Project Summaries

12-183  Project Manager: D. C. Jones


Lori Hinze, USDA-ARS

SSR marker data is available from a pilot study of 96 diverse accessions and a larger study of ~2,200 representative accessions from the U.S. National Cotton Germplasm Collection (NCGC).  These studies included a range of tetraploid and diploid species. The SSRs used in these previous studies were a core set of 105 markers distributed as two markers per chromosome arm on all 26 chromosomes of the cotton tetraploid genome.  From these preliminary analyses, we observed that many of the SSRs did not amplify a PCR product in the diploid accessions.  In addition, those SSRs that did amplify in diploid accessions did not show as much variation as the same SSRs revealed in the tetraploid accessions.  Many of the SSRs used were developed from the tetraploid species, and as such, are inherently optimized to identify polymorphisms among tetraploid accessions.  The diploid species have evolved separately and likely have unique polymorphisms that cannot be detected by markers developed in tetraploid species.  To that end, G. arboreum was identified as a diploid species of present interest to the cotton community (particularly in the evaluation of the cotton leaf curl virus in Pakistan) that would benefit from further characterization with SSR markers. The SSR markers selected for the current proposal will include approximately half of those in the core set since the diploid genome would contain only half of the chromosomes of the tetraploid genome. The levels of polymorphism of this smaller group of markers within G. arboreum will be evaluated, and, if necessary, additional markers will be identified that are putatively more informative for discriminating among G. arboreum accessions.

The objectives of this project include examining the patterns or structure of geographic and race relationships and diversity within the A-genome diploid cotton species, Gossypium arboreum (A2); discovering the transferability of G. hirsutum derived SSRs to the G. arboreum diploid species; and revealing sources of unique molecular variation within G. arboreum. These objectives are in reference to G. arboreum as it is represented in the U.S. National Cotton Germplasm Collection.

A set of ~200 SSRs potentially having polymorphism among G. arboreum accessions were identified.  These SSRs were selected from published studies that characterized small subsets of G. arboreum accessions in the U.S. NCGC.  SSRs were also selected from a mapping study in a G. arboreum F2 population and from an unpublished study using SSR markers to distinguish G. arboreum germplasm in a Chinese germplasm collection. All SSRs are putatively single locus, have referenced map positions on chromosomes 1-13, and have primer sequences available in public databases (i.e., CottonGen).

Approximately 760 G. arboreum accessions were chosen for genotyping based on their passport data (i.e. including a range of cultivars from research institutions in different countries and non-cultivated race types). For all accessions, water agar media was used to germinate seeds until root tips ~1cm in length emerged. Ten of these root tips per accession were bulked and used to obtain a representative DNA sample.
A preliminary molecular analysis of 145 G. arboreum accessions showed somewhat distinct clusters of accessions from India, China, and Russia. To capitalize on this apparent diversity, accessions were chosen from each of these groups along with representatives from Pakistan and Uzbekistan to form a panel for screening the previously identified 200 SSRs for polymorphism. The diversity panel included seven G. arboreum accessions and the G. hirsutum genetic standard, TM-1. The diversity panel was screened with 72 SSR markers and this data is in the process of being reviewed to determine SSR polymorphism.

The G. arboreum diversity panel will be used for continued screening of SSR markers to identify polymorphic SSRs for large-scale genotyping. A minimum of 52 polymorphic SSRs that are single locus, easy to amplify, and distributed four per chromosome in G. arboreum will be identified. From these primers, multiplex sets will be created to allow for more efficient PCR and electrophoresis procedures. The 760 previously selected G. arboreum accessions will be genotyped with 52 SSR markers, results will be analyzed, and findings will be reported in a peer-reviewed manuscript.


Project Year: 2012

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