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Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.).

Sharma RK, Bhardwaj P, Negi R, Mohapatra T, Ahuja PS - BMC Plant Biol. (2009)

Bottom Line: Therefore, they can serve as efficient and cost effective alternative markers in such species.Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels.Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biotechnology Division, Institute of Himalayan Bioresource Technology, (CSIR), Post Box 6, Palampur, Himachal Pradesh, 176061, India. mrk_sharma@yahoo.com

ABSTRACT

Background: Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.

Results: Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (HE) and observed (Ho) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

Conclusion: UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.

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Sequence alignment of cross species amplicons. Cross-species amplicons obtained with TUGMS3, TUGMS28, TUGMS34, TUGMS73 markers in different Camellia spp. are indicated by species names. The shaded nucleotide highlights the conservation of microsatellite motifs in different species and arrow indicates the respective primer sequences.
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Figure 4: Sequence alignment of cross species amplicons. Cross-species amplicons obtained with TUGMS3, TUGMS28, TUGMS34, TUGMS73 markers in different Camellia spp. are indicated by species names. The shaded nucleotide highlights the conservation of microsatellite motifs in different species and arrow indicates the respective primer sequences.

Mentions: To validate the conservation of SSRs across the varieties and species, at least one amplicon from different genotypes/species and multiple amplicons from the same genotypes were sequenced. Multiple amplicons from single genotype were selected to determine the orthology and paralogy of the sequence. When a locus wise DNA sequences data in each case was compared, it showed electromorphic size variation 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. Even at the multiple amplicons from the diploid genotypes similar situation was noticed. As illustrated in Figure 3, the size of the multiple amplicons having (GA)n motif and consumed primer sites were 95, 89, 82 bp longer in case of genotype UPASI-10 for marker TUGMS27. Similarly, for the Kangra Jat genotype amplicon size 124, 138 and 151 bp were obtained for TUGMS46 that amplified TC repeats. Similar situation was observed with identical amplicon size, and repeat motifs for allelic amplicons from different genotypes as in case of TUGMS3 and TUGMS53, respectively. Further, in order to confirm DNA polymorphism and cross-transferability at the sequence level, selected amplicons from C. lutescens, C. irrawadiensis and C. japonica (RF: red flower & WF; white flower) were sequenced for three UGMS primers namely TUGMS3, TUGMS-34 and 73. The presence of the target microsatellites were observed in all the cases (Figure 4).


Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.).

Sharma RK, Bhardwaj P, Negi R, Mohapatra T, Ahuja PS - BMC Plant Biol. (2009)

Sequence alignment of cross species amplicons. Cross-species amplicons obtained with TUGMS3, TUGMS28, TUGMS34, TUGMS73 markers in different Camellia spp. are indicated by species names. The shaded nucleotide highlights the conservation of microsatellite motifs in different species and arrow indicates the respective primer sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2693106&req=5

Figure 4: Sequence alignment of cross species amplicons. Cross-species amplicons obtained with TUGMS3, TUGMS28, TUGMS34, TUGMS73 markers in different Camellia spp. are indicated by species names. The shaded nucleotide highlights the conservation of microsatellite motifs in different species and arrow indicates the respective primer sequences.
Mentions: To validate the conservation of SSRs across the varieties and species, at least one amplicon from different genotypes/species and multiple amplicons from the same genotypes were sequenced. Multiple amplicons from single genotype were selected to determine the orthology and paralogy of the sequence. When a locus wise DNA sequences data in each case was compared, it showed electromorphic size variation 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. Even at the multiple amplicons from the diploid genotypes similar situation was noticed. As illustrated in Figure 3, the size of the multiple amplicons having (GA)n motif and consumed primer sites were 95, 89, 82 bp longer in case of genotype UPASI-10 for marker TUGMS27. Similarly, for the Kangra Jat genotype amplicon size 124, 138 and 151 bp were obtained for TUGMS46 that amplified TC repeats. Similar situation was observed with identical amplicon size, and repeat motifs for allelic amplicons from different genotypes as in case of TUGMS3 and TUGMS53, respectively. Further, in order to confirm DNA polymorphism and cross-transferability at the sequence level, selected amplicons from C. lutescens, C. irrawadiensis and C. japonica (RF: red flower & WF; white flower) were sequenced for three UGMS primers namely TUGMS3, TUGMS-34 and 73. The presence of the target microsatellites were observed in all the cases (Figure 4).

Bottom Line: Therefore, they can serve as efficient and cost effective alternative markers in such species.Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels.Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biotechnology Division, Institute of Himalayan Bioresource Technology, (CSIR), Post Box 6, Palampur, Himachal Pradesh, 176061, India. mrk_sharma@yahoo.com

ABSTRACT

Background: Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.

Results: Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (HE) and observed (Ho) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

Conclusion: UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.

Show MeSH
Related in: MedlinePlus