s genetic similarity index

The dendrogram created for all 48 individual samples in the present analysis clearly

indicated that all the samples from a particular location grouped in one cluster (Fig. 3) that

was similar to overall pattern of population grouping as depicted in Fig.2.

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Fig 3. Dendrogram of 48 samples from 10 populations of Salampanja representing clustering of populations in four major groups on the basis of Nei’s genetic similarity index

Table 6. Nei's genetic similarity index among populations of Salampanja

The coefficient of gene differentiation among populations within the species (Gst) was

0.7898. Based on this Gst value, the estimated extent of gene flow (Nm) among populations

Sangla Chhitkul Rangrik Shakoli Bathardh Tosh Mane Koma Sichling Shego Giyu

Sangla 1.0 0.968 0.691 0.946 0.613 0.884 0.783 0.769 0.754 0.842

Chhitkul 1.0 0.679 0.973 0.573 0.919 0.766 0.739 0.745 0.829

Rangrik 1.0 0.674 0.584 0.639 0.726 0.701 0.808 0.747

Shakoli 1.0 0.543 0.959 0.768 0.739 0.732 0.791

Bathardh 1.0 0.492 0.667 0.628 0.614 0.720

Tosh 1.0 0.735 0.712 0.683 0.723

Mane Koma 1.0 0.973 0.871 0.924

Sichling 1.0 0.868 0.905

Shego 1.0 0.888

Giyu 1.0

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is 0.1331, ranging from 0.008 (between populations from Bathard and Tosh) to 2.88

(between populations from ManeKoma and Sichling). The pair-wise differences (Fst)

between populations (calculated based on allele sharing) varied from 0.05 to 0.878 (Table

8). A migration rate of 0.5 is considered sufficient to overcome the diversifying effects of

random drift in the populations. In this study, the estimated gene flow was lower than the

average value (2.85) reported for out crossed animal-pollinated species and that of mixed

mating species. Based on AMOVA and gene flow between populations, it can be concluded

that Salampanja populations have mainly been distributed to various sites by anthropogenic

methods and its method of propagation is mainly clonal as there is lower gene flow and

population differentiation, resulting in genetically similar populations.

Based on this study it can be said that there might exist four different introductions/major

ecotypes of Salampanja in Himachal Pradesh which probably are getting propagated

predominantly through clonal means.

Table 7. Nei’s gene diversity (h), Shannon’s Information Index (I), Effective number of alleles (Ne) and percent polymorphic loci for each population of Salampanja

Population h I ne PPL (%)

Sangla 0.0536+0.1342 0.0812+0.1981 1.0891+0.2379 15.79

Chhitkul 0.0302+0.1092 0.0445+0.1582 1.0529+0.2003 7.89

Rangrik 0.0738+0.1572 0.1107+0.2284 1.1268+0.2876 21.05

Shakoli 0.0302+0.1092 0.0445+0.1582 1.0529+0.2003 7.89

Bathardh 0.0612+0.1450 0.0908+0.2138 1.1022+0.2474 15.79

Tosh 0.0193+0.0885 0.0286+0.1278 1.0343+0.1682 5.26

Mane Koma 0.0987+0.1715 0.1492+0.2501 1.1669+0.3139 28.95

Sichling 0.0363+0.1033 0.0580+0.1586 1.0554+0.1716 13.16

Shego 0.0363+0.1138 0.0549+0.1678 1.0608+0.2041 10.53

Giyu 0.0795+0.1597 0.1215+0.2303 1.1361+0.2966 26.32

Independent analysis of pair-wise distances (Fst) and gene flow (Nm) indicated that the

relationship between the populations differs irrespective of geographic closeness. The

Mantel test (Fig. 5) did not show a significant correlation between geographic and genetic

distances (r = 0.002).

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Fig 5. Test of correlation between genetic and geographic distances among 10 populations of Salampanja

Table 8. Nei’s gene diversity (h), Shannon’s Information Index (I), Population differentiation Gst and Phi statistics (Fst), heterozygosity, and estimate of gene flow (Nm) among ten Salampanja populations

Pop1 Pop2 h I Ht Gst Fst Nm

(Gst)

Nm

(Fst)

Sangla Chhitkul 0.0542±0.134 0.0826±0.199 0.0533±0.0174 0.2143 0.206 1.8335 0.966

Sangla Rangrik 0.1989±0.2162 0.2933±0.3027 0.1989±0.0467 0.6798 0.744 0.2355 0.086

Chhitkul Rangrik 0.1986±0.2210 0.2901±0.3094 0.1979±0.0495 0.7374 0.783 0.1781 0.069

Sangla Shakoli 0.0655±0.1493 0.0982±0.2178 0.0645±0.0215 0.3507 0.370 0.9258 0.425

Chhitkul Shakoli 0.0384±0.1060 0.0607±0.1640 0.0384±0.0112 0.2153 0.259 1.8224 0.714

Rangrik Shakoli 0.2075±0.2296 0.2982±0.3214 0.2070±0.0533 0.7488 0.786 0.1677 0.068

Sangla Bathardh 0.2363±0.2254 0.3418±0.3152 0.2363±0.0508 0.7571 0.798 0.1604 0.063

Chhitkul Bathardh 0.2455±0.2336 0.3497±0.3273 0.2462±0.0556 0.8144 0.841 0.1139 0.047

Rangrik Bathardh 0.2613±0.2307 0.37470.3193 0.2613±0.0532 0.7416 0.784 0.1742 0.069

Shakoli Bathardh 0.2600±0.2348 0.3692±0.3280 0.2609±0.0559 0.8249 0.851 0.1062 0.044

Sangla Tosh 0.0932±0.1818 0.1356±0.2586 0.0920±0.0320 0.6042 0.636 0.3276 0.143

Chhitkul Tosh 0.0647±0.1527 0.0961±0.2197 0.0647±0.0233 0.6178 0.667 0.3094 0.125

Rangrik Tosh 0.2255±0.2305 0.3246±0.3212 0.2247±0.0533 0.7929 0.821 0.1306 0.055

Shakoli Tosh 0.0480±0.1335 0.0712±0.1939 0.0480±0.0178 0.4849 0.467 0.5312 0.286

Bathardh Tosh 0.2850±0.2342 0.4030±0.3254 0.2857±0.0553 0.8591 0.878 0.082 0.035

Sangla Mane Koma 0.1723±0.2035 0.2578±0.2896 0.1723±0.0414 0.5582 0.622 0.3957 0.152

Chhitkul Mane Koma 0.1678±0.2122 0.2462±0.2006 0.1652±0.0449 0.61 0.669 0.3197 0.123

Rangrik Mane Koma 0.2062±0.2199 0.3009±0.3097 0.2062±0.0483 0.5817 0.648 0.3596 0.136

Shakoli Mane Koma 0.1734±0.2180 0.2524±0.3071 0.1709±0.0471 0.6229 0.666 0.3027 0.125

y = 0.002x + 5.0223 R² = 0.0004

0.000

5.000

10.000

15.000

20.000

0.000 50.000 100.000 150.000 200.000 250.000

GD

GGD

GGD vs GD

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Bathardh Mane Koma 0.2268±0.2222 0.3302±0.3111 0.2268±0.0494 0.6474 0.707 0.2723 0.104

Tosh Mane Koma 0.1896±0.2218 0.2750±0.3127 0.1868±0.0484 0.6842 0.719 0.2308 0.098

Sangla Sichling 0.1561±0.2085 0.2299±0.2949 0.1561±0.0435 0.7121 0.741 0.2022 0.087

Chhitkul Sichling 0.1523±0.2154 0.2197±0.3041 0.1535±0.0477 0.7834 0.813 0.1383 0.058

Rangrik Sichling 0.1900±0.2173 0.2796±0.3039 0.1900±0.0472 0.7102 0.757 0.2041 0.080

Shakoli Sichling 0.1549±0.2124 0.2257±0.3001 0.1561±0.0461 0.7869 0.813 0.1354 0.058

Bathardh Sichling 0.2272±0.2306 0.3268±0.3212 0.2272±0.0532 0.7853 0.808 0.1367 0.059

Tosh Sichling 0.1678±0.2288 0.2389±0.3183 0.1686±0.0531 0.8351 0.849 0.0987 0.044

Mane Koma Sichling 0.0792±0.1305 0.1306±0.2038 0.0792±0.0170 0.1479 0.050 2.8813 4.750

Sangla Shego 0.1455±0.1995 0.2172±0.2840 0.1458±0.0406 0.692 0.757 0.2226 0.080

Chhitkul Shego 0.1445±0.2102 0.2101±0.2965 0.1445±0.0442 0.7701 0.822 0.1493 0.054

Rangrik Shego 0.1366±0.1996 0.2015±0.2853 0.1355±0.0397 0.5938 0.655 0.342 0.131

Shakoli Shego 0.1554±0.2210 0.2224±0.3098 0.1554±0.0489 0.7862 0.830 0.1359 0.051

Bathardh Shego 0.2242±0.2253 0.3246±0.3155 0.2249±0.0516 0.7833 0.815 0.1383 0.057

Tosh Shego 0.1811±0.2353 0.2552±0.3282 0.1811±0.0553 0.8467 0.877 0.0906 0.035

Mane Koma Shego 0.1204±0.1792 0.1847±0.2594 0.1175±0.0314 0.4257 0.485 0.6746 0.265

Sichling Shego 0.0947±0.1687 0.1451±0.2435 0.0954±0.0293 0.6195 0.649 0.3071 0.135

Sangla Giyu 0.1308±0.1897 0.1995±0.2686 0.1304±0.0361 0.4899 0.622 0.5207 0.152

Chhitkul Giyu 0.1263±0.1875 0.1907±0.2698 0.1248±0.0360 0.5608 0.681 0.3916 0.117

Rangrik Giyu 0.1796±0.1962 0.2745±0.2769 0.1821±0.0393 0.5791 0.703 0.3635 0.106

Shakoli Giyu 0.1396±0.1916 0.2116±0.2744 0.1392±0.0375 0.6062 0.726 0.3249 0.095

Bathardh Giyu 0.1954±0.2095 0.2915±0.2942 0.1974±0.0448 0.6436 0.737 0.2769 0.089

Tosh Giyu 0.1703±0.2100 0.2511±0.2984 0.1711±0.0465 0.7115 0.798 0.2028 0.063

Mane Koma Giyu 0.1177±0.1542 0.1910±0.2312 0.1202±0.0248 0.2587 0.330 1.4327 0.508

Sichling Giyu 0.1088±0.1706 0.1679±0.2499 0.1065±0.0285 0.4565 0.506 0.5953 0.244

Shego Giyu 0.1020±0.1682 0.1572±0.2462 0.0992±0.0271 0.4167 0.552 0.6999 0.203

Overall 0.2466+0.1686 0.3837+0.2302 0.2469+0.0298 0.7898 - 0.1331 -

V. Publications: A publication on “Characterization of novel polymorphic microsatellite

markers in Dactylorhiza hatagirea – a critically endangered orchid species from western Himalayas” was published in Conservation Genet Resour (Reprint printed below). Another manuscript on “Molecular diversity in the ecotypes of Dactylorhiza hatagirea from North Western Himalyas” is under preparation.

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Objective 3: Development of efficient in vitro protocols for rapid multiplication of quality planting material of Dactylorhiza hatagirea

a. Initiation of in vitro cultures from leaf, tuber and root cuttings: The work on in vitro culture was initiated by using leaf cuttings, tuber segments, stem cutting, root segments, epidermal layers of shoot segments, shoot tips and seeds as ex plants. Different combinations of hormones were tried for direct and indirect plantlet regeneration using different basal media. The data generated are presented in tables 9-14. Leaf, root and tuber cutting didn’t show any promise as leaf and tuber cutting exhibited browning with no response at all, whereas root segments/cuttings survived but didn’t exhibit any response to the used auxins towards callusing (Table 9 and 11). Most of the leaf and tuber cuttings turned brown and didn’t respond to any of the given treatments ultimately browning and dying after about a month of culturing (Plates 10-11).

Plate 10. In vitro cultured leaf segment of Dactylorhiza hatagirea and their response

Plate 11. In vitro culture of tuber segment of Dactylorhiza hatagirea

b. In vitro cultures of epidermal layers of shoots and shoot segments: In vitro cultures of epidermal layers of shoots and/ shoot segments cultured on various combinations of hormones i.e. NAA, 2,4-D and BA did not show much promise in our experiments except shoot tips as such which exhibited slight growth and callus initiation on terrestrial orchid medium supplemented with 1 mg/l BA (Table 12-13). Most of the epidermal segments of shoots exhibited browning against general expectations that these tissues are usually considered more amenable to dedifferentiation and cell division.

c. In vitro mature seed cultures: Mature seeds harvested from field grown plants were also used to initiate in vitro seed culture and achieve seed germination. Mature seeds

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were cultured on Terrestrial Orchid Medium in the months of the September/October, 2013-14. The seeds were kept under growth room conditions, both under light and dark but seeds didn’t germinate even after 4-5 months of cultures (Plate 12).

Plate 12. Response of in vitro seed cultures of Dactylorhiza hatagirea

d. In vitro shoot tip cultures: Shoot tips cultured on different media and hormones could be established successfully in vitro although the level of contamination by the fungus was quite high (Plates 13-14). High contamination may be due to symbiotic association of this species with fungi as reported in the literature. Cultured shoot tips exhibited response in form of formation of protocorm like bodies (PLBs) from the base of the shoot tips on Orchid multiplication medium supplemented with 1mg/l BA. PLBs further responded on the same medium leading to formation of 2-3 new shoots per shoot tip cultured and developed successfully into complete plantlets although the rate of growth was quite slow under in vitro conditions (Plates 15-16). Overall both rate of multiplication and growth achieved through shoot tip cultures was low (2-3X) to claim it a successful multiplication protocol for this species. However, this lead can be used for further refinement of this protocol to make it more efficient and economical along with further possibilities of focussing on other promising explants such as seeds.

Plate 13. Successful in vitro culture of shoot tips of Dactylorhiza hatagirea

Plate 14. Shoot tips of Dactylorhiza hatagirea established successfully at larger scale

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Plate 15. Shoot tips of Dactylorhiza hatagirea exhibiting limited multiplication in vitro

Plate 16. Protocorm like bodies(PLBs)/new shoots developing from cultured shoot tips of Dactylorhiza hatagirea under in vitro conditions

Table 9. Various combinations of PGRs and their effect on in vitro cultures of leaf cuttings of Salampanja

Treatment 2,4-D mg/l

NAA mg/l TDZ mg/l

2ip mg/l

BAP mg/l

Response

T - 0 - - - - browning T - 1 - 0.5 - - browning T - 2 - 1.0 - - browning T - 3 - 2.0 - - browning T 0.5 4 - - - - browning T 1.0 5 - - - - browning T 1.5 6 - - - - browning T - 7 1.0 - - - browning T - 8 2.0 - - - browning T - 9 3.0 - - - browning T - 10 1.0 - 2.0 - browning T - 11 0.66 - 2.0 - browning T - 12 1.0 - 3.0 - browning T - 13 1.5 - 3.0 - browning T - 14 1.0 - - 3.0 browning T - 15 1.5 - - 3.0 browning T - 16 2.5 - - 5.0 browning T - 17 1.6 - - 5.0 browning T - 18 - - 2.0 - browning T - 19 - - 3.0 - browning T - 20 - - - 3.0 browning T - 21 - - - 5.0 browning T ROM 22 - - - - browning

Shoot tips exhibiting multiplication 2-3 PLBs per shoot tip cultured

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Table 10. Effect of different concentration and combinations of growth regulators on in vitro establishment of cultures of Salampanja through tuber segments

*Basal medium used is Orchid multiplication media Table 11. In vitro establishment and response cultures of root segment of Salampanja *Basal medium used is Orchid multiplication media Table 12. In vitro establishment and response of epidermal tissues of shoots of Salampanja on different hormonal combinations *Basal medium used is Orchid multiplication media Table 13. In vitro establishment and response of shoot segments of Salampanja on different hormonal combinations *Basal medium used is terrestrial Orchid medium

Treatment BA mg/l

TDZ mg/l

2ip mg/l

NAA mg/l 2,4-D mg/l Response

DTS1* - - - - - browning DTS2 - - - 2.0 - browning DTS3 - - - - 1.5 browning DTS4 - 1.5 - - - browning DTS5 - - 1.0 - - browning DTS6 2.0 - - - - browning

Treatment NAA mg/l

2,4-D mg/l

Response

DRS1* - 1.0 Tissues sustained but no further response/growth

DRS2 - 2.0 Tissues sustained but no further response/growth

DRS3 1.0 - Tissues sustained but no further response/growth

DRS4 2.0 - Tissues sustained but no further response/growth

Treatment NAA mg/l

2,4-D mg/l

Response

DSE1* - 2.0 Browning and no further response DSE2 - 4.0 Browning and no further response DSE3 - 6.0 Browning and no further response DRS5 2.0 - Browning and no further response DRS6 4.0 - Browning and no further response

Treatment NAA mg/l

2,4-D mg/l

BA mg/l

Response

DSS1* - 2.0 - No growth and callusing DSS2 - 4.0 - Proliferation of inner rings of shoot DSS3 - 6.0 - No growth and callusing DSS5 2.0 - - No growth and callusing DSS6 4.0 - - No growth and callusing DSS7 - - 1.0 Slight growth and callus initiation

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Table 14. Effect of different concentration and combinations of growth regulators on in vitro establishment of shoot tips cultures of Salampanja

*Basal medium used is Orchid multiplication media

iii) Has the progress been according to original plan of work and towards achieving the objective, If not, state reasons: The work has been completed as per the original plan and completed successfully. Diversity in the existing populations has been established both at molecular and morphological level. Fifteen new SSR primer sets have been developed as new genomic resources for this species and the data made available in public domain through a publication. A preliminary multiplication protocol from shoot tips has been established successfully which can further be refined and used for large scale multiplication of this endangered species. iv) Please indicate the difficulties, if any, experienced in implementing the project:

Nil v) If project has not been completed, please indicate the approximate time by which it is likely to be completed. A summary of the work done for the period (Annual Basis) may please be sent to the Commission on a separate sheet : The project has been completed successfully as per the proposed work plan. vi) If the project has been completed, please enclose a summary of the findings of the study. Two bound copies of the final report of work done may also be sent to the Commission: The summary has been enclosed in the prescribed format and two bound copies of the final report also forwarded to the Commission. vii) Any other information which would help in evaluation of work done on the project. At the completion of the project, the first report should indicate the output, such as (a) Manpower trained (b) Ph. D awarded (c) Publication of results (d) other impact, if any: The major outputs of the project have been i. a publication in Conservation Genetic Resources, an international journal of

repute,

Treatment BA mg/l

TDZ mg/l

2ip mg/l

NAA mg/l

2,4-D mg/l

Response

DST1* 1.0 - - - - Protocorm like bodies (PLBs) & shoot formation

DST2 2.0 - - - - Shoot elongation DST3 4.0 - - - - Slow shoot growth DST4 - 1.0 - - - Shoot elongation DST5 - 2.0 - - - Shoot elongation DST6 - 3.0 - - - Shoot elongation

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ii. development and validation of new genomic resources for D. hatagirea in form of 15 new SSR primer sets,

iii. assessment and documentation of the level of diversity in Dactylorhiza hatagirea from north western Himalayan region at molecular level, and

iv. development of a multiplication protocol for D. hatagirea using shoot tips as ex plants.

Outputs in pipeline

iv. a manuscript on “Molecular diversity in the ecotypes of Dactylorhiza hatagirea from North Western Himalyas” is under preparation.

Signature of the Principal Signature of Co PI Investigator

(Signature of the Head of the Department)

Signature of the Director of Research, CSK HPKV Palampur, (HP) - 176 062

Documents

UNIVERSITY GRANTS COMMISSION BAHADUR SHAH ZAFAR … · KSSR-17. iv. Molecular diversity analysis: For deducing relationship among individual plants and/ or populations, each band