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Construction and Mapping

Construction and mapping of introgression lines.

To produce the four sets of NILs, we will backcross, into the elite cultivars, BC1-derived lineages at random until any backcross lineage has only a few introgressed segments still segregating, such that one can self and expect to find progeny that contain only one introgressed segment. We will maintain a sufficient number of different backcross lineages such that the introgressed segments are expected to provide 3x coverage of the donor genome. With a genome of 4500 cM and about 175 cM per each of 26 chromosomes, a BC1-derived BC7 family is expected to segregate for an average of 4 introgressed segments of about 16 cM each, with about 1/16 individuals expected to contain one and only one introgressed segment that can be rendered homozygous by a single generation of selfing. A total of 192 BC1-derived lines are needed to provide 3x genome coverage. Both wild genotypes (Tx2094 and K101) are photoperiodic; however, to provide for meaningful comparisons of expression profiles and developmental programs at parallel stages of fiber development, we plan to allow natural selection to eliminate the genomic regions associated with photoperiodism by conducting backcrossing under temperate conditions. The BC7 generation is a realistic goal for the two intraspecific crosses. In the reciprocal Maxxa/Pima interspecifics, we know from pilot studies of similar genotypes that donor chromatin is retained at a substantially lower frequency, and the BC4 generation will likely be realistic for NIL isolation. Anticipating lower retention of introgressed chromatin, 192 BC4-derived lines will be retained here as well. By growing plants in containers in the greenhouse, we can turn about 2 generations per year, starting from BC1 plants produced prior to the start of the funding period.

Genotyping will include the following steps: (1) for the BC6 parent of the BC7 progenies (BC4 for interspecifics), we will genotype each plant at ca. 5 cM intervals to determine the portions of the donor genome that are still segregating; (2) a small subset of markers diagnostic of still-segregating chromosomal segments within each family (i.e. two markers at opposite termini of each introgressed segment) will need to be evaluated on each individual in each BC6 (or 4)-derived family; (3) once NILs are selected, each will be characterized to the limit of resolution of the reference map to define the introgressed segments as precisely as possible, anticipating that we will be able to delimit termini to approximately 1-3 cM intervals.

Important to the expedient genotyping is an enhanced cotton SSR resource. To provide genome-wide, fine-scale (ca. 1-3 cM) determination of the genomic coverage of introgressed segments, and particularly to accommodate the lower polymorphism rates associated with the intraspecific populations, we estimate that about 1 SSR per cM will be needed, or ~5,000 across the cotton genome. About 2000 are available, including ~1000 mapped by collaborator J. Yu on an interspecific RIL population, about 600 mapped by collaborator P. Chee on a new G. hirsutum x G. mustelinum population, and smaller sets mapped in France and China. Collaborators (U. Reddy, West VA; M. ur-Rahman, Pakistan) also plan to add about 1000 SSRs to the reference genetic map, from which we will choose markers, bringing the expected total to about 3,000. Accordingly, in the early part of the project while populations are being developed, we plan to enhance the reference map with ~2000 new SSRs, derived from several sources including published SSR primers that have been characterized for polymorphism rates but not genetically mapped, new SSRs from ESTs generated herein and elsewhere, and (Sanger-based) genomic sequence generated in the JGI pilot project. We have budgeted for mapping about 300 SSRs that already appear on other maps, selected to improve the resolution at which we can align our reference map to other published maps. Using improved methods, we will then be able to deduce 'consensus' maps that integrate this suite of resources into a single unified platform of widespread applicability to a host of applications, including our own. This will also foster the ability to extend meta-analysis in cotton genomics, building on our early efforts and permitting us to better utilize the results of prior studies in the interpretation of the results generated here.

We welcome your comments and suggestions.