Chapter-4

Results

Results

----------------------------------------------------------- 4.1 Isolation and characterization of bamboo specific microsatellite markers

Microsatellites or simple sequence repeats (SSRs) are tandem repeats of 1–6

nucleotides which frequently show variation in the number of repeats at a locus. The

ubiquity of these markers in eukaryotic genomes and their usefulness as genetic markers

has been well established over the last decade. Microsatellites are mainly characterized by

high frequency, co-dominance, multi-allelic nature, reproducibility and ease of detection

by polymerase chain reaction with unique primer pairs that flank the repeat motif (Gupta

and Varshney 2000). Amenability of these markers for automation and high-throughput

genotyping has been very well explored in number of crop and forest plants (Parida et al.

2006; Zhang et al. 2003). Due to all these desirable characteristics microsatellites have

become the most suitable markers for various molecular genetic studies and routinely

utilized for genetic diversity, population genetic structure, establishing phylogenetic

relationships, construction of high-density linkage maps, gene mapping, comparative

mapping and marker-assisted selection in larger number of plants species (Bruford and

Wayne 1993; Wang et al. 2012; Tsukazaki et al. 2010; Wu and Tankseley 1993; Gonzalo

et al. 2005; Baldwin et al. 2008).

In general, SSRs are identified from either genomic DNA or cDNA sequences. The

standard method for development of SSR markers involves the creation of small insert

genomic DNA libraries, followed by a subsequent DNA hybridization selection by probing

them either with radioactively labeled probes or trapping them with biotinylated SSR

motifs, and clone sequencing (Paneigo et al. 2002; Lowe et al. 2004). These processes are

time consuming and labour intensive. Availability and continuous enrichment of expressed

sequence tags (ESTs) database at http://www.ncbi.nlm.nih.gov in most of the crop species

can serve as an alternative strategy for identification and development of microsatellite

markers. SSRs can be directly sourced from such databases, thereby reducing time and cost

for microsatellite development. However, non-availability of sufficient sequence

information and other genomic resources and redundancy that yield multiple set of markers

at the same locus are among the major drawbacks of EST derived microsatellite markers.

However, more recently unique gene sequences (unigenes) have been developed via

clustering of overlapping EST sequences, which overcome the problem of redundancy in

EST database and detect variation in the functional genome with unique identity and

45

position (Varshney et al. 2005). Parida et al. (2006) identified and characterized

microsatellite motifs in the unigenes available in five cereal crops (rice, wheat, maize,

sorghum, barley) and arabidopsis. These unigene derived microsatellite (UGMS) markers

are expected to possess high inter specific transferability as they belong to relatively

conserved regions of the genome. Such a data base and genomic information were either

limited or not available in bamboo at the time this dissertation proposed. However, during

this research work small public ESTs database were created. Therefore, both standard

(identification of microsatellite through nucleotide sequencing of positive clones derived

from bamboo enriched specific genomic libraries) and mining of SSRs from public EST

datasets of different bamboo species, approaches were utilized in the current study.

4.1.1 SSRs identification from expressed sequence data sets

Expressed sequence data available in public domain for bamboo was mined for

identification of microsatellite markers. In total, 329 public ESTs of B. oldhamii and

Phyllostachys edulis were mined. These ESTs were subsequently clustered into 55 unique

clusters (contigs/ unigenes) using SeqMan DNA Star lasergene version 7.1. Non-redundant

EST sequences were screened for identification of SSRs containing sequences using

repeatmasker software (http://www.repeatmasker.org/) with search criteria to mask ≥ 20bp

Table: 4.1: Characteristics of EST-SSR markers of B.oldhamii, cross-amplified in

Different bamboo species

46

SSRs. A total of 16 such SSR containing contigs were identified and 13 were utilized

successfully for primer designing using Generunner 3.05 version software.

Each primer pair was used to amplify DNA from accessions representing 23 different

species of Bambusoideae (Table 3.1). Of the 13 newly identified EST-SSRs primers, 10

(76.9 %) could produce repeatable amplifications in most of the tested species of bamboo.

All the EST-SSR markers found to be moderately to highly polymorphic (Table 4.1). The

number of alleles amplified ranged from 2-10 alleles per locus. The average polymorphism

information content (PIC) value calculated was 0.273. The marker wise transferability rate

varied from 60 % to 100 % in 23 bamboo species (Table 2). Three (30 %) locus namely

Boes-3, Boes-4, Boes-7 found to be conserved in Bambusoideae. The validated EST-SSRs

with their properties are shown in Table 4.1.

4.1.2 Identification of Microsatellites from enriched genomic library

Amongst the various molecular markers available, simple sequence repeats are the

most efficient and reliable due to their high informativeness, co-dominant expression and

multiallelic nature. However their occurrence in plants is at much lesser frequency than

mammals (Wang et al. 1994). To overcome this hurdle, the microsatellite enrichment

protocols have been applied with various modifications (Edwards et al. 1996; Fischer and

Bachmann 1998; Hamilton et al. 1999). There has been a noteworthy (40-60%) increase in

the efficiency of enriched libraries over conventional libraries and has resulted in a great

progress towards plant genetic studies (Gupta and Varshney 2000; Zane et al. 2002

4.1.2.1 Microsatellites Enriched library construction

In the current investigation a modified enrichment protocol using streptavidin

magnetic beads based on separation of biotinylated DNA containing SSR motifs was used

(Kijas et al. 1994). Three biotinylated probes namely (GA)n, (GT)n and (CAA)n repeats

were used to construct small insert genomic libraries from the nuclear DNA isolated from

bamboo accession of D. hamiltonii. In total, 1152 putative positive clones (selected in blue/

white selection and PCR secondary enrichment) were selected for nucleotide sequencing.

Secondary enrichment through PCR helped in not only confirming the presence of repeated

region but also helped in determination of size and position of microsatellite hence reduced

the cost and time in selecting the positive clones for sequencing.

47

4.1.2.2 Characterization of Microsatellites

A total of 772 high quality and non-redundant sequences were identified from

nucleotide sequencing of 1152 positive clones. Contings assembly was done using SeqMan

module of DNA Star lasergene version 7.1 generated Non-redundant (NR) sequence data

set which represented ~ 0.302 Mb genome of bamboo species namely D. hamiltonii. Three

hundred fourteen of these were found to be containing one or more targeted and non-

targeted microsatellite motifs, hence suggested 40.6% enrichment. A significant

predominance of perfect motifs 280 (89.17%) was observed in the NR dataset, while only

34 (10.82%) were compound. Details of the clones selected, nucleotide sequencing, and

microsatellite identified along with their class types has been depicted in Table 4.2.

Table 4.2: Summarization of the microsatellite enrichment information in the present

study

* SSRs with repeat length ≥ 20 nucleotides; nts** SSRs with repeat length >10 nts to < 20 n

Among the perfect repeats, sequences containing di-repeats (180; 64.28%) were

prominent followed by 97 (34.64 %) tri-repeats. However, a few sequences containing

tetra- (3; 1.07%) were also detected. Among the di-nucleotide repeats the (GA)n motifs

were most abundant (67.2%) followed by (CT)n. Among the microsatellites containing tri-

Type of Sequences No.

Total colonies picked and cultured 1536

Clones sequenced 1152

Sequences rejected after sequencing due to poor quality 380

Sequences processed for SSR identification 772

SSR containing sequences 314

Perfect 280

Compound 34

Perfect Di-repeats 180

Perfect Tri-repeats 97

Perfect Tetra-repeats 3

Repeat containing sequences with flanking region 123

Class I* 80

Class II** 43

Primer synthesized 90

Primers Validated 43

Polymorphic 38

Monomorphic 05

48

repeats, (GTT)n (35.7 %) and (CAA)n (30 %) were the maximum. However, three tetra

repeats were interestingly containing three different repeat motifs. The different types of

repeats and class captured through enrichment process are given in Table 4.3.

* SSRs with repeat length ≥ 20 nucleotides; nts

** SSRs with repeat length >10 nts to < 20 nts

SSR details No. of

primers

designed

Successful

primers

Repeat Type No. Repeat Motif No. of SSRs

identified

Class-

I*

Class-

II**

Di-repeat

(Perfect)

121 (GA)n 37 28 7 32 7

37 (CT)n 15 9 6 12 6

7 (CA)n 5 4 1 03 2

14 (GT)n 7 4 3 05 2

1 (TA)n 1 1 - - -

Di-repeat

(Compound)

22 (GA)n(GT)n 2 2 - 1 1

14 (CA)n(GA)n 2 1 1 1 1

1 (CA)n(CT)n 1 - 1 1 1

7 (GT)n(GA)n 2 2 - 1 1

2 (CT)n(GT)n 1 - 1 1 1

9 (GA)n(CA)n 3 1 2 1 1

1 (AT)n(GT)n 1 1 - 1 1

1 (TA)n(GT)n 1 1 - 1 1

1 (TG)n(TA)n 1 - 1 1 1

3 (GA)n(GC)n(GA)n 2 1 1 2 2

1 (GA)n(CT)n(CA)n 1 1 - 1 1

6 (CT)n TT (CT)n 3 3 - 1 1

5 (GA)n CC (GA)n 1 - 1 1 1

Tri-repeat

(Perfect)

13 (CAA)n 7 3 4 5 3

15 (GTT)n 9 9 2 8 -

4 (CAT)n 1 - 1 1 1

1 (CAG)n 1 1 - - -

5 (GAT)n 2 1 1 1 1

1 (GCT)n 3 1 2 2 2

1 (CTG)n 1 - 1 1 1

1 (CGG)n 1 - 1 1 1

1 (GAA)n 1 1 - - -

Tri-repeat

(Compound)

1 (CAA)n(CA)n 1 1 - - -

1 (GTT)n(GGT)n 1 1 - - -

3 (CAG)n (CTG)n 2 1 1 - -

2 (GCT)n...(GCT)n 1 - 1 1 1

7 (GTT)n(GGT)n 1 - 1 1 1

1 (CAA)n(GAA)n 1 1 - 1 1

1 (GAT)n..(GAT)n 1 1 - - -

Tetra-repeat

(Perfect)

1 (CTTT)n 1 - 1 - -

1 (GTTT)n 1 - 1 1 -

1 (GGCT)n 1 - 1 1 -

Total 314 123 80 43 90 43

Table 4.3: Types and frequency of repeat motifs in genomic libraries enrichment

with (GA)n, (GT)n and (CAA)n probes

newly identified SSR markers was checked

49

4.1.2.3 Primer design and amplification validation

Of the 314, 123 (39.2 %) microsatellite containing sequences were having

sufficient flanking regions for primer designing. Primers could not be designed for the rest

191 SSR containing sequences because of either insufficient flanking sequence (occurrence

of SSR near or/at either end of the contings) or inability to fulfil the criteria for primer

design. Ninety primer pairs flanking to microsatellite repeat containing sequences could be

designed and validated in 32 accessions of D.

Hamiltonii (DH) species. Of these, 43 (47.7 %)

primer pairs produced repeatable and reliable

amplifications, while 47 (52.2 %) primer pairs

either completely failed or led to weak

amplifications and thus were excluded from

further analysis. Among the validated primer

pairs, 21 and 22 were of class I and class II,

respectively. Microsatellites frequency and

distribution of different repeat motifs of both

types of SSR markers are represented

graphically in Figure 4.1.

4.1.2.4 Polymorphic potential of DHGMS markers

Across the 32 DH accessions used in the study, the microsatellite loci resulted in 243

fragments with an average of 5.65 alleles per locus. The minimum numbers of observed

fragments were 3 corresponding to four primers (DHGMS43, DHGMS49, DHGMS66 and

DHGMS70) and maximum 13 corresponding to DHGMS45. The Polymorphic

information content (PIC) ranged from 0.2297 (DHGMS82) to 0.5 for two primers

(DHGMS70; DHGMS85) with an average PIC values of 0.4071 (Table 4.5.). A

representative profile revealed by primer pair DHGMS45 is shown in Figure 4.2.

Figure 4.1: Frequency of repeat and

Class types (Class I and Class

II) of validated SSRs markers

50

4.1.2.5 Genetic variation and cluster analysis

Novel 43 DHGMS markers identified in the present study were utilized for

evaluation of genetic variation in D. hamiltonii accessions collected from different sites,

broadly representing two different regions namely Kangra and Mandi of Himachal

Pradesh, India. Fifteen genotypes were collected from each region of Himachal Pradesh

and two accessions of Mizoram (a north eastern state), India were included for comparison

purpose. Overall genetic diversity (GD)

obtained in these thirty two accessions was

32 %, while Kangra populations revealed

little higher GD (30 %) than populations

collected from Mandi, wherein recorded GD

was 27 %. Further, Analysis of Molecular

Variance (AMOVA) showed that only 16%

variation resides among populations while

84% variation was found within population,

which indicated that high level gene flow

between populations prevailing in Himachal

Pradesh, India (Table 4.4; Figure 4.3).

Source Df SS MS Est. Variance % variance

Among Populations 2 80.750 40.375 2.890 16%

Within Populations 29 424.500 14.638 14.638 84%

Total 31 505.250 17.528 100%

Df: Degree of freedom, SS: sum of squares, MS: mean square

Table 4.4: Summary of Analysis of molecular variance (AMOVA)

Figure 4.3: Graphical representation of

partitioned genetic variation

within and among populations

Figure 4.2: Amplification profile generated with primer DHGMS-45 in 32

accessions D. hamiltonii. Lanes 1-32 represent accessions of DH

presented in Table 2.2; M: 50 bp DNA ladder (MBI fermentas) as

size standards

51

S.

No. Primer Name Sequence(5'-3') Repeat Motif Ta Na

Size

Range

(bp)

Unique

Loci Size PIC

Gene Bank

Accession

No.

1. DHGMS-2 F-AGAGAGAGGTGAGATGGG

R-CCATGATCGTATAATGAAAC

(GA)6GC

(GA)2 47°C 7 320-380 1 330 0.3545 JX409669

2. DHGMS-8 F-TGTACAGATACATGATGGGG

R-GCGGGAATTCGATTAGA (CT)6 45°C 6 250-350 1 350 0.3246 JX409670

3. DHGMS-09 F-CAGCACCCTCATTGTTGTTG

R-CCCCCGCGAATTTGTTTAT (GA)13 50°C 1 120 0 - 0 JX409671

4. DHGMS-12 F-TACTGTCAATCAGGCCTTCG

R-AGAGAGAGAGAGAGAGAGGTATACAGA (TG)4(TA)2 53°C 1 150 0 - 0 JX409672

5. DHGMS-13 F-AGATCCCAGATGTTGTAGG

R-CGAGAAGAAGAGAGAGAGAG (CT)30 50°C 1 103 0 - 0 JX409673

6. DHGMS-17 F-ATATTTTAAACGCGGCCTGA

R-GGCGGCTAGCTAAATATTCG (GA)16 49°C 10 210-250 3

240, 230,

210 0.2498 JX409674

7. DHGMS-19 F-GAGCCCGTACCTCTCCTCTT

R-CCGAAATACCTTGAGGATCG (CT)2TT(CT)7 53°C 6 120-190 - - 0.4793 JX409675

8. DHGMS-31 F-CCTCGGATGTAAGGGCATAA

R-GTGGAAATGGCACTGTTGTG (CA)4 (CT)5 53°C 9 150-210 2 208, 200 0.439 JX409676

9. DHGMS-32 F-GCAGAGAGATAGAGAGAGAAAGG

R-TAGACCGTGTGCGACTGAC (GA)8 54°C 4 130-170 - - 0.4986 JX409677

10. DHGMS-33 F-CTGCTGCTGCTGGCAATA

R-CCGTGGGTCCTCTTACAATG

(GCT)3..

(GCT)3 51°C 4 175-190 - - 0.4382 JX409678

11. DHGMS-37 F-ACAAGAAGCCGCAGTTGTTT

R-GGGCCCACTAACCCTACAGT (CT)8 51°C 4 190-230 - - 0.4245

JX409679

12. DHGMS-41 F-GCGGCATTACTGGTTGTTAG

R-CATGGCCCTCCTCATAGAAA (CAA)4 53°C 8 160-240 - - 0.4034 JX409680

13. DHGMS-42 F-ACCCCAATAAAGCCTCAGGT

R-GAAAATCGCGTGACTTGTGA (CA)5(GA)13 51°C 9 140-410 1 140 0.3977 JX409681

14. DHGMS-43 F-CGTGATCGTCTCCACACCTA

R-ACTCGTACTAGCGGGCGTAA (CA)12 55°C 3 330-350 - - 0.4632 JX409682

15. DHGMS-44 F-GTGCTCCTTCATGGTGTGAA

R-CAAAACAGCAGCCACCATAG (GCT)5 53°C 5 240-320 - - 0.42 JX409683

16. DHGMS-45 F-ATTTCGTGCGTTGGTACTCC

R-CCTGTGAACACTTAGGAAAGCA (GA)20 53°C 13 160-220 2 165, 160 0.2585 JX409684

17. DHGMS-46 F-TACTGGGCAACGTATGTGGA

R-CGCCCTATTGCTAGGAGTGA (AT)5(GT)7 53°C 12 210-400 2 355, 350 0.375 JX409685

18. DHGMS-48 F-TGATCACGGTAGCAGTTGGA (GT)5 53°C 4 180-260 - - 0.4793 JX409686

Table 4.5: Characteristics of forty three SSR markers amplified and validated in thirty two accessions of Dendrocalamus hamiltonii

52

R-GGCCACACTCCCTAGACTCA

19. DHGMS-49 F-CAATGGTGCTCCCTTTCTGT

R-GAAGTTGTCTTGGTGGAGACG (CT)5 53°C 3 200-230 1 230 0.4315 JX409687

20. DHGMS-50 F-GAAGCATAGGACCGATCCAC

R-GTGCCATCCTCACCTTCAAT (GTT)2(GGT)3 53°C 6 250-280 1 250 0.4338 JX409688

21. DHGMS-51 F-GAGGTGGAGGCGATAGTGAA

R-CCTTGGCTCCATATCTTCCA (GAT)5 53°C 7 130-220 2 175, 150 0.3738 JX409689

22. DHGMS-54 F-CTCGGCGTTTGTTTCTTCAG

R-GGCCTCAAAAGAGAGGTTCC (CTG)6 53°C 6 145-170 1 160 0.4445 JX409690

23. DHGMS-55 F-AGCACAACACACAGGGCTTA

R-TGTGCATAGTTGGTTCAGAGC (CA)12 53°C 6 170-216 - - 0.4403 JX409691

24. DHGMS-56 F-CCCTCATAACAATGGGGAAT

R-TTGGGGATGGGAAAGTGATA (GCT)6 51°C 4 180-240 1 180 0.3929 JX409692

25. DHGMS-58 F-AATGCCTCAGGTCGGTTGT

R-TCTGGTCAAGCAGTGTTTCG (GA)6.. (GA)3 52°C 10 225-365 2 310, 225 0.3499 JX409693

26. DHGMS-59 F-AATTGTCAGACACCGGCAGT

R-TTGGGTGATTCCAACAACAA (GA)11 49°C 5 370-450 1 385 0.3695 JX409694

27. DHGMS-60 F-AGCAGTGAGCAAAGGGAAAA

R-AAAGGAGCCTTGTGTTCACTC (GA)12 51°C 5 110-180 1 150 0.2828 JX409695

28. DHGMS-66 F-AACACCGACACAAAAGATA

R-CTCTCTTTTTTTGTCTCTCTC (GT)8(GA)14 46°C 3 240-250 - - 0.4939 JX409696

29. DHGMS-67 F-CGCTCACTCTCGCTCTCTC

R-ACGCCAGTGCTACGGTTATT (CT)14 53°C 4 240-280 - - 0.4641 JX409697

30. DHGMS-69 F-GAGGCTCGTTTGGCATGTAG

R-ACCACATACCATGAGGCAAT (GTT)39(GT)22 51°C 5 140-230 2 230, 210 0.4921 JX409698

31. DHGMS-70 F-TGCTCTTCAGTGTGCTCCAG

R-CCAACACACAAGGATGCACT (CAA)7 53°C 3 170-205 1 205 0.5 JX409699

32. DHGMS-71 F-AATCTCCTCGCCAGTCAGAA

R-TTGAGCCAATTTTGTCATCG (CT)7 49°C 4 205-230 2 230, 215 0.4994 JX409700

33. DHGMS-75 F-ACCCATCGCCTTGCAAATAG

R-AAAGCTCAACAAAAAGCCAAA (GA)7 49°C 5 500-580 - - 0.4342 JX409701

34. DHGMS-76 F-ACACACCAGAGAGAGAGAGAG

R-GCTGTGTGTGTGTGAGAGAG

(GA)41(CT)6

(CA)6 55°C 1 117 0 - 0 JX409702

35. DHGMS-77 F-ACGGGTAGGAGACCCGTTAG

R-CACATGCTTCTTGGGAGGA (CT)3(GT)4 52°C 7 230-280 1 280 0.4275 JX409703

36. DHGMS-81 F-TCCCAGGAGTATAGAATCATTTTC

R-TAGTGCCTAGGCGCCATAAT (TA)12(GT)29 53°C 11 230-390 - - 0.2596 JX409704

37. DHGMS-82 F-GTCATTGATGGAAGGCCACT

R-ACCGCTCGACATTAGCTTGT (GT)6 53°C 5 250-380 - - 0.2257 JX409705

53

Ta: Annealing temperature, Na: Number of alleles, PIC: Polymorphism Information Content

38. DHGMS-83 F-CAAAAGGCTTTGTTGTTGTTG

R-GTCCAATGCGAACCATCC (GTT)3(GT)25 50°C 8 450-680 - - 0.4445 JX409706

39. DHGMS-84 F-CAACAACTGCAACTACAAGAACG

R-GCCAGAACCATGAGCTTGA

(CAA)4

(GAA)5 52°C 7 240-300 2 275, 250 0.2636 JX409707

40. DHGMS-85 F-CCGGTGGAGAGATCTGTAGC

R-AGCGCGAGGAATAAAAACCT (CGG)5 51°C 6 200-240 - - 0.5 JX409708

41. DHGMS-86 F-AGTTGCTTGGCTTTGCTCAT

R-CACACTCACACCCTTGAGGA (GTT)20(GT)15 51°C 6 400-530 1 440 0.4445 JX409709

42. DHGMS-89 F-TGACTACAACAACACCTACAAC

R-GCGAAAGAGAAGTGATAAAG (CAA)13 59°C 1 205 0 - 0 JX409710

43. DHGMS-90 F-GTGAATGGATTGGAGGTGCT

R-TATTGGCAATGACAGCTTCC (CAT)4 51°C 8 100-130 1 126 0.4997 JX409711

Total 243 32

Mean 6.263 0.8421 0.4071

54

According to the dendrogram the accessions were divided into two major clusters.

All the accessions were randomly distributed with no clear differentiation between

populations collected from two regions. However, it is evident from the cluster analysis

that these populations were dominated by two populations and also supported a high level

of anthropogenic activity between populations with tremendous level of out-crossing

between the accessions. While, two accessions from north east India remained as out

group. A dendrogram constructed based on Jaccard’s similarity coefficient using

unweighted pair grouped mean of average (UPGMA) is shown in Figure 4.4.

Although propagation through seed formation in this species seems rare event as

propagation methods are dominated by vegetative means, somehow this sexually

propagating method has played role in exchange of allele between the gene pools of local

regional populations which resulted in admixture of available gene pool as a result

branching pattern of two populations of Kangra and Mandi showed the presence of both

Figure 4.4: Genetic relationships among 32 accessions of D.hamiltonii based on the

43 DHGMS primers identified in the present study. Bootstrap values of

greater than 60 are indicated

I

II

Out groups

55

types of alleles in them showing their common ancestry and shared gene pool. On the other

hand third group representing north eastern region showed major divergence and seem to

be completely different population having different gene pool. Thus, from the cluster

analysis and AMOVA we can conclude that there exists two gene pools for this bamboo

species in the country and as the north western gene pool seem to be largely duplicated, the

northeastern gene pool of D. hamiltonii need to be characterized at large scale so that the

majority of available genetic diversity in this species can be captured to make proper

conservation and management strategies and to identify the useful elite germplasm lines of

this species which can benefit the bamboo sector in future. Cluster analysis results also

proves the effectiveness of this set of SSR markers as they were able to discriminate

between two existing gene pools of D. hamiltonii.

4.2 Cross-transferability of microsatellite markers in bamboo

Simple sequence repeat (SSR) markers are valuable tools for many purposes, such

as phylogenetic, fingerprinting and molecular breeding studies. However, only a few SSR

markers are known and available in bamboo species of the tropics. Based on the fact that

sequence analyses of SSR loci of several grass species have shown high homology in their

flanking regions (Saha et al. 2004), SSR primer pairs developed from one species could be

used to amplify SSRs in related species (Dirlewanger et al. 2002; Kuleung et al. 2004; Yu

et al. 2004). Therefore, microsatellite markers developed in D. hamiltonii in the current

dissertation and public microsatellite markers of rice and sugarcane were evaluated in

different bamboo species to determine their utility in genetic diversity and phylogenetic

analysis of tropical bamboo germplasm. In total, 177 microsatellite markers (59 of

bamboo, 98 of rice and 20 of sugarcane) were evaluated for cross-transferability studied in

23 species of Bamboo.

4.2.1 Cross transferability of bamboo microsatellite markers

Fifty nine bamboo (49 genomic SSRs, 10 EST-SSRs) microsatellite markers were

used for cross transferability studies. Species wise transferability rate varied from 47.37 %

(P. pubescens) to 89.4 % (D. giganteus, B. balcooa). These SSR loci recorded high level of

overall cross-transferability rate in Dendrocalamus and Bambusa species. Among five

Dendrocalamus species transferability rate varied from 81.6% (D. Asper) to 89.4% (D.

giganteus) with average of 86 %, while an average of 84.7 % transferability recorded

among nine Bambusa species (Table 4.6).

56

Marker wise intra-generic transferability and conservation of these marker loci

showed that at least 13 (22 %) bamboo markers shown amplification across all the 23

tested species (100 %). However, DHGMS-56 revealed least transferability in 7 bamboo

species (30.4 %). DH microsatellite primers namely, DHGMS9, DHGMS12, DHGMS13,

DHGMS31, DHGMS50, DHGMS54, DHGMS70, DHGMS81, DHGMS89, DHGMS90

and EST–SSRs Boes3, Boes4 and Boes7 were transferred all 23 bamboo species (Table

4.2). However, none of the Bambusa arundinacea SSR primers was transferred to P.

pubescens and P. heteroclada (temperate bamboo species collected from China).

4.2.2 Cross-transferability of rice and sugarcane microsatellite markers

To determine the transferability from related genera, 98 mapped SSR primers

representing 12 linkage groups of rice (Oryza sativa) and 20 EST derived sugarcane

(Saccharum spp.) SSR primers were evaluated for transferability to 23 bamboo species. Of

the 118 SSR primers tested 59 (44 of rice and 15 of sugarcane) could produce repeatable

amplification in at least one bamboo species, amounting to 44.8 % (rice) and 75 %

(sugarcane) transferability to Bambusoideae.

Fig. 4.5: The amplification profiles generated with sugarcane primer,

MCSA180E02 (A) and rice primer RM129 (B). Lanes 1-23 different

bamboo species Table 2.1; M and M*: 100 bp ladder plus and 50 bp

DNA ladder (MBI Fermentas, Lithuania)

M 1 23 M

M 1 23 M

57

At the species level, transferability ranged from a minimum of 23.4 % in P. pubescens

(with rice SSR primers) to a maximum of 83.3 % in seven species namely D. hamiltonii,

D. giganteus, D. membranaceous, B. vulgaris, B. ventricosa, B. multiplex and B.

polymorpha. The maximum transferability with rice and sugarcane SSR primers was 37.7

% and 65 %, respectively (Table 4.6; Fig 4.6). Interestingly, 10 primers (7 of rice and 3 of

sugarcane) could be transferred to all the 23 species of bamboo indicating that they may

represent a set of well conserved loci across the taxa. Details of amplification pattern and

transferability of the SSR primers across the 23 species of bamboo are given in Table 4.7.

The higher rate of transferability of sugarcane EST-SSRs than rice genomic SSRs reported

in this present study was most likely due to the SSRs derived from transcribed regions

remaining more conserved during evolution.

58

Table 4.6: Transferability details of bamboo, rice and sugarcane SSR primers

Donor species of SSR primers

Bamboo (D. hamiltonii)

enriched genomic SSR

Bamboo(Bambusa

arundinacea) genomic SSR

Rice (Oryza sativa) mapped

genomic SSR

Bamboo (Bambusa

oldhamii) EST-SSR

Sugarcane (Saccharum

spp.) EST-SSR

Number of SSR 43 6 98 10 20

Number of SSR 43 (100%) 6 (100 %) 44 (44.9 %) 10 (100%) 15 (75 %)

Primers transferred

(Species wise)

Number

Percentage (%)

Number

Percentage (%)

Number Percentage (%)

Number

Percentage (%)

Number Percentage (%)

Dendrocalamus 43 100 5 83.3 36 36.7 10 100 13 65

D. asper 31 81.6 4 66.7 37 37.7 8 80 13 65

D. strictus 33 86.8 3 50 31 31.6 7 70 12 60

D. hookeri 34 89.5 3 50 32 32.6 8 80 10 50

D. giganteus 34 89.5 5 83.3 30 30.6 8 80 13 65

D. membranaceus 33 86.8 5 83.3 31 31.6 10 100 12 60

Bambusa bambos 31 81.6 4 66.7 29 36.7 8 80 9 45

B. vulgaris 32 84.2 5 83.3 34 34.6 8 80 10 50

B. multiplex 32 84.2 5 83.3 34 34.6 8 80 13 65

B. ventricosa 32 84.2 5 83.3 32 32.6 9 90 13 65

B. polymorpha 33 86.8 5 83.3 33 33.6 7 70 10 50

B. nutans 33 86.8 4 66.7 27 27.5 7 70 11 55

B. nana 32 84.2 3 50 28 28.5 7 70 9 45

B. tulda 31 81.6 4 66.7 34 34.6 8 80 13 65

B. balcooa 34 89.5 4 66.7 32 32.6 9 90 11 55

Phyllostachys nigra 19 50.0 2 33.3 32 32.6 7 70 13 65

P. aurea 22 57.9 4 66.7 33 33.6 10 100 12 60

P. pubescens 18 47.4 - - 23 23.4 3 30 8 40

P. heteroclada 26 68.4 - - 30 30.6 4 40 11 55

Ochlandra 20 52.6 3 50 33 33.6 8 80 13 65

O. scriptoria 25 65.8 3 50 26 26.5 8 80 7 35

Sasa auricoma 30 78.9 1 16.7 31 31.6 7 70 11 55

Melocanna baccifera 28 73.7 2 33.3 33 33.6 7 70 12 60

59

Species

Locus dh da ds dho dg dm bb bv bm bvt bp bnt bn bt bbl pn pa pp ph ot os sa mbf %

Transferability

DHGMS-02 18 (78.26%)

DHGMS-08 20 (86.96%)

DHGMS-09 23 (100%)

DHGMS-12 23 (100%)

DHGMS-13 23 (100%)

DHGMS-17 10 (43.48%)

DHGMS-19 21 (91.30%)

DHGMS-31 23 (100%)

DHGMS-32 21 (91.30%)

DHGMS-33 13 (56.52%)

DHGMS-37 19 (82.81%)

DHGMS-41 16 (69.57%)

DHGMS-42 9 (39.13%)

DHGMS-43 21 (91.30%)

DHGMS-44 16 (69.57%)

DHGMS-45 20 (86.96%)

DHGMS-46 9 (39.13%)

DHGMS-48 15 (65.22%)

DHGMS-49 17 (73.91%)

DHGMS-50 23 (100%)

DHGMS-51 12 (52.17%)

DHGMS-54 23 (100%)

DHGMS-55 20 (86.96%)

DHGMS-56 8 (34.78%)

DHGMS-58 18 (78.26%

DHGMS-59 20 (86.96%)

DHGMS-60 18 (78.26%

Table 4.7: Cross species amplification details of 118 transferred SSR markers. The green shade indicates amplification by

respective primer

60

DHGMS-66 13 (56.52%)

DHGMS-67 16 (69.57%)

DHGMS-69 20 (86.96%)

DHGMS-70 23 (100%)

DHGMS-71 21 (91.30%)

DHGMS-75 13 (56.52%)

DHGMS-76 23 (100%)

DHGMS-77 22 (95.65%

DHGMS-81 23 (100%)

DHGMS-82 14 (60.87%)

DHGMS-83 22 (95.65%

DHGMS-84 22 (95.65%

DHGMS-85 22 (95.65%

DHGMS-86 17 (73.91%)

DHGMS-89 23 (100%)

DHGMS-90 23 (100%)

Ba10 3 (13.04%)

Ba14 21 (91.30%)

Ba18a 13 (56.52%)

Ba18b 19 (82.81%)

Ba20 5 (21.74%)

Ba58 19 (82.81%)

RM259 21 (91.30%)

RM129 19 (82.81%)

RM34 22 (95.65%

RM237 20 (86.96%)

RM128 23 (100%)

RM265 20 (86.96%)

RM154 23 (100%)

RM236 10 (43.48%)

RM29 4 (17.39%)

RM262 4 (17.39%)

RM341 22 (95.65%

RM106 18 (78.26%

RM112 22 (95.65%

RM125 23 (100%)

61

RM36 11 (47.82%)

RM251 23 (100%)

RM135 18 (78.26%

RM142 17 (73.91%)

RM252 6 (26.09%)

RM241 2 (8.70%)

RM280 18 (78.26%

RM131 22 (95.65%

RM574 19 (82.81%)

RM178 2 (8.70%)

RM31 21 (91.30%)

RM225 8 (34.78%)

RM136 22 (95.65%

RM30 23 (100%)

RM340 18 (78.26%

RM103 18 (78.26%

RM118 23 (100%)

RM248 22 (95.65%

RM51 18 (78.26%

RM152 21 (91.30%)

RM126 22 (95.65%

RM281 23 (100%)

RM242 18 (78.26%

RM215 22 (95.65%

RM205 6 (26.09%)

RM244 10 (43.48%)

RM286 18 (78.26%

RM 229 14 (60.87%)

RM247 19 (82.81%)

Boes-3 23 (100%)

Boes-4 23 (100%)

Boes-5 19 (82.81%)

Boes-6 18 (78.26%

Boes-7 23 (100%)

Boes-8 15 (65.22%)

Boes-10 15 (65.22%)

62

Boes-11 14 (60.87%)

Boes-12 14 (60.87%)

Boes-13 12 (52.17%)

MCS003B02 7 (30.43%)

MCS005C04 2 (8.70%)

MCS014E10 20 (86.96%)

MCSA053C10 20 (86.96%)

MCSA062B06 23 (100%)

MCSA077C02 19 (82.81%)

MCSA116D08 23 (100%)

MCSA175A08 21 (91.30%)

MCSA175G03 16 (69.57%)

MCSA176C01 7 (30.43%)

MCSA176C03 22 (95.65%

MCSA180E02 23 (100%)

MCSA205C07 22 (95.65%)

YCS02.047 22 (95.65%)

YCS24.043 12 (52.17%)

To

tal

10

7 (

60

.11

%)

99

(5

5.6

1%

)

92

(5

1.6

8%

)

92

(5

1.6

8%

)

96

(5

3.9

3%

)

96

(5

3.9

3%

)

89

(5

0%

)

94

(5

2.8

0%

)

97

(5

4.4

9%

)

97

(5

4.4

9%

)

94

(5

2.8

0%

)

88

(4

9.4

3%

)

86

(4

8.3

1%

)

95

(5

3.3

7%

)

95

(5

3.3

7%

)

77

(4

3.2

5%

)

87

(4

8.8

8%

)

58

(3

2.5

8%

)

77

(4

3.2

5%

)

81

(4

5.5

1%

)

75

(4

2.1

3%

)

86

(4

8.3

1%

)

88

(4

9.4

4%

)

63

Of 177 microsatellite markers, 118 (59 of bamboo, 44 of rice and 15 of sugarcane)

could produce repeatable amplification in at least one bamboo species, amounting to locus

wise 100 % (bamboo), 44.8% (rice), and 75 % (sugarcane) transferability. At the species

level, transferability ranged from a minimum of 16.6 % in P. pubescens (with B.

arundinacea SSR primers) to a maximum of 100 % in three species namely D. hamiltonii,

D. membranaceus and P.nigra (with Bambusa oldhamii EST-SSRs). The maximum

transferability with rice and sugarcane SSR primers was 37.7 % and 65 %, respectively

(Table 4.1). Graphical representation of transferred SSR markers has been shown in Fig

4.6.

Fig 4.6: Transferability of bamboo, rice and sugarcane SSR primers to 23 bamboo

species

4.2.3 Polymorphic potential of transferred markers

The transferred primers amplified a total of 1062 fragments out of which 1055

(99.34 %) were polymorphic. Individually bamboo markers amplified 452 fragments, 423

were amplified by rice primers and 187 by sugarcane EST-SSR primers. The number of

fragments amplified by polymorphic primers ranged from 2 to 23 with an average of 7.8

fragments per primer (Table 4.8). At the genera level, bamboo SSR primers amplified 809

fragments in Bambusa, 452 fragments in Dendrocalamus, 206 fragments in Phyllostachys,

111 fragments in Ochlandra, 77 fragments in Sasa and 66 fragments in Melocanna.

Likewise rice and sugarcane SSR primers amplified 173 fragments in Bambusa, 161 in

Dendrocalamus, 125 in Phyllostachys and 76 in Ochlandra. The genera Melocanna and

Sasa had 74 and 46 fragments, respectively. The polymorphism information content

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

dh da ds dho dg dm bb bv bm bv bp bnt bn bt bbl pn pa pb ph ot os sa mbf

% t

ran

sfe

rab

ilit

y

Bamboo species

dh+ba+os+bol+so D.hamiltonii B.arundinacea O.sativa B.oldhamii S.officinarum

64

(PIC) ranged from 0.077 to 0.500 with an average of 0.263 (Table 4.8). No

significant difference in average PIC values was recorded between bamboo, rice and

sugarcane SSR primers. Ten primers of bamboo (DHGMS32, DHGMS33, DHGMS37,

DHGMS54, DHGMS69, DHGMS77, DHGMS83, DHGMS85, DHGMS90 and Ba18b),

Four primers of rice (RM 29, RM 178, RM 242 & RM 259) and two of sugarcane

(MCSA116D08 and YCS02.047) with PIC values ≥ 0.40 were identified as most

informative and thus would be useful in further genetic characterization of bamboo

germplasm. All the transferred EST-SSR primers (Bamboo and Sugarcane) amplified an

average of 9.8 fragments per primer pair whereas genomic SSR primers (Bamboo and

Rice) amplified 8.77 fragments per primer pair. Number of fragments detected by each

bamboo SSR primers was low (7.6 fragments per primer) as compared to rice (9.6

fragments per primer) and sugarcane (12.4 fragments per primer). In bamboo SSR primers

majority of the transferred SSR primers amplified dinucleotide repeats (43.5 %) followed

by compound repeats (39.2 %) and least commonly amplified were tri repeats (20.9%).

Transferred SSR primers of rice and sugarcane showed amplification of majority of Di

repeats (59%) followed by tri (27%) and compound repeats (15.9%).

Table 4.8: Amplification pattern and polymorphism information content (PIC) of

bamboo, rice (genomic) and sugarcane (EST) SSR primers in bamboo

germplasm S. No. Primers Alleles Size

(bp)

No of

fragments

P

or

M

Size

Range

(bp)

Unique

fragments

PIC

Bamboo (Dendrocalamus hamiltonii) genomic 1. DHGMS-02 7 85 7 P 320-380 1 0.317 2. DHGMS-08 6 77 6 P 250-350 1 0.309

3. DHGMS-09 1 120 1 M 120 - - 4. DHGMS-12 1 150 1 M 150 - -

5. DHGMS-13 1 103 1 M 103 - -

6. DHGMS-17 10 177 10 P 210-250 1 0.194 7. DHGMS-19 6 159 15 P 110-225 - 0.363

8. DHGMS-31 9 159 16 P 150-210 2 0.315 9. DHGMS-32 4 131 4 P 130-170 - 0.497

10. DHGMS-33 4 150 4 P 175-190 - 0.499

11. DHGMS-37 4 181 5 P 185-230 - 0.499 12. DHGMS-41 8 157 11 P 150-300 3 0.327

13. DHGMS-42 9 183 11 P 150-180 1 0.262 14. DHGMS-43 3 246 12 P 330-350 - 0.300

15. DHGMS-44 5 228 7 P 240-310 - 0.352 16. DHGMS-45 13 186 15 P 160-220 - 0.227

17. DHGMS-46 12 173 18 P 210-270 2 0.210

18. DHGMS-48 4 181 8 P 180-240 2 0.359 19. DHGMS-49 3 162 5 P 180-230 1 0.295

20. DHGMS-50 6 205 10 P 240-270 1 0.283

21. DHGMS-51 7 171 10 P 130-220 1 0.265

65

22. DHGMS-54 6 156 8 P 150-170 - 0.401

23. DHGMS-55 6 208 10 P 170-260 - 0.302 24. DHGMS-56 4 179 9 P 180-240 4 0.153

25. DHGMS-58 10 253 14 P 230-280 2 0.229 26. DHGMS-59 5 247 11 P 370-410 - 0.212

27. DHGMS-60 5 150 9 P 110-200 2 0.189

28. DHGMS-66 3 97 9 P 240-250 1 0.237 29. DHGMS-67 4 237 10 P 240-280 - 0.241

30. DHGMS-69 5 247 8 P 140-230 - 0.406 31. DHGMS-70 3 169 13 P 175-205 1 0.291

32. DHGMS-71 4 196 10 P 190-270 1 0.321 33. DHGMS-75 5 185 7 P 480-580 - 0.326

34. DHGMS-76 1 117 1 M 117 - -

35. DHGMS-77 7 210 7 P 238-280 1 0.490 36. DHGMS-81 11 192 21 P 175-390 - 0.187

37. DHGMS-82 5 169 6 P 250-380 1 0.205 38. DHGMS-83 8 153 8 P 450-680 - 0.486

39. DHGMS-84 7 183 15 P 160-300 - 0.206

40. DHGMS-85 6 185 8 P 180-240 1 0.434 41. DHGMS-86 6 167 7 P 400-530 2 0.383

42. DHGMS-89 1 205 1 M 205 - - 43. DHGMS-90 8 180 9 P 95-130 1 0.470

Total 243 378 33

Mean 5.65 8.79 0.76 0.316

Bamboo (Bambusa arundinacea) genomic

44. Ba10a 13 146 1 P’ 210 - 0.13 45. Ba14 1 237 1 P’ 200 - 0.09

46. Ba18a 2 166 3 P 170- 190 - 0.36 47. Ba18b 1 146 5 P 100-150 - 0.50

48. Ba20 12 169 4 P 250-700 - 0.27

49. Ba58 1 187 1 P’ 190 - 0.16 Total 30 15 -

Mean 5 2.5 0.252

Rice (Oryza sativa) mapped genomic

50. RM 29 2 250 6 P 200-1000 6 0.450 51. RM 30 5 105 14 P 100-2500 5 0.205

52. RM 31 8 140 17 P 190-1500 10 0.157

53. RM 34 1 161 16 P 199-2000 8 0.125 54. RM 36 1 112 3 P 500-1000 1 0.316

55. RM 51 5 142 1 M 300 - 0.34 56. RM 103 6 336 11 P 150-1000 7 0.180

57. RM 106 2 297 10 P 280-1000 3 0.260

58. RM 112 2 128 20 P 120-2500 7 0.136 59. RM 118 3 156 20 P 160-2500 2 0.189

60. RM 125 3 127 12 P 390-1500 3 0.295 61. RM 126 3 171 13 P 400-2100 3 0.148

62. RM 128 4 157 3 P 380-500 - 0.316 63. RM 129 3 205 20 P 275-2200 6 0.216

64. RM 131 4 215 13 P 100-2200 4 0.203

65. RM 135 3 131 20 P 120-2200 3 0.183 66. RM 136 3 101 23 P 200-2500 5 0.126

67. RM 142 5 240 5 P 100-1031 1 0.31 68. RM 152 4 151 5 P 150-500 3 0.356

69. RM 154 5 183 10 P 330-1500 1 0.268

70 RM 178 4 117 2 P 300-1900 2 0.450 71. RM 205 7 122 2 P 400-600 - 0.31

72. RM 215 5 148 19 P 210-1500 1 0.195

73. RM 225 5 140 4 P 300-900 1 0.24 74. RM 229 8 116 9 P 150-1200 - 0.375

75. RM 236 2 191 5 P 300-700 2 0.23 76. RM 237 6 130 8 P 100-1500 4 0.304

66

77. RM 241 7 138 2 P 150-500 2 0.08

78. RM 242 8 225 4 P 300-1500 1 0.40 79. RM 244 4 163 3 P 200-700 1 0.29

80. RM 246 8 225 9 P 300-1800 - 0.249 81. RM 247 10 131 6 P 150-1500 - 0.278

82. RM 248 8 102 12 P 120-2000 3 0.139

83. RM 251 8 147 1 M 300 - 0 84. RM 252 6 216 4 P 400-1031 1 0.16

85. RM 259 8 160 8 P 180-2000 - 0.42 86. RM 262 NA 154 2 P 300-800 1 0.16

87. RM 265 3 106 11 P 150-1200 3 0.227 88. RM 280 5 155 8 P 250-1500 2 0.196

89. RM 281 5 183 12 P 270-1500 4 0.360

90. RM 286 6 110 8 P 400-1500 - 0.165 91. RM 340 4 163 11 P 350-2500 1 0.225

92. RM 341 4 172 16 P 100-2400 - 0.240 93. RM 574 1 574 15 P 150-2300 2 0.152

Total 204 423 109 -

Mean 4.64 9.61 2.47 0.241

Bamboo (Bambusa oldhamii) EST-

94. Boes-03 - - 2 P 140-180 0 0.076 95. Boes-04 - - 5 P 240-280 4 0.347

96. Boes-05 - - 4 P 310-350 1 0.384 97. Boes-06 - - 10 P 210-250 3 0.217

98. Boes-07 - - 8 P 280-300 2 0.313

99. Boes-08 - - 5 P 90-95 1 0.380 100. Boes-10 - - 7 P 200-260 2 0.268

101. Boes-11 - - 5 P 300-340 1 0.246 102. Boes-12 - - 5 P 240-260 1 0.268

103. Boes-13 - - 8 P 150-170 2 0.233

Total - - 59 17 Mean - - 5.9 1.7 0.273

Sugarcane (Saccharum spp.) EST- 104. MCS003B02 2 250 4 P 300-800 - 0.14

105. MCS005C04 1 386 2 P 1500- 2 0.05 106. MCS014E10 2 112 14 P 100-2500 1 0.29

107. MCSA053C10 3 152 17 P 150-700 4 0.270

108. MCSA062B06 2 161 21 P 100-2000 3 0.197 109. MCSA077C02 4 144 11 P 150-2500 - 0.21

110. MCSA116D08 3 174 8 P 100-2000 - 0.49 111. MCSA175A08 3 122 9 P 500-2500 - 0.398

112. MCSA175G03 2 114 17 P 70-600 4 0.098

113. MCSA176C01 1 258 11 P 320-900 - 0.230 114. MCSA176C03 2 243 17 P 100-600 1 0.260

115. MCSA180E02 5 162 20 P 110-2500 2 0.250 116. MCSA223B07 2 201 21 P 120-2100 3 0.120

117. YCS02.047 2 154 3 P 150-180 - 0.480 118. YCS24.043 2 198 12 P 400-1600 9 0.077

Total 36 187 29 -

Mean 2. 4 12.47 1.93 0.237

Overall mean 4.42 7.85 1.71 0.263

NA = Not available; P/ M = polymorphic/ Monomorphic, P’ = Polymorphism based on absence of

amplification (null allele) with tested primer across the test array.

67

More particularly, greater success was obtained with GA/TC repeat primers in case

of all SSR primers including bamboo, rice and sugarcane, which are reported to be the

most abundant in plant genomes (La Rota et al. 2005; Lee et al. 2004). Maximum number

of fragments were detected with SSR containing compound repeat motifs, e.g. the rice

SSR primer RM 136 [(CTT)8T3(CTT)14] that amplified 23 fragments. From bamboo SSR

primers maximum fragments were detected by compound di repeat [(TA)12(GT)29] which

were amplified by primer DHGMS- 81with 21 amplicons. Among the transferred EST-

SSRs of sugarcane, the CGG/GCC motif (38.5 %) was most common and the maximum

number of fragments were detected with primers amplifying the (CAG)6 (MCSA223BO7)

and (CGG)8 (MCSA062BO6) motifs.

4.2.4 Sequence comparison of SSR locus

To validate the paralogs and orthologs conservation of SSRs, multiple amplicons from the

same genotypes and at least one amplicon from different species were sequenced. When a

locus wise DNA sequences data in each case was compared, it showed that in general

electromorphic size variation with species was solely attributed either due to expansion/

contraction of the SSRs, or due to interruptions in the SSR regions. This was most notable

among different alleles where the size differences resulted from either simple or complex

variation in SSR motifs. Sequence comparisons among different Dendrocalamus species

was also broadly due to presence of SSRs, however, few variations were also recorded in

flanking regions. As illustrated in Fig 4.7 , the size of the multiple amplicons having

(GA)n motif and consumed primer sites were 169, 173, 177, 179 bp longer selected

accessions of D. hamiltonii for marker DHGMS-45. Similarly, amplicon size 159, 161, 169

bp were obtained for DHGMS-31 that amplified repeats.

Further, in order to confirm DNA polymorphism and cross-transferability at the

sequence level, selected amplicons from four bamboo species namely B.balcooa, B.nutans,

D.longispathus, G. albociliata were sequenced for two SSR loci DHGMS- 31 and

DHGMS- 45. The presence of the target microsatellites were observed in all the cases,

however, few indels were also observed in flanking region. Four bases indel (TGTT)

recorded in G.albociliata with DHGMS- 45, while 2 bp indels observed in B.nutans and a

single bp indel in D. logispathus and B. balcooa was recorded in case of DHGMS- 31

(Figure 4.8).

68

DHGMS-45

DHGMS-31

Figure 4.7: Sequence alignments of different amplicons of D. hamiltonii are

indicated by a1, a2, a3 and a4.These alleles were amplified by two

primers namely DHGMS-45 and DHGMS-31. The shaded nucleotides

highlight the SSR motifs and variation in nucleotides which resulted

in length variation

69

DHGMS-45

DHGMS-31

Figure 4.8: Sequence alignment of different amplicons from five different species are

indicated by species names. These alleles were amplified by two primers

namely DHGMS45 and DHGMS31. The shaded nucleotides highlight

the SSR motifs and variation in nucleotides which resulted in length

variation

70

4.3. Evaluation of genetic diversity and phylogenetic analyses in bamboo

Sustainable utilization of plant genetic resources requires a strategic action plan to

conserve the available germplasm at the national level. Bamboo is one of such a wonderful

bioresource, which has multiple commercial and domestic importance. However, most of

bamboo resources remain uncharacterized. In the present study, 224 accessions of five

commercially important bamboo species namely, Dendrocalamus hamiltonii, D. strictus,

Bambusa nutans, B. bambos and B. balcooa collected from different geographical regions

of India, were characterized using AFLP markers. Of the twenty five primer combinations

screened, 8 AFLP primer combinations (E-AAC+ M-CTG, E-ACT+ M-CTG, E-AGG+ M-

CTC, E-AGG+ M-CTA, E-AGG+ M-CTG, E-AGG+ M-CAC, E-AGC+ M-CTT) detected

informative and reproducible profiles in representative accessions were extrapolated for

genetic diversity evaluation in targeted bamboo species. Eight primer combinations

detected 2095 polymorphic loci with an average of 261.87 loci per primer combination.

The mean polymorphism information content (PIC) value was 0.187. Highest and lowest

marker index (MI) of 63.08 and 34.87 was observed for the primer combinations E-

AAC/M-CAC and E-AGG/M-CTA, respectively. Species wise genetic diversity estimates

and phylogenetic inferences drawn from AFLP analysis are discussed in ensuing pages.

4.3.1 Polymorphic potential of AFLP markers

D. hamiltonii has countrywide distribution and widely cultivated in Himachal Pradesh

and north eastern states of India. Eight AFLP primer combinations amplified 1474

polymorphic fragments in 111 accessions of D. hamiltonii (Table 4.9). Total fragments

amplified by each primer combinations varied from minimum of 135 by E-AGG/ M-CTA

to maximum of 231 (E-AAC/ M-CAC) with an average of 184 fragments. Polymorphisim

information content (PIC) and marker index (MI) were calculated individually for each

primer combination. Overall PIC values revealed by each primer combination were not

significantly different and average PIC remained 0.2429. Effective multiplex ratio (E)

ranged 135 (E-AGG/ M-CTA) to 231 (E-AAC/ M-CAC) with an average value of 184.25.

Marker index (MI), which is another attribute of a marker, and varied was ranged from

31.09 to 54.56 with mean value of 44.83. Primer combinations namely, E-AAC/ M-CAC

and E-AGG/ M-CTA exhibited 31.09 (minimum) and 54.56 (maximum) MI, respectively.

Unique loci detected by each primer combination varied from 47 (E-AGG/ M-CTC) to 77

71

(E-AAC/ M-CAC) with an average of 58.37 (12.49%). A representative profile of AFLP

and sizing of fragments by Genemapper is shown in Figure 4.9A and B.

Figure 4.9a: A representative AFLP profile of bamboo samples .Green fragments

represents detected fragments while red peaks are the marker

fragments indicating size

Figure 4.9b: Bins and sizing of AFLP fragments done with the help of Genemapper

after setting best fit parameters in analysis method editor

72

Dendrocalamus strictus, AFLP genotyping of 40 accessions with 8 primer

combinations resulted in 985 polymorphic fragments. Maximum (141) and minimum (95)

fragments were amplified by primer combination E-AAC/ M-CAC and E-AGG/ M-CTA,

respectively. The average fragments amplified by each primer combination were 123. The

mean PIC was 0.33, which ranged from 0.29 (E-ACT/ M-CTG) to 0.38 (E-AGG/ M-CTG).

Marker index ranged from 36.2 to 50.6 with an average of 41.2, while, mean unique loci

remained 45.6 and varied from 59 to 32 with primer combinations E-AGG/ M-CAC and

E-AGG/ M-CTA, respectively.

Forty four accessions of B.nutans produced 1327 fragments with a mean value of

165.8 fragments per primer combination. PIC values ranged from 0.264 to 0.297 with

average value of 0.275. Marker index values showed that primer combination E-AAC/ M-

CAC is the most informative with MI value of 64.6. Total unique fragments detected 8

AFLP primer combinations were 447 with an average of 55.8.

In similar studies, 17 accessions of B. bambos and detected 736 fragments with 8

AFLP primer combinations and recorded an average of 92 fragments per primer

combination. Primer combination E-AAC/ M-CAC produced maximum (117) fragments,

while lowest (80) fragments amplified by primer combination E-AGG/ M-CTC. The mean

PIC recorded was 0.40, wherein, E-AGG/ M-CTG exhibited highest PIC (0.428) and E-

AGG/ M-CTC (0.373) had the lowest. An average marker index recorded was 36.9, while

unique loci were 38.2 and ranged from 27 to 53 with primer combination E-AAC/ M-CAC

and E-AGG/ M-CTA, respectively.

In B.balcooa, 8 AFLP primer combinations generated 706 polymorphic fragments

in 12 accessions and an average numbers of fragments remained 88.2. Highest (103)

numbers of fragments were amplified by primer combination E-AGG/ M-CTG and lowest

(77) by E-AAC/ M-CTG. PIC values ranged from 0.30 to 0.47 with an average of 0.39 per

primer combination. Marker index values detected were ranged from ranged 28.9 to 43.7.

(Mean, a total of 277 with a mean of 34.6 unique fragments were detected in AFLP

analysis).

Species wise consolidated information about the number of fragment, PIC and

unique bands detected in AFLP analysis are summarized in Table 4.9.

73

PB: Polymorphic Band, PIC: Polymorphism Information Content, MI: Marker Index, UB: Unique Band

D. hamiltonii D. strictus B. nutans B. bambos B.balcooa

Primer PB PIC MI UB PB PIC MI UB PB PIC MI UB PB PIC MI UB PB PIC MI UB

E-AAC+ M-CTG 157 0.255 44.31 53 133 0.324 43.19 40 162 0.269 43.62 63 85 0.419 35.68 38 67 0.473 31.71 18

E-ACT+ M-CTG 218 0.249 57.28 72 138 0.291 40.20 57 198 0.264 52.29 62 96 0.379 36.47 44 98 0.400 39.20 46

E-AGG+ M-CTC 156 0.225 37.5 47 111 0.326 36.23 34 139 0.275 38.24 46 80 0.373 29.88 33 95 0.305 28.97 50

E-AGG+ M-CTA 135 0.230 32.76 49 95 0.304 28.90 32 129 0.269 34.70 41 77 0.377 29.04 27 99 0.324 32.12 43

E-AGG+ M-CTG 209 0.249 55.11 68 122 0.382 46.71 43 183 0.272 49.80 52 101 0.428 43.24 38 103 0.424 43.73 44

E-AGG+ M-CAC 192 0.233 49.03 52 133 0.318 42.35 59 150 0.275 41.33 49 81 0.417 33.78 29 80 0.415 33.26 27

E-AAC+ M-CAC 231 0.236 60.64 77 141 0.359 50.63 57 217 0.297 64.62 80 117 0.408 47.74 53 86 0.443 38.17 24

E-AGC+ M-CTT 176 0.264 48.68 49 112 0.376 42.12 43 149 0.278 41.49 54 99 0.398 39.43 44 78 0.403 31.44 25

Total 1474 467 985 365 1327 447 736 306 706 277

Mean 184.2 48.16 0.242 123.1 0.335 41.29 45.6 165.8 0.275 45.76 55.8 92 0.400 36.91 38.2 88.2 0.398 34.82 34.6

Table 4.9: Number of fragments, polymeric rates and uniques fragments detected by AFLP primer combinations

0 0.2

12

3

4

5

67

8

9

10

1112

1314

15

16

17

18

19

20

21

2223

24

25

26

27

2829

30

31

32

33

3435

36

37

38

39

40

41

4243

4445

4647

48

49

50

51

52

5354

55

56

57

58

59

60

61

62

6364

65

66

67

68

69

70

71

7273

74

75

76

77

78

79

80

81

8283

8485

86

87

8889

9091

92

93

94

95

96

9798

99

100

101

102103

104

105

106107

108109

110

111

94

70

68

54

52

90

56

6980

88

5858

84

99

58100

80

7457

50

78

60

50

6494

63

54

95

61

50

Group-I

Group-II

4.3.2 Genetic diversity and cluster analysis

To established genetic relationships, AFLP data generated with 8 primer combinations

in 224 accessions was utilized separately in five commercially important bamboo species viz;

D. hamiltoni, D. strictus, B. nutans, B. bambos, B. balcooa for cluster analysis. Two distance

based methods namely principal coordinates analysis (PCoA) and methods based on Jaccards’

similarity coefficient and Unweighted Pair Group Method with Arithmetic mean (UPGMA)

were applied. In addition, Bayesian model implemented in STRUCTURE was also used for

clustering.

Dendrocalamus hamiltonii

Genetic diversity (GD) in

111 accessions of D. hamiltonii

ranged from 56% to 73% with an

overall GD of 64 % in this

species. Fst values for each cluster

inferred by STRUCTURE were

then used to assess gene flow

(Nm) in these clusters. The range

of mean Fst and Nm values in

these two populations was from

0.24 to 0.50 (mean 0.37) and 0.24

to 0.77(mean 0.50), respectively.

Both PCoA and NJ

analysis clustered 111 D.

hamiltonii accessions into two

major groups on the basis of their

geographical origin with minor

subgroups. The accessions

belonging from the North-eastern

states were clearly separated in

one group (Figure 4.10).

Figure 4.10: Neighbour-Joining tree showing

clustering of 111 accessions of D.hamiltonii.

The bootstrap values >= 50% were shown

75

PCoA analysis complemented the NJ clustering and grouped the all the accessions

into two groups (Figure 4.11). Percentages of variation in first three axes were 32.31%,

17.80% and 15.71% successively. Neighbour- Joining tree complemented PCA and structure

analysis and clustered all the accessions into two major groups on the basis of their

geographical origin with minor subgroups.

Figure 4.11: Principal coordinate analysis plot for 111 accessions of Dendrocalamus

hamiltonii. Each colour represents accession from two different

populations

Further, STRUCTURE analysis comparison with the NJ based tree and PCoA revealed

considerable congruence and revealed that 111 accessions of D. hamiltonii were contributed

with two populations and grouped broadly in accordance to defined geographical groups with

few exceptions. Although individual accessions clustered in each group were sharing the

genomic proportions, majority of accessions in both the clusters were belonging to two

geographically isolated populations. Many accessions were shown with admixture to different

extents. The percentages of accessions belonging to pure ancestry (accessions with

membership probabilities ≥ 0.80 %) were not significantly different in cluster I (73.6 %) &

76

cluster II (72.8%), respectively. However, in total ~ 26% accessions recoded mixed ancestry at

various levels (Figure 4.12).

Figure 4.12a: Two genetic clusters inferred by STRUCTURE for 111 accessions of D.

hamiltonii

D. strictus

A total of 985 polymorphic AFLP fragments based GD estimates in 40 accessions of D.

strictus ranged from of 52% to 75% with an average 61%. NJ and PCoA analysis

complemented each other and clustered 40 DS accessions into three major populations (Figure

4.13) PCoA the percentage of variation explained by first three axes was 29.05 %, 21.21 %

and 15.68 % respectively. The genetic differentiation (fst) values of these populations were

Figure 12b: A bar plot of 111 accessions of D. hamiltonii. Each vertical line

represents the single accession and the two colors shows the two

genetic stocks for each population

77

III

I

II Co

ord

. 2

Coord. 1

Pop1

Pop2

Pop3

0 0.2

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

3536

37

38

39

40

0.159, 0.589 and 0.534 likewise gene flow (Nm) was 1.3, 0.17 and 0.21 recorded for

population 1, 2 and 3 respectively.

The STRUCTURE analysis also inferred 3 populations (K = 3 with greatest value of

probability which indicate that 40 accessions of D. strictus were probably contributed by

three different gene pools of this species and these prevailing in all the sampling regions. The

percentages of accessions showing pure ancestry were 25%, 36.4% and 36% in the three

inferred populations (Fugure 4.14).

Figure 4.14: A bar plot of 40 accessions of D. strictus showing three clusters.

Each vertical line represents the single accession and different

colors shows contribution of each population

Figure 4.13: Cluster analysis of 40 accession of D. strictus . (A) An un-rooted

neighbor-joining tree; (B) Principal coordinate plot

B)

78

B. nutans

Overall diversity detected among 44 accessions of B. nutans was little higher as

compared to Dendrocalamus species. Genetic diversity estimates varied from 60% to 82%

with an average of 71 %. Phylogenetic tree showed two major groups with each group having

minor subgroups, which was also confirmed in PCoA analysis (Figure 4.15). These two

populations showed genetic differentiation (Fst) and gene flow (Nm) values of 0.49, 0.15 and

0.25, 1.34 respectively.

Two populations detected in STRUCTURE analysis and all the accessions grouped

into two clusters with 30 and 14 individuals in each cluster (Figure 4.16).The percentage of

pure ancestry in each cluster varied from 53.33 % to 71.42 %.

0 0.5

1

23

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

3738

39

40

41

42

43

44

I

II

Factorial analysis: Axes 1 / 2

-.25 -.2 -.15 -.1 -.05 .05 .1 .15 .2 .25 .3 .35

.35

.3

.25

.2

.15

.1

.05

-.05

-.1

-.15

-.2

-.25

-.3

-.35

bnt-IHB/BT/100

bnt-IHB/BT/101

bnt-IHB/BT/102

bnt-IHB/BT/103

bnt-IHB/BT/104

bnt-IHB/BT/105

bnt-IHB/BT/106

bnt-IHB/BT/107

bnt-IHB/BT/108

bnt-IHB/BT/109

bnt-IHB/BT/110

bnt-IHB/BT/111

bnt-IHB/BT/112

bnt-IHB/BT/113

bnt-IHB/BT/114

bnt-IHB/BT/115

bnt-IHB/BT/116

bnt-IHB/BT/117

bnt-IHB/BT/118

bnt-IHB/BT/119bnt-IHB/BT/120

bnt-IHB/BT/121

bnt-IHB/BT/122

bnt-IHB/BT/123

bnt-IHB/BT/124

bnt-IHB/BT/125

bnt-IHB/BT/126

bnt-IHB/BT/127

bnt-IHB/BT/128

bnt-IHB/BT/129

bnt-IHB/BT/130

bnt-IHB/BT/09-98a

bnt-IHB/BT/132

bnt-IHB/BT/133

bnt-IHB/BT/134

bnt-IHB/BT/135

bnt-IHB/BT/136

bnt-IHB/BT/137

bnt-IHB/BT/138

bnt-IHB/BT/139

bnt-IHB/BT/140

bnt-IHB/BT/141

bnt-IHB/BT/142

bnt-IHB/BT/128

Figure 4.16: Forty four accessions of B. nutans assigned into two

populations by STRUCTURE

Figure 4.15: Cluster analysis of 44 accession of B.nutans. (A) An un-rooted neighbor-

joining tree; (B) Principal coordinate plot

(A) (B)

79

B. bambos

NJ and PCoA based GD estimates

among 17 accessions of B. Bambos was

67.5, while the clusters wise it varied

from 81% (I) and 54 % (II) (Figure

4.17). Genetic differentiation and gene

flow for these two populations were 0.23

to 0.44 and 0.30 to 0.81 respectively.

Cluster analysis of B. balcooa showed

two groups (Figure 4.18), one for each

population inferred in Bayesian model

based cluster analysis using

STRUCTURE.

B. balcooa

In similar studies, cluster analysis of B.

balcooa with NJ, PCoA and Structure

analysis showed two groups (Figure

4.18). Genetic diversity estimates

recorded in these species are also not significantly different and GD recorded in two clusters

ranged from 54% to 82 % with an average of 68%. PCoA analysis explained 36.60 %, 15.96

% and 15.09 % genetic variation in first three axes. Genetic differentiation and gene flow for

these two populations were 0.34 to 0.42 and 0.33 to 0.47 respectively.

To summarize, overall average genetic diversity (GD) recorded in five species of

bamboo was 66.9 %. Species wise GD inferences revealed by AFLP markers were not

significantly different. The overall average GD varied from 63 % recorded in D. strictus to 71

% witnessed in B. nutans. GD recorded in D. hamiltonii, B. bambos and B. balcooa were 65%,

67.5 % and 68 %, respectively. Further, NJ based hierarchical clustering, PCoA and structure

Figure 4.17: An unrooted NJ tree of 17

accesions of B. bambos

Figure.4.18: An unrooted NJ tree of 12

accesions of B. balcooa

80

analysis revealed two populations among the nationwide collections of D. hamiltonii,

B.bambos, B.balcooa, B. Nutans, while three populations were detected in D. strictus.

4.3.2.1 Analysis of Molecular Variance (AMOVA)

Based on the inferences on cluster analysis derived in three different methods (NJ,

PCoA and STRUCTURE), species wise the partition of genetic variation within and among

populations was studied with the help of Analysis of Molecular Variance (AMOVA).

AMOVA detected high level of within population and low level of genetic variation among

the populations in all the tested species. Hence suggest high level of gene flow between

different populations. Genetic variations within the population varied from 71 % recorded in

B.balcooa to 86 % witnessed in D. hamiltonii. Within population genetic variations recorded

in B. bambos, D. strictus and B. nutans were 79%, 81 % and 83 %, respectively. The observed

values of within and among population variation of each species is given in Table 4.9

*degree of freedom, **sum of squares, ***Mean of square

Source Df* Ss** MS*** Est. Var. % variance

D. hamiltonii

Among population 1 843.821 843.821 16.007 14%

Within population 109 11057.404 101.444 101.444 86%

D. strictus

Among population 2 587.120 293.560 19.938 19%

Within population 37 3134.205 84.708 84.708 81%

B. nutans

Among population 1 546.907 546.907 22.706 17%

Within population 42 4764.048 113.430 113.430 83%

B. bambos

Among population 1 230.914 230.914 23.223 21%

Within population 15 1332.615 88.841 88.841 79%

B.balcooa

Among population 1 313.398 313.398 37.805 29%

Within population 10 928.686 92.869 92.869 71%

Table 4.9: Analysis of molecular variance of in five bamboo species

81

4.3.3 Phylogenetic Analysis

The phylogenetic relationship among the 23 species of bamboo was established by the

combined molecular data generated with 8 AFLP and 43 DHGMS SSR markers identified in

current dissertation. A total of 2438 fragments (AFLP +SSR) used to calculate phylogenetic

inferences. The cluster analysis

grouped the 23 species into three

major groups with bootstrap

values higher than 80 % for each

major group (Figure 4.19). The

group I included all the species

of Bambusa. All the species

belonging to genus

Dendrocalamus were clustered

together in Group II, while

group III clustered other species

including species of

Phyllostachys, Melocanna

baccifera, O. travancorica and

O. scriptoria.

This classification was in

accordance with the existing

taxonomical classification

(Ohrnberger 1999). Further, all

accessions within D.

hamiltonii were clustered

together without any ambiguity.

Thus, present results by and

large also supported the

clustering at the sub-tribe level.

All the species representing Dendrocalamus and Bambusa genus and belonging to subtribe

Bambusinae were grouped together.

Figure 4.19: Phylogenetic tree of 23 species of

bamboo constructed using AFLP and SSR data;

Bootstrap values greater than 60 % are

indicated