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Molecular Characterization and polymorphism analysis of collected

bamboo germplasms by SSR markers

Submitted To: Indian Council of Agricultural Research

ICAR Research Complex for NEH Region Arunachal Pradesh Centre, Basar, India

Submitted By:GCC BIOTECH (INDIA) PVT. LTD.

e-mail tech.support@gccbiotech.co.in website: www.gccbiotech.co.in

1 | P a g e

CONTENT

Project Aim………………….……………………………………………………..2

Materials and Methods

Plant Materials………………………………………………………………2

DNA Extraction………………………………………………………...........2

Primer Screening…………………………………………………….............2

PCR Amplifications …………………………………………………...........2

Capillary Electrophoresis ……………………………………………...........3

Results and Discussions ……………………………………………………………3

References……………………………………………………………………….....4

Annexures

Appendix-I

Appendix-II

III-Appendix

VI-Appendix

2 | P a g e

Molecular Characterization and polymorphism analysis of collected

Bamboo germplasms by SSR markers

Project Aim

DNA isolation, purification and quality check of bamboo genotypes (leaf samples)

PCR standardization and agarose gel analysis

o PCR of 50 (50 X 38) data points (PCR reactions) and analyzed by Agarose gel electrophoresis

o Approximately 30 highly polymorphic markers selection among the pool of 50 markers

Signature allele identification and sequencing on 3730XL genetic analyzer

o Sequencing of unique bands which can serve as signature for unique germplasm

Bioinformatics analysis

Materials and Methods

Plant Materials

A group of 40 genotypes belonging to different bamboo species for SSR genotyping analysis was

received. The leaves of these genotypes were subjected to total DNA isolation.

DNA Extraction

Total DNA was extracted from young leaves according to the manufacture’s instruction of

GSure Plant Mini Kit with WLN Buffer (Cat. No.: G4620W, GCC Biotech, India). DNA was

dissolved in nuclease free water. DNA samples with absorbance ratios above 1.8 were used for

the analysis in this experiment and stored at −20°C until needed.

Primer Screening

Primers were selected from EST-SSRs (please see Reference) developed by different school of

thoughts. Among those 78 polymorphic markers were used to amplify DNA to evaluate the size

and the band pattern in agarose gel (2.5%). Multi-locus markers with fragment size between 70

bp to 1500 bp were analyzed.

PCR Amplifications

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For capillary electrophoresis, PCR reaction included the two specific primers (0.3 µM) plus a

labeled M13F primer (CAC GAC GTT GTA AAA CGA C, 0.13 µM), approximately 30 ng of

template DNA, 0.2 mM dNTPs, 1× PCR buffer, 2 mM magnesium chloride, and 1 U of G9 Taq

DNA polymerase (G7115, GCC Biotech, India). PCR was performed at 95°C for 5 min;

followed by 35cycles of 95°C for 30 sec, 55-58°C for 30 sec, and 72°C for 45 sec; and one final

cycle of 72°C for 10 min, on GeneAmp 9700 and Veriti Thermal Cycler(Applied Biosystems,

USA).

Capillary Electrophoresis

An aliquot of 0.5–2 µl of PCR product (depending on the performance of amplification of each

primer pair) was mixed with 10 µl of formamide and denatured at 95°C for 5 min. Sequencing

was performed on 3730XL Genetic Analyzer (Applied Biosystems, USA).

Result and Discussion

The following report shows a distinguishing polymorphism representation among the different

bamboo genotypes through SSR markers. Approximately 30 polymorphic markers are selected

after this extensive analysis and further confirm the desired bioinformatics analysis for

biodiversity study.

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References

Sixteen novel microsatellite markers developed for Dendrocalamus sinicus (Poaceae), the

strongest woody bamboo in the world.

Dong YR, Zhang ZR, Yang HQ.

Am J Bot. 2012 Sep;99(9):e347-9. doi: 10.3732/ajb.1200029. Epub 2012 Aug 29.

Identification, cross-taxon transferability and application of full-length cDNA SSR markers in

Phyllostachys pubescens.

Lin Y, Lu JJ, Wu MD, Zhou MB, Fang W, Ide Y, Tang DQ.

Springerplus. 2014 Aug 29;3:486. doi: 10.1186/2193-1801-3-486. eCollection 2014.

Development of polymorphic microsatellite markers for Phyllostachys edulis (Poaceae), an

important bamboo species in China.

Jiang WX, Zhang WJ, Ding YL.

Appl Plant Sci. 2013 Jun 21;1(7). pii: apps.1200012. doi: 10.3732/apps.1200012. eCollection

2013.

Assessment of Genetic Diversity of Kau ayan tin ik (Bambusa blumeana Schultes f.) from Five

Plantations in the Philippines through Transferability of SSR Markers.

Maria Theresa A. Delos Reyes, Ph.D., Jen Daine L. Nocum, Joshua Israel V. Sumague and Aida

C. Baja-Lapis, Ph. D. 1 0th World Bamboo Congress, Korea 2015

___________ END of DOCUMENT _____________

Sample Context (Lane-1 to Lane-25 in agarose gel analysis)

1. 100bp ladder 13. 50bp Ladder 2. 33 14. 33 3. 30 15. 30 4. 27 16. 27 5. 22 17. 22 6. 17 18. 17 7. 11 19. 11 8. 6 20. 6 9. 1 21. 1 10. 40 22. 40 11. 36 23. 36 12. NTC 24. NTC

25. 100bp Ladder

Appendix-I

A

Gba18a Gba18b

Gba20 Gba25

Gba58 Gba202

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

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

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

I

Gba10 Gba14

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

Phe-01 Phe-34

Phe-51 Phe-185

1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25

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

II

PBM-031 PBM-039

PBM-052 PBM-088

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

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

PBM-035 PBM-043

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

III

PBM-051 PBM-060

PBM-071 PBM-081

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

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

PBM-094 Phe-10

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

IV

Phe-28 Phe-35

Phe-44 Phe-98

Phe-139 Phe-163

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

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

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

Phe-167 RM-10

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

V

RM-154 RM-125

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

RM-118 RM-162

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

PBM-032 PBM-033

PBM-034 PBM-036

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

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

VI

PBM-032 PBM-033

PBM-034 PBM-036

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

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

PBM-046

Den-006 Den-020

1 2 3 4 5 6 7 8 9 10 11 12 13

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

VII

PBM-037 PBM-038

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

PBM-040 PBM-041

PBM-042

PBM-044 PBM-045

1 2 3 4 5 6 7 8 9 10 11 12 13

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

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

Appendix-II

Capillary Sequencing Report

Score E

Sequences producing significant alignments: (bits) Value

AJ507487|AJ507487.1 Bambusa bambos microsatellite DNA, clone Ba14. 100 2e-18

AJ507488|AJ507488.1 Bambusa bambos microsatellite DNA, clone Ba18. 84 1e-13

AP015040|AP015040.1 Vigna angularis var. angularis DNA, chromoso... 38 7.1

AP015037|AP015037.1 Vigna angularis var. angularis DNA, chromoso... 38 7.1

AP015035|AP015035.1 Vigna angularis var. angularis DNA, chromoso... 38 7.1

LT594792|LT594792.1 Theobroma cacao genome assembly, chromosome: V. 38 7.1

JX586664|JX586664.1 Gossypium hirsutum clone NBRI_GE20537 micros... 38 7.1

>AJ507488|AJ507488.1 Bambusa bambos microsatellite DNA, clone Ba18.

Length = 546

Score = 119 bits (60), Expect = 2e-24

Identities = 66/68 (97%)

Strand = Plus / Minus

Query: 80 gccagacccgagagaattctaccgggtagagcctatataaagaattgagcagtgaaacga 139

|||||||||||| |||||||||||||||||||||||||||||| ||||||||||||||||

Sbjct: 417 gccagacccgagtgaattctaccgggtagagcctatataaagagttgagcagtgaaacga 358

Query: 140 cctggctt 147

||||||||

Sbjct: 357 cctggctt 350

Score = 75.8 bits (38), Expect = 3e-11

Identities = 54/58 (93%), Gaps = 1/58 (1%)

Strand = Plus / Minus

Bamboo-SSR

50bp 33 30 27 22 17 11 6 1 40 36 NTC 100bp

Query: 10 ggttaggtaggccgtgctgcga-tacctaacaacggtagagttcactcgaagcggacg 66

||||||| ||||||||||||| | |||||||||||||||||||||||||||||||||

Sbjct: 526 ggttaggcaggccgtgctgcgggtgcctaacaacggtagagttcactcgaagcggacg 469

>AJ507487|AJ507487.1 Bambusa bambos microsatellite DNA, clone Ba14.

Length = 519

Score = 115 bits (58), Expect = 4e-23

Identities = 67/70 (95%)

Strand = Plus / Minus

Query: 85 acccgagagaattctaccgggtagagcctatataaagaattgagcagtgaaacgacctgg 144

||||||| |||||||||||||||||||||||||||||| ||||||| |||||||||||||

Sbjct: 320 acccgagtgaattctaccgggtagagcctatataaagagttgagcaatgaaacgacctgg 261

Query: 145 cttccttaac 154

||||||||||

Sbjct: 260 cttccttaac 251

Score = 63.9 bits (32), Expect = 1e-07

Identities = 48/52 (92%), Gaps = 1/52 (1%)

Strand = Plus / Minus

Query: 10 ggttaggtaggccgtgctgcg-atacctaacaacggtagagttcactcgaag 60

||||||| |||||||||| || || |||||||||||||||||||||||||||

Sbjct: 418 ggttaggcaggccgtgctacggatgcctaacaacggtagagttcactcgaag 367

Score E

Sequences producing significant alignments: (bits) Value

AJ507488|AJ507488.1 Bambusa bambos microsatellite DNA, clone Ba18. 119 2e-24

AJ507487|AJ507487.1 Bambusa bambos microsatellite DNA, clone Ba14. 115 4e-23

AP014957|AP014957.1 Oryza sativa Japonica Group DNA, chromosome ... 44 0.11

AP003342|AP003342.3 Oryza sativa Japonica Group genomic DNA, chr... 44 0.11

JX409694|JX409694.1 Dendrocalamus hamiltonii microsatellite DHGM... 44 0.11

FN595754|FN595754.1 Vitis vinifera cv. PN40024, annotated scaffo... 38 6.6

>AJ507488|AJ507488.1 Bambusa bambos microsatellite DNA, clone Ba18.

Length = 546

Score = 119 bits (60), Expect = 2e-24

Identities = 66/68 (97%)

Strand = Plus / Minus

Query: 80 gccagacccgagagaattctaccgggtagagcctatataaagaattgagcagtgaaacga 139

|||||||||||| |||||||||||||||||||||||||||||| ||||||||||||||||

Sbjct: 417 gccagacccgagtgaattctaccgggtagagcctatataaagagttgagcagtgaaacga 358

Query: 140 cctggctt 147

||||||||

Sbjct: 357 cctggctt 350

Score = 75.8 bits (38), Expect = 3e-11

Identities = 54/58 (93%), Gaps = 1/58 (1%)

Strand = Plus / Minus

Query: 10 ggttaggtaggccgtgctgcga-tacctaacaacggtagagttcactcgaagcggacg 66

||||||| ||||||||||||| | |||||||||||||||||||||||||||||||||

Sbjct: 526 ggttaggcaggccgtgctgcgggtgcctaacaacggtagagttcactcgaagcggacg 469

>AJ507487|AJ507487.1 Bambusa bambos microsatellite DNA, clone Ba14.

Length = 519

Score = 115 bits (58), Expect = 4e-23

Identities = 67/70 (95%)

Strand = Plus / Minus

Query: 85 acccgagagaattctaccgggtagagcctatataaagaattgagcagtgaaacgacctgg 144

||||||| |||||||||||||||||||||||||||||| ||||||| |||||||||||||

Sbjct: 320 acccgagtgaattctaccgggtagagcctatataaagagttgagcaatgaaacgacctgg 261

Query: 145 cttccttaac 154

||||||||||

Sbjct: 260 cttccttaac 251

Score = 63.9 bits (32), Expect = 1e-07

Identities = 48/52 (92%), Gaps = 1/52 (1%)

Strand = Plus / Minus

Query: 10 ggttaggtaggccgtgctgcg-atacctaacaacggtagagttcactcgaag 60

||||||| |||||||||| || || |||||||||||||||||||||||||||

Sbjct: 418 ggttaggcaggccgtgctacggatgcctaacaacggtagagttcactcgaag 367

Bamboo_SSR

Gba-14

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

Gba-18b

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

Gba-25

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

Gba-58

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 100bp

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

PBM-039

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

PBM-040

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

PBM-041

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

PBM-048

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

PBM-049

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

PBM-053

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

100bp 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

100bp 35 36 37 38 39 40

1-DEN020-F

TTGTATTTGCAGAGCACCTTATGCTAGAGTCCATTTCTGAGTTACAGAAAAAGGTAACCAAGCTGCAACCTTGACT

GCAGTAACAAATAACGTGGTGTGAGTGTGTCTATAGGCACTTGCACCGTACCATATCTAAACTAAATCATATTACC

AAACTGCATTCAGGAGAAACATGCATGCATGC

1-PBM-088-F

GGCAGGCACCCTTCTCCTTCCCCAAAACCCTTTTCCTCTTTCTCTCCCCCCAAGAACTTCACCTCAAACTCAAACCGT

CTCCTGCTGC

2-DEN-020-R

CTTAAGATATAGTAGTTCAAGTGACCATACATACACAACAGTGACAGTTGCAGGGTAGGTTGCAGCTTGGTTACCT

TTTTCTGTAGCTCAGAAATGGACTCCAGCAGAAGGTGGCTCTGCAAATACAAGTATGTTACACAGTTAGTCATCAA

TTAGAAATATGCCCCAGAAGACAAAGGCGACCC

5-Phe-28-F

GATTAGATCTCTCTTTCTCTTAGTTTCGTGTGTGTTCTTTGAATCTTTTTTCCTCCCTCCCAAAGAAAGCTTTTCCTGTT

CTTCAACAACCAAGTGTGCCCAAGGACCCGATGCGCTCTGTAATGAAAAATTGTGCAGCTGAGATTCCTAGCTATA

CTTAGTTACTGACAACCCGGTTACTTACTGAATGGAAATTTGCACATTATTACTCCTGTTGCCTTTTTAAACACAAAA

AATGAGAAAAATACTCTCCTAACTTCTGAATCCGCATCTCTCACCATGTCAGATTCGGTTCTCCATGCTATGTGCTCA

TCGGGCCTAACCAAAATTACAGTTTTCCTAGTTATCTGACACATCTCCCACCATGAAGTAGCAGATGCCCTAGGTAT

TTCCTCAACATCGGCATAGTTT

5-Phe-167-F

AAACTAAATAGACAGTCATCTCACTGATAAGGAGCTCAACAAATCAGGTTTTGACTAAATTGAGACACCATGACTG

ATAACTAACAAACTGACAGATATAACAACTCTCCGGGGATGTTCCTTCAACCGAGTCAGATAACAATTCTCAGGCC

TTATCCTTCTGCCTCTCCCATGTCCGCTTAACCTAGTCAAGTCGACCCTCGCTCGTTGCCATGGAGAGGAACTTGGT

GCGGAGCTTTGACTTCATGAACAACTGTGAGGCGTAGTAGTACTCGTTGCTTCCTTTGGTAGCTCCTGTGTCGTTCA

CCAACTTCATGAGTCTCATGAACTCTTGGCTC

6-DEN-86-F

CCCTGTTGTTCCTCACAATTGTGTGTGTGTGTGTAGATGTTGATGAAAATAGAAG

6-Gba-58-R

GTGGGTTCTGTCGTTCCAGGATTCTATTGAATGTTTGTCCGTGTGTGTGTGCATGTTTATGCCTTGCAGGAGCACG

AATCGTTCGGGAGGAGGAGTGGGATGCCTTCATGAGTGTGCTTC

6-PBM-84-F

TCTTCATTTCTGATCTCTAACAATTATCCATCATAACTATTTTTAAATCCACCGAAATCATTCGTATCAGGACTTTCCA

ACAATTCAATCCTTTCTGTTATTGATCCAAAACCATGGTTTGCTCTTCCAATAATCAACCTTATTTTCCATTATCTATC

TATAACTAATTCACTATATACTTCCACCAAAAATATAATTGACCAAGATCGCAGAAAGTG

11-Phe-163-F

TCTTTACATGCAACAACACATCCTTAAAATCAGCATCAAAAGCATATTGTCCTTTAATAGTTTCAAGACCAAAAATG

CGACAATCAAGTTGAGACAACATGGTATAGCGTCGAGACAAAAACAGAGGGTGG

13-PBM-39-F

ATTCCCAGCTCGCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTGTGTCTACCTGAGCTTCTTCTTCTTC

13-PBM-84-R

AGAGATCAGAAGTGAAGAGTTGATGAATAGCAACCACAAGATTAGATGGTTAGAAATTTAGAACACAGAAAATAT

TGGTGGATCTAAATTAAAGGATACTGAATTTCCATTAGACTAAGGAACTGATCAACTTGGTAAAATAATGAACTGA

GATTGGATCTATGAATTGGTGGTAAATATTTGAATTCATATGAGAGTAGCTGACCCCATGAGAAG

18-PBM-48-R

GGTGAGGAGACATGGAGTCAAGTGCGCTCTTTTTCAAACGAGTTGTGTCACCGTCACCAGTCAAGTGCGATCTTTT

GCATCTAAACAGGAGGTCTAGAGCCTCCTCTAGGGGGCATCATCACGATCCTACTCTTTATCCTTTCTTCTTATCCTC

ATCTTCCGAATCGTCAAGTGCGCTCTT

22-PBM-55-F

CTCCACCATCCATGTGTACACTAGGTACATGCATGCAGCTTCGTACGTAGTGGTTCATGTGAGAGATCGAGGAGTT

GAGCCACCTCGAGTACTAGTCTTTGTGCA

28-PBM-40-F

ACATGCTATTTATCTCTAGATCTCTCTCTCTCTCTGTCTCTCCTTTTGTTACTTCTCTCTCTCTCTCTCTCTCTCCTCACT

ACTAAGCCAAATGCAATGCAA

9-DEN-20-F

AAGCTGCAACCTTGACTGCAGTAACGAATAACGTGGTGTGTGTGTCTATAGGCACTTGCACTG

9-Phe-163-R

CAATATGCTTTTGATGCTGATTTTAAGGATGTGTTGTTGCATTGTAAAGAGGGACAAGCATGGAATAAATTCATGA

TCAATGATG

21-PBM-88-R

CCTCCAAGCTATATCATCATTAGAGACGAGAAGTGGAGGACGAGACGGAGAGAGGAAAAGCTG

21-PBM-90-F

GTAAAACACTCCACCACAACAGGTTGCAATGTGTAGTATAGACTCGAAGCGTATGAAATCGAG

27-GBA-14-F

ATACCTAACCCAACTACTCAACCTCCTGACTTCTCAACACCTGTAGAGTTTCAATCGAGTT

Bioinformatics Data analysis

The allele frequency module reads a text-based file of genotypes at one or more loci, counts the number

of occurrences of each allele at each locus, and using these data calculates various statistics about each locus.

These include expected heterozygosity, polymorphic information content (PIC). The Bandsize-Binary program was

used to calculate the genetic parameters, including allele number (A), heterozygosities (H). All the genotypes were

scored for the presence and absence of the SSR bands. And the data were entered into a binary matrix as discrete

variables, 1 for presence and 0 for absence of the character and this data matrix was subjected to further analysis.

The Excel file containing the binary data was imported into NT Edit of NTSYS-pc 2.02J. The 0/1 matrix was used to

calculate Similarity as Jaccard’s coefficient using SIMQUAL subroutine in SIMILARITY routine. The resultant

similarity matrix was employed to construct dendrograms using Sequential Agglomerative Hierarchical Nesting

(SAHN) based Unweighted Pair Group Method with Arithmetic Means (UPGMA) to infer genetic relationships and

phylogeny. In addition, Principal Component Analysis (PCA) based clustering was also done using the subroutine

EIGEN.

S. No. Primer Allele No (A) Min Allel Size Max Allele Size Heterozygosity (H)

1 Den006 7 219 643 0.8

2 Den020 3 172 240 0.58

3 Gba14 2 209 245 0.5

4 Gba18b 3 103 140 0.67

5 Gba25 9 104 800 0.8

6 Gba58 4 168 205 0.67

7 PBM-039 3 141 177 0.65

8 PBM-040 4 148 172 0.72

9 PBM-041 5 146 194 0.72

10 PBM-064 3 147 168 0.66

11 PBM-071 2 97 135 0.47

12 PBM-081 4 106 134 0.7

13 Phe-28 2 223 230 0.32

14 Phe-35 4 108 569 0.65

15 Phe-51 2 130 143 0.49

16 PBM-048 3 81 145 0.45

17 PBM-053 5 76 162 0.76

18 PBM-055 5 146 213 0.56

19 PBM-068 9 120 850 0.75

20 PBM-075 2 110 490 0.5

21 PBM-084 4 190 310 0.6

22 PBM-090 4 120 160 0.67

23 PBM-098 5 129 1552 0.53

25 PBM-056 3 121 361 0.54

26 DEN-036 5 291 410 0.63

27 DEN-086 4 99 140 0.62

28 PBM-049 3 199 351 0.43

29 PBM-061 5 179 588 0.72

30 Phe-167 6 197 696 0.75

31 PBM-088 3 191 353 0.59

Phylogenetic Tree For Bamboo

Phylogetic tree for bamboo genotypes

Dice Coefficient

0.47 0.60 0.73 0.85 0.98

1

27

34

35

2

21

22 3

4

7

26

12

37

9

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10

13

17

18

28

16

6

8 36

38

19

25

24

32

14

20

15

30

33

5

23

39

29 31

40

3D Plot For Eigen Vector

3D Plot For Eigen Vector- Bamboo

40

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2322

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1918

1716

15 14

13

1211

10

9

8

7

65

43

2

1

0.610.61

0.400.40

0.180.18

Dim-2Dim-2

-0.03-0.03

-0.24-0.24-0.34-0.34

0.560.56

0.630.63

0.710.71

Dim-1Dim-1 0.790.79

0.860.86

-0.17-0.17

Dim-3Dim-3

-0.01-0.01

0.160.16

0.320.32

2D Plot For Eigen Vector- Bamboo

Dim-1

0.56 0.63 0.71 0.79 0.86

Dim-2

-0.24

-0.03

0.18

0.40

0.61

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