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Development and Characterization of Genic SSR Markers from Indian Mulberry Transcriptome and Their Transferability to Related Species of Moraceae

View Article: PubMed Central - PubMed

ABSTRACT

Improving mulberry leaf production with enhanced leaf quality holds the key to sustain the ever increasing demand for silk. Adoption of modern genomic approaches for crop improvement is severely constrained by the lack of sufficient molecular markers in mulberry. Here, we report development and validation of 206 EST derived SSR markers using transcriptome data generated from leaf tissue of a drought tolerant mulberry genotype, Dudia white. Analysis of transcriptome data containing 10169 EST sequences, revealed 1469 sequences with microsatellite repeat motifs. We designed a total of 264 primers to the most appropriate repeat regions, of which 206 were locus specific. These markers were validated with 25 diverse mulberry accessions and their transferability to closely related species belonging to family Moraceae was examined. Of these markers, 189 revealed polymorphism with up to 8 allelic forms across mulberry species, genotypes and varieties with a mean of 3.5 alleles per locus. The markers also revealed higher polymorphic information content of 0.824 among the accessions. These markers effectively segregated the species and genotypes and hence, can be used for both diversity analysis and in breeding applications. Around 40% of these markers were transferable to other closely related species. Along with the other genic and genomic markers, we report a set of over 750 co-dominant markers. Using these markers we constructed the first genetic linkage map of mulberry exclusively with co-dominant markers.

No MeSH data available.


Characterization of EST-SSR and their classification based on repeat motifs.(A) Microsatellite diversity among the EST sequences (B) Microsatellite diversity among the SSR markers identified. Note: Of the 264 sequences identified to be the most appropriate, 304 repeat motifs were identified.
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pone.0162909.g001: Characterization of EST-SSR and their classification based on repeat motifs.(A) Microsatellite diversity among the EST sequences (B) Microsatellite diversity among the SSR markers identified. Note: Of the 264 sequences identified to be the most appropriate, 304 repeat motifs were identified.

Mentions: The cDNA library developed from drought stressed leaf tissues of mulberry genotype, Dudia white, contains 10190 ESTs (NCBI-SRA SRP047446). Initially, the ESTs were searched at NCBI against nr database with a stringent E-value and 10169 EST sequences were selected and analyzed for sequence redundancy. A set of 7813 unique ESTs were selected for identifying SSR motifs. Further, this set was analyzed using Mreps and Gramene to discover 1469 sequences with SSR motifs. These sequences were found to harbor 2028 repeat regions among which TNRs were found to be the most abundant accounting for 57% (1158) (Fig 1) followed by 14% of DNR and 13% TtNR motifs. Penta, Hexa and longer than hexa-nucleotide repeats (PNR, HNR and LHNRs) were relatively less frequent among the repeat motifs. However, not all these sequences or repeat motifs could be developed into useful markers. Hence, the criteria for developing scorable SSR markers such as repeat motif size and the total amplicon size were applied while identifying putative sequences. This analysis led to the discovery of 828 sequences harboring 1010 microsatellites (Table 2). These sequences were further examined using the software Primer3 to identify the possible priming regions and manually selected the most appropriate sequences to design primers.


Development and Characterization of Genic SSR Markers from Indian Mulberry Transcriptome and Their Transferability to Related Species of Moraceae
Characterization of EST-SSR and their classification based on repeat motifs.(A) Microsatellite diversity among the EST sequences (B) Microsatellite diversity among the SSR markers identified. Note: Of the 264 sequences identified to be the most appropriate, 304 repeat motifs were identified.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036888&req=5

pone.0162909.g001: Characterization of EST-SSR and their classification based on repeat motifs.(A) Microsatellite diversity among the EST sequences (B) Microsatellite diversity among the SSR markers identified. Note: Of the 264 sequences identified to be the most appropriate, 304 repeat motifs were identified.
Mentions: The cDNA library developed from drought stressed leaf tissues of mulberry genotype, Dudia white, contains 10190 ESTs (NCBI-SRA SRP047446). Initially, the ESTs were searched at NCBI against nr database with a stringent E-value and 10169 EST sequences were selected and analyzed for sequence redundancy. A set of 7813 unique ESTs were selected for identifying SSR motifs. Further, this set was analyzed using Mreps and Gramene to discover 1469 sequences with SSR motifs. These sequences were found to harbor 2028 repeat regions among which TNRs were found to be the most abundant accounting for 57% (1158) (Fig 1) followed by 14% of DNR and 13% TtNR motifs. Penta, Hexa and longer than hexa-nucleotide repeats (PNR, HNR and LHNRs) were relatively less frequent among the repeat motifs. However, not all these sequences or repeat motifs could be developed into useful markers. Hence, the criteria for developing scorable SSR markers such as repeat motif size and the total amplicon size were applied while identifying putative sequences. This analysis led to the discovery of 828 sequences harboring 1010 microsatellites (Table 2). These sequences were further examined using the software Primer3 to identify the possible priming regions and manually selected the most appropriate sequences to design primers.

View Article: PubMed Central - PubMed

ABSTRACT

Improving mulberry leaf production with enhanced leaf quality holds the key to sustain the ever increasing demand for silk. Adoption of modern genomic approaches for crop improvement is severely constrained by the lack of sufficient molecular markers in mulberry. Here, we report development and validation of 206 EST derived SSR markers using transcriptome data generated from leaf tissue of a drought tolerant mulberry genotype, Dudia white. Analysis of transcriptome data containing 10169 EST sequences, revealed 1469 sequences with microsatellite repeat motifs. We designed a total of 264 primers to the most appropriate repeat regions, of which 206 were locus specific. These markers were validated with 25 diverse mulberry accessions and their transferability to closely related species belonging to family Moraceae was examined. Of these markers, 189 revealed polymorphism with up to 8 allelic forms across mulberry species, genotypes and varieties with a mean of 3.5 alleles per locus. The markers also revealed higher polymorphic information content of 0.824 among the accessions. These markers effectively segregated the species and genotypes and hence, can be used for both diversity analysis and in breeding applications. Around 40% of these markers were transferable to other closely related species. Along with the other genic and genomic markers, we report a set of over 750 co-dominant markers. Using these markers we constructed the first genetic linkage map of mulberry exclusively with co-dominant markers.

No MeSH data available.