2013 Annual Report
(2) A comparison of G. hirsutum (AtDt) loci with corresponding loci in the progenitor diploid G. herbaceum (A) and G. raimondii (D) genomes revealed that soon after formation of the AtDt tetraploid, unidirectional DNA exchanges between homeologous chromosomes were the predominant mutational type, far outnumbering random mutations. At to Dt conversion, creating four copies of the Dt allele, is far more abundant than Dt to At conversion. Additionally, At to Dt conversions are more common in heterochromatin and closely associated with GC content and transposon distribution. Dt to At conversion is abundant in euchromatin and in genes, frequently reversing losses of gene function. Eventually, unidirectional exchanges between homeologs subsided, and random mutation became the predominant mutation type. A manuscript describing these findings has been submitted to GENOME RESEARCH.
(3) ARS scientists are producing draft assemblies of various diploid cotton species using both ab initio and reference guided assembly approaches. The reference guided approach, in which sequences are assembled based on comparison with the G. raimondii genome sequence, will permit high resolution comparisons between genomes. Alignment of ab initio assemblies with reference guided assemblies should permit detection of chromosomal rearrangements between species; such rearrangements would not necessarily be discovered by examination of reference guided assemblies alone.
(4) Alignment of the various Gossypium genome sequences with the G. raimondii reference sequence has allowed identification of single-nucleotide polymorphisms (SNPs). SNPs are useful molecular markers permitting high resolution genetic mapping.
(5) The repetitive DNA contents of the various Gossypium genomes are being compared as a means of exploring mechanisms underlying Gossypium genome divergence. With regard to the AtDt tetraploids, comparison of repeat sequence contents is providing insight into the effects of allopolyploidy and domestication on repeat contents.
(6) We have continued work on the upland cotton pest, Rotylenchulus (R.) reniformis (reniform nematode). The R. renifomis work has involved close collaboration between this ARS group and the USDA-ARS Precision Agriculture Unit at Mississippi State University. For instance, we have constructed a high quality transcriptome for reniform nematode and obtained gene expression data from the various life stages of the worm. A manuscript describing the transcriptome is near completion, while a manuscript on differential gene expression is in progress.
(7) We conducted a small proteomics study in which R. reniformis peptides were queried against protein databases for the model nematode Caenorhabditis elegans, the root knot nematode (Meloidogyne hapla), and cDNA/gene assemblies for R. reniformis. This has allowed us to identify candidate parasitism genes. A manuscript describing this work is in progress.
(8) We used flow cytometry to estimate the genome size of R. reniformis and found its genome size to be considerably larger than previous estimates. M. hapla and C. elegans nuclei were used among the control species utilized. A manuscript re-evaluating genome size estimates in parasitic nematodes is in progress.
(9) An earlier attempt at producing a genome sequence for R. reniformis yielded unsatisfactory results. Of note, the DNA used in sequencing was obtained from a pool of thousands of individual worms. Heterogeneity in R. renformis is extensive, and thus assembly of a quality genome from high coverage sequencing proved disappointing. However, ARS scientists subsequently isolated DNA from a single R. reniformis egg. The DNA was amplified and relatively low depth sequencing was conducted. Not surprisingly, the assemblies from the single egg were orders of magnitude better than the assemblies from the heterogenous population of R. reniformis. Additional sequencing of amplified DNA from the single egg is currently underway.