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