Location: Genomics and Bioinformatics Research2012 Annual Report
1a. Objectives (from AD-416):
Collaborate with ARS researchers to provide germplasm for Cotton Leaf Curl Virus (CLCuV) screening and transfer resistance identified into adapted cotton lines. Transferring traits from diploid to cultivated tetraploid cotton is a difficult process. ARS at Stoneville, MS, and Texas A&M University, College Station, TX, are the two locations in the U.S. successfully transferring genes from diploid lines into tetraploid cotton. These groups will cooperate to transfer the resistance genes as quickly and efficiently as possible. The lines will be advanced more rapidly by using DNA markers to verify successful transfer of the resistance gene(s) in each generation of the introgression process. The efficiency will be improved by cytogenetic analyses to confirm the lines produced are cytogenetically stable with a complete set of chromosomes.
1b. Approach (from AD-416):
The university collaborator will work with ARS to increase seed of exotic diploid species and provide technical support for increase of germplasm and preparation of seed for CLCuV resistance screening. Lines identified as resistant will be used as donor parents in crosses to transfer the resistant trait (genes) into adapted cotton lines. The transfer of the trait will be tracked using DNA markers and resistant progeny from the crosses will be cytogenetically evaluated to select lines with a complete set of chromosomes and no chromosomal abnormalities.
3. Progress Report:
We received a visiting scientist from Pakistan in mid-April, and initiated exchange and training at that time. He has participated in all aspects of our program, from greenhouse, to field, to data handling, to molecular analysis. He also visited the National Cotton Germplasm Collection, met with the curator and saw the low-temperature/low humidity storage facilities and living plant collections. He participated exceptionally well in all lab-wide activities, including lab meetings, so his exposure to our science and culture is extensive. In June, he traveled to the Stoneville site for several days of additional experiences. We also sequenced a test set of cDNAs of two alien species (G. longicalyx) introgression products to see if we can readily localize alien sequences that might be of interest for single nucleotide polymorphism (SNP) development for marker-assisted selection and alien gene localization. If so, this approach could be expended to related early generation partially introgressed germplasm that contains relatively large amount of genes from a Cotton Leaf Curl Virus (CLCuV)-resistant species such as G. longicalyx and number of other diploid species. Early-generation introgression products, on average and overall, contain more comprehensive coverage of an alien genome, and in this case, are more likely to contain genes (or gene combinations) that confer resistance to CLCuV. Given that our pilot cDNA sequencing runs above showed that our methods of normalization and sequencing library preparation were successful, remaining genotypes for which samples were previously prepared but not submitted for sequencing, pending results with the initial pilot set, were queued for normalized cDNA sequencing (sequencing runs are expected sometime over the summer 2012). We planted 100+ seed for each of two tetraploid mapping tri-species populations involving a synthetic tetraploid A2D1 hybrid created from gossypiom (G.) arboreum (2n=2x=26; A2-genome) and G. thurberi (2n=2x=26; D1-genome)) and G. hirsutum line TM1. Given resistance or non-host characteristics of G. arboreum, the synthetic b- and tri-species hybrids are expectedly CLCuV-resistant, and their progeny will segregate. Given the "distance" between the genomes and the presence of a complex ancestral chromosome translocation, we expect to observe relative rates of recombination and recovery of recombinants will differ considerably among regions of the A-genome. Nonetheless, the observed segregations will provide a guide as to the rates that we can expect in other crosses involving A2 genomes that have been or are being introgressed. The two seed populations involve self and backcross progeny: [A2D1xTM1]F1S1 seed and [TM1*(TM1*A2D1)]BC1F1 seed. In the following quarter, we will attempt to germinate these seed and establish mapping populations of plants, from which we can collect samples for DNA extraction, genotyping and subsequent breeding. We planted seed from backcrossed tetraploid Gossypium hirsutum germplasm that contains varying amounts of G. longicalyx germplasm. The germplasm was derived by introgression via a synthetic tri-species hybrid (G. longicalyx, G. armourianum, and G. hirsutum), analogous to the above. The African wild species G. longicalyx is also resistant to CLCuV, according to previous tests conducted in Pakistan. So that we can screen this germplasm in Pakistan next year, we expect to increase some of it here this summer in a low-outcrossing environment, and try to establish an Material Transfer Agreement (MTA) for handling the germplasm, values of which could be diverse, and present a further opportunity for collaboration as a bi-product of our main goal, namely CLCuV-resistant G. hirsutum lines. Because some of our in-hand materials are complex populations at moderately advanced stages of backcrossing, they could provide a quick road to success, if they harbor CLCuV-resistance. We planted seed from BC2 and BC4 introgression levels for single-plant increases.