James Frelichowski

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Genetic diversity of the two commercial tetraploid cotton species in the Gossypium Diversity Reference Set

A diversity reference set has been constructed for the Gossypium accessions in the US National Cotton Germplasm Collection to facilitate more extensive evaluation and utilization of accessions held in the Collection. A set of 105 mapped simple sequence repeat markers was used to study the allelic diversity of 1933 tetraploid Gossypium accessions representative of the range of diversity of the improved and wild accessions of G. hirsutum and G. barbadense. The reference set contained 410 G. barbadense accessions and 1523 G. hirsutum accessions. Observed numbers of polymorphic and private bands indicated a greater diversity in G. hirsutum as compared to G. barbadense as well as in wild-type accessions as compared to improved accessions in both species. The markers clearly differentiated the 2 species. Patterns of diversity within species were observed but not clearly delineated, with much overlap occurring between races and regions of origin for wild accessions and between historical and geographic breeding pools for cultivated accessions. Although the percentage of accessions showing introgression was higher among wild accessions than cultivars in both species, the average level of introgression within individual accessions, as indicated by species-specific bands, was much higher in wild accessions of G. hirsutum than in wild accessions of G. barbadense. The average level of introgression within individual accessions was higher in improved G. barbadense cultivars than in G. hirsutum cultivars. This molecular characterization reveals the levels and distributions of genetic diversity that will allow for better exploration and utilization of cotton genetic resources.

Diversity analysis of cotton (Gossypium hirsutum L.) germplasm using the CottonSNP63K Array

BackgroundCotton germplasm resources contain beneficial alleles that can be exploited to develop germplasm adapted to emerging environmental and climate conditions. Accessions and lines have traditionally been characterized based on phenotypes, but phenotypic profiles are limited by the cost, time, and space required to make visual observations and measurements. With advances in molecular genetic methods, genotypic profiles are increasingly able to identify differences among accessions due to the larger number of genetic markers that can be measured. A combination of both methods would greatly enhance our ability to characterize germplasm resources. Recent efforts have culminated in the identification of sufficient SNP markers to establish high-throughput genotyping systems, such as the CottonSNP63K array, which enables a researcher to efficiently analyze large numbers of SNP markers and obtain highly repeatable results. In the current investigation, we have utilized the SNP array for analyzing genetic diversity primarily among cotton cultivars, making comparisons to SSR-based phylogenetic analyses, and identifying loci associated with seed nutritional traits.ResultsThe SNP markers distinctly separated G. hirsutum from other Gossypium species and distinguished the wild from cultivated types of G. hirsutum. The markers also efficiently discerned differences among cultivars, which was the primary goal when designing the CottonSNP63K array. Population structure within the genus compared favorably with previous results obtained using SSR markers, and an association study identified loci linked to factors that affect cottonseed protein content.ConclusionsOur results provide a large genome-wide variation data set for primarily cultivated cotton. Thousands of SNPs in representative cotton genotypes provide an opportunity to finely discriminate among cultivated cotton from around the world. The SNPs will be relevant as dense markers of genome variation for association mapping approaches aimed at correlating molecular polymorphisms with variation in phenotypic traits, as well as for molecular breeding approaches in cotton.

Genome-wide divergence, haplotype distribution and population demographic histories for Gossypium hirsutum and Gossypium barbadense as revealed by genome-anchored SNPs

Use of 10,129 singleton SNPs of known genomic location in tetraploid cotton provided unique opportunities to characterize genome-wide diversity among 440 Gossypium hirsutum and 219 G. barbadense cultivars and landrace accessions of widespread origin. Using the SNPs distributed genome-wide, we examined genetic diversity, haplotype distribution and linkage disequilibrium patterns in the G. hirsutum and G. barbadense genomes to clarify population demographic history. Diversity and identity-by-state analyses have revealed little sharing of alleles between the two cultivated allotetraploid genomes, with a few exceptions that indicated sporadic gene flow. We found a high number of new alleles, representing increased nucleotide diversity, on chromosomes 1 and 2 in cultivated G. hirsutum as compared with low nucleotide diversity on these chromosomes in landrace G. hirsutum. In contrast, G. barbadense chromosomes showed negative Tajima’s D on several chromosomes for both cultivated and landrace types, which indicate that speciation of G. barbadense itself, might have occurred with relatively narrow genetic diversity. The presence of conserved linkage disequilibrium (LD) blocks and haplotypes between G. hirsutum and G. barbadense provides strong evidence for comparable patterns of evolution in their domestication processes. Our study illustrates the potential use of population genetic techniques to identify genomic regions for domestication.

The U.S. National Cotton Germplasm Collection – Its Contents, Preservation, Characterization, and Evaluation

The early history of the cotton industry in the U.S. was, for the most part, a story of importation and adaptation of cottons from Mexico, Guatemala, and the tropics of the Western Hemi‐ sphere. By the 19th century there were two distinctive types of upland cotton, with distinctive origins, being grown in the U.S. One type, known as green-seeded cotton, was from southern Mexico. The other dominant type of upland, white-seeded cotton, had its origins in the central plateau of Mexico [1,2]. Although these cottons were grown extensively, no coordinated efforts were made to maintain the original stocks or their progeny. Modern, systematic collection and preservation of cotton in the U.S. only began in response to the outbreak of the boll weevil in the 1880’s [3]. Three collection trips between 1902 and 1906 were responsible for the introduc‐ tion of two cottons, Acala and Kekchi, which would contribute significantly to the develop‐ ment of modern U.S. cultivars [4]. From these initial collecting trips and ensuing trips, federal, university and state experiment stations began to assemble the germplasm collections. Also from these efforts there sprang collaborative efforts that were formalized into U.S. regional projects involving scientists and members from all aspects of the U.S. cotton industry. The first Regional Research Project was S-1 in 1950 (succeeded by many) and it established a priority of acquiring and studying diverse germplasm for cotton improvement [5-7]. Increased organization and centralization of germplasm activities was necessary to manage and increase

Gossypolhemiquinone, a dimeric sesquiterpenoid identified in cotton (Gossypium).

The report that the cotton leaf perforator, Bucculatrix thurberiella, is one of the few insect herbivores to attack Gossypium thurberi prompted an investigation of the terpenoids present in the leaves of this wild species of cotton. Members of Gossypium produce subepidermal pigment glands in their leaves that contain the dimeric sesquiterpenoid gossypol as well as other biosynthetically related terpenoids. In addition to gossypol, a previously unknown dimeric sesquiterpenoid, gossypolhemiquinone (GHQ), was identified in trace amounts in G. thurberi, a member of the D genome. Other members of the D genome of Gossypium were subsequently found to contain this compound, but GHQ was not detected in commercial cotton cultivars. When fed to Helicoverpa zea in an artificial diet, GHQ delayed days-to-pupation, reduced pupal weights, and survival to adulthood to a lesser or equal extent than gossypol in comparison to the control diet. However, GHQ had a synergistic effect on survival and days-to-pupation when combined with gossypol at the highest dosage tested (0.18%; 15.5:84.5 GHQ:gossypol). Because gossypol exhibits anti-cancer activity, GHQ was also evaluated for its anti-cancer activity against the National Cancer Institutes 60-Human Tumor Cell Line Screen. Significant inhibitory activity against most of these cell lines was not observed, but the results may offer some promise against leukemia cancer cell lines.