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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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Phylogenetic tree construction. Genetic relationships among 34 accessions of Camellia spp. based on the 61 UGMS primers identified in the present study.(a) UPGMA clustering based on Jaccard's coefficient of similarity; (b) Neighbour Joining tree based on Nei and Li distance. Tree branched with bootstrap values greater than 60% are indicated. The scale bar represents simple matching distance.
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Figure 5: Phylogenetic tree construction. Genetic relationships among 34 accessions of Camellia spp. based on the 61 UGMS primers identified in the present study.(a) UPGMA clustering based on Jaccard's coefficient of similarity; (b) Neighbour Joining tree based on Nei and Li distance. Tree branched with bootstrap values greater than 60% are indicated. The scale bar represents simple matching distance.

Mentions: The phenetic analysis of the UGMS data by two methods showed distinct groups and subgroups (Figure 5a &5b). The cluster analysis with Jaccard's similarity matrix corresponded well with the Nei and Li's matrix. Though minor changes were evident within the subclusters of the major varietal types, the relative position of the major clusters remained preserved. The neighbour joining (NJ) tree was more precise in differentiating the closely related accessions with high bootstrap values (Figure 5b). Clustering of thirty four accessions of genus Camellia into three major groups was strongly supported by high bootstrap values (≥ 90%). However, accession of C. lutescens remained isolated as a single solitary genotype with 100% bootstrap value and defined as outgroup. All the China accessions were clustered together in group I. However, two accessions namely UPASI 6 (Assam) and C-6017 (Cambod) were also clustered in this group. Majority of Assam and Cambod tea accession clustered together in group II with bootstrap values of 65%. All but one (TV-19), TV series accessions representing either Assam or Cambod also clustered together in group II. Interestingly, two accessions namely UPASI 13 and UPASI 9 known for excellent spread and are the source of good quality tea, remained together as intermediates between groups I and II. Accession 124/48/8, an extreme Cambod type with broad-elliptic leaves without distinct marginal veins with pink pigmentation at the petiole base, along with TV-19 (Cambod) clustered as an intermediate group between ornamentals and cultivated tea accessions. As expected, all the three species (C. irrawadiensis, C. lutescens, C. japonica with white and red flower) clustered separately in the present case.


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)

Phylogenetic tree construction. Genetic relationships among 34 accessions of Camellia spp. based on the 61 UGMS primers identified in the present study.(a) UPGMA clustering based on Jaccard's coefficient of similarity; (b) Neighbour Joining tree based on Nei and Li distance. Tree branched with bootstrap values greater than 60% are indicated. The scale bar represents simple matching distance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Phylogenetic tree construction. Genetic relationships among 34 accessions of Camellia spp. based on the 61 UGMS primers identified in the present study.(a) UPGMA clustering based on Jaccard's coefficient of similarity; (b) Neighbour Joining tree based on Nei and Li distance. Tree branched with bootstrap values greater than 60% are indicated. The scale bar represents simple matching distance.
Mentions: The phenetic analysis of the UGMS data by two methods showed distinct groups and subgroups (Figure 5a &5b). The cluster analysis with Jaccard's similarity matrix corresponded well with the Nei and Li's matrix. Though minor changes were evident within the subclusters of the major varietal types, the relative position of the major clusters remained preserved. The neighbour joining (NJ) tree was more precise in differentiating the closely related accessions with high bootstrap values (Figure 5b). Clustering of thirty four accessions of genus Camellia into three major groups was strongly supported by high bootstrap values (≥ 90%). However, accession of C. lutescens remained isolated as a single solitary genotype with 100% bootstrap value and defined as outgroup. All the China accessions were clustered together in group I. However, two accessions namely UPASI 6 (Assam) and C-6017 (Cambod) were also clustered in this group. Majority of Assam and Cambod tea accession clustered together in group II with bootstrap values of 65%. All but one (TV-19), TV series accessions representing either Assam or Cambod also clustered together in group II. Interestingly, two accessions namely UPASI 13 and UPASI 9 known for excellent spread and are the source of good quality tea, remained together as intermediates between groups I and II. Accession 124/48/8, an extreme Cambod type with broad-elliptic leaves without distinct marginal veins with pink pigmentation at the petiole base, along with TV-19 (Cambod) clustered as an intermediate group between ornamentals and cultivated tea accessions. As expected, all the three species (C. irrawadiensis, C. lutescens, C. japonica with white and red flower) clustered separately in the present case.

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