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Title: Mining and validating grape (Vitis L.) ESTs to develop EST-SSR markers for genotyping and mapping

Author
item HUANG, HONG - FLORIDA A&M UNIVERSITY
item LU, JIANG - FLORIDA A&M UNIVERSITY
item REN, ZHONGBO - FLORIDA A&M UNIVERSITY
item Hunter, Wayne
item DOWD, SCOT - Spiro Stat Technologies
item Dang, Phat

Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/19/2010
Publication Date: 8/1/2011
Citation: Huang, H., Lu, J., Ren, Z., Hunter, W.B., Dowd, S., Dang, P.M. 2011. Mining and validating grape (Vitis L.) ESTs to develop EST-SSR markers for genotyping and mapping. Molecular Breeding. 28(2):241-254.

Interpretive Summary: Ninety-six new primer pairs, used to identify disease resistance in grapes were created. The newly identified EST-derived SSRs are being used to conduct analyses of grape functional diversity, grape breeding, and in the development of grape genetic maps. The EST-SSR genetic markers are very useful in screening grape plants quickly to see if they contain disease resistant genes. Vitis shuttleworthii grape has been using extensively in several grape breeding programs for improving disease resistance. To develop genetic markers for marker-assisted grape breeding and genome typing, we identified new markers by using a method to identify Simple Sequence Repeats, SSR’s, within grape sequences. A total of 6, 447 ESTs containing SSRs were identified out of 215,609 available grape sequences. These included 194,200 sequences from Vitis vinifera, 10,704 from Vitis shuttleworthii, 2,177 from Vitis aestivalis, 6,533 from Vitis hybrids (Vitis rupestris 'A. de Serres' x V. spp. 'b42-26'), and 1,995 Vitis riparia. The SSR frequency and distribution analysis showed that EST-derived SSRs were present in about 3 % of the total investigated grape sequences. The dominant repeat types were the trinucleotides (AGG/CCT and AAG/CCT) followed by the dinucleotide (AG/CT). All the identified SSRs were annotated by BLASTX analyses which produced 575 markers with similarities known proteins. Ninety-six of these were directly linked to disease resistance and were selected for further evaluation to conduct comparisons between eight grape varieties/species. These genetic markers are very useful in screening plants quickly to see if they contain disease resistance genes while they are still seedlings, before they are planted out into the fields. This saves time and money in the production of new grape varieties.

Technical Abstract: Ninety-six new primer pairs, used to identify disease resistance in grapes were created. The newly identified EST-derived SSRs are being used to conduct analyses of grape functional diversity and in the development of grape SSR-EST genetic and physical maps. A pipeline including several computational tools was used to analyze the simple sequence repeats (SSRs) frequency, distribution and diversity in grape expressed sequence tags (ESTs) retrieved from NCBI. A total of 6, 447 ESTs containing SSRs were identified from out of 215,609 grape ESTs. These included 194,200 from Vitis vinifera, 10,704 from Vitis shuttleworthii, 2,177 from Vitis aestivalis, 6,533 from Vitis hybrids (Vitis rupestris 'A. de Serres' x V. spp. 'b42-26'), and 1,995 Vitis riparia. The SSR frequency and distribution analysis disclosed that EST-derived SSRs were present in about 3 % of the total investigated grape ESTs. The dominant repeat types were the trinucleotides (AGG/CCT and AAG/CCT) followed by the dinucleotide (AG/CT). Different cultivar-based non-redundant ESTs from Vitis vinifera comparison indicated that Chardonnay is highly abundant in the AG/TC repeat representing 19.5% of total Chardonnay SSRs and the AG/CT repeat from Cabernet represents up to 25% of the total Cabernet SSRs. Trinucleotide repeats are the dominant SSR type among all Vitis species making up 45 to 55 % of their SSRs. The 6,447 EST-SSR were reduced to 1,701 non redundant sequence sets post assebly. All EST-derived SSRs were annotated by BLASTX which produced 575 of 1,701 sequences with homologous to known proteins. Ninety-six pairs of primers developed from those SSR - ESTs were linked to putative disease resistance and were selected for polymorphisms observed during comparison among eight grape varieties/species. The primers present in high polymorphisms identified homozygous and heterozygous genotypes. These newly identified EST-derived SSRs can be used for analysis of grape functional diversity and development of grape SSR-EST genetic and physical maps.