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

BACKGROUND: Cotton 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. RESULTS: The 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. CONCLUSIONS: Our 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.

Inheritance and QTL Mapping of Leaf Nutrient Concentration in a Cotton Inter-Specific Derived RIL Population.

Developing and deploying cotton cultivars with high nutrient uptake, use efficiency and tolerance to nutrient related soil stresses is desirable to assist sustainable soil management. Genetic variation, heritability, selection response and quantitative trait loci (QTLs) were investigated for five macronutrients (P, K, Ca, Mg, S) and five micronutrients (Fe, Mn, B, Zn, and Cu) in a recombinant inbred line (RIL) population from an inter-specific cross between Gossypium hirsutum cv. Guazuncho 2, and G. barbadense accession VH8-4602. Na and K/Na ratio were also studied as the imbalance between Na and other nutrients is detrimental to cotton growth and development. The concentrations of nutrients were measured for different plant parts of the two parents and for leaf samples of the whole population collected at early to peak flowering in field experiments over two years in a sodic Vertosol soil. Parental contrast was large for most nutrient concentrations in leaves when compared with other plant parts. Segregation for leaf nutrient concentration was observed within the population with transgression for P, K, K/Na ratio and all micronutrients. Genotypic difference was the major factor behind within-population variation for most nutrients, while narrow sense heritability was moderate (0.27 for Mn and Cu, and 0.43 for B). At least one significant QTL was identified for each nutrient except K and more than half of those QTLs were clustered on chromosomes 14, 18 and 22. Selection response was predicted to be low for P and all micronutrients except B, high for K, Na and B, and very high for K/Na ratio. Correlations were more common between macronutrients, Na and K/Na ratio where the nature and strength of the relations varied (r=-0.69 to 0.76). We conclude that there is sufficient genetic diversity between these two tetraploid cotton species that could be exploited to improve cotton nutrient status by introgressing species-unique favourable alleles.

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.

Distribution and differentiation of wild, feral, and cultivated populations of perennial upland cotton (Gossypium hirsutum L.) in Mesoamerica and the Caribbean.

Perennial forms of Gossypium hirsutum are classified under seven races. Five Mesoamerican races would have been derived from the wild race 'yucatanense' from northern Yucatán. 'Marie-Galante', the main race in the Caribbean, would have developed from introgression with G. barbadense. The racial status of coastal populations from the Caribbean has not been clearly defined. We combined Ecological Niche Modeling with an analysis of SSR marker diversity, to elucidate the relationships among cultivated, feral and wild populations of perennial cottons. Out of 954 records of occurrence in Mesoamerica and the Caribbean, 630 were classified into four categories cultivated, feral (disturbed and secondary habitats), wild/feral (protected habitats), and truly wild cotton (TWC) populations. The widely distributed three first categories cannot be differentiated on ecological grounds, indicating they mostly belong to the domesticated pool. In contrast, TWC are restricted to the driest and hottest littoral habitats, in northern Yucatán and in the Caribbean (from Venezuela to Florida), as confirmed by their climatic envelope in the factorial analysis. Extrapolating this TWC climatic model to South America and the Pacific Ocean points towards places where other wild representatives of tetraploid Gossypium species have been encountered. The genetic analysis sample comprised 42 TWC accessions from 12 sites and 68 feral accessions from 18 sites; at nine sites, wild and feral accessions were collected in close vicinity. Principal coordinate analysis, neighbor joining, and STRUCTURE consistently showed a primary divergence between TWC and feral cottons, and a secondary divergence separating 'Marie-Galante' from all other feral accessions. This strong genetic structure contrasts strikingly with the absence of geographic differentiation. Our results show that TWC populations of Mesoamerica and the Caribbean constitute a homogenous gene pool. Furthermore, the relatively low genetic divergence between the Mesoamerican and Caribbean domesticated pools supports the hypothesis of domestication of G. hirsutum in northern Yucatán.

Deep sequencing reveals differences in the transcriptional landscapes of fibers from two cultivated species of cotton.

Cotton (Gossypium) fiber is the most prevalent natural product used in the textile industry. The two major cultivated species, G. hirsutum (Gh) and G. barbadense (Gb), are allotetraploids with contrasting fiber quality properties. To better understand the molecular basis for their fiber differences, EST pyrosequencing was used to document the fiber transcriptomes at two key development stages, 10 days post anthesis (dpa), representing the peak of fiber elongation, and 22 dpa, representing the transition to secondary cell wall synthesis. The 617,000 high quality reads (89% of the total 692,000 reads) from 4 libraries were assembled into 46,072 unigenes, comprising 38,297 contigs and 7,775 singletons. Functional annotation of the unigenes together with comparative digital gene expression (DGE) revealed a diverse set of functions and processes that were partly linked to specific fiber stages. Globally, 2,770 contigs (7%) showed differential expression (>2-fold) between 10 and 22 dpa (irrespective of genotype), with 70% more highly expressed at 10 dpa, while 2,248 (6%) were differentially expressed between the genotypes (irrespective of stage). The most significant genes with differential DGE at 10 dpa included expansins and lipid transfer proteins (higher in Gb), while at 22 dpa tubulins, cellulose, and sucrose synthases showed higher expression in Gb. DGE was compared with expression data of 10 dpa-old fibers from Affymetrix microarrays. Among 543 contigs showing differential expression on both platforms, 74% were consistent in being either over-expressed in Gh (242 genes) or in Gb (161 genes). Furthermore, the unigene set served to identify 339 new SSRs and close to 21,000 inter-genotypic SNPs. Subsets of 88 SSRs and 48 SNPs were validated through mapping and added 65 new loci to a RIL genetic map. The new set of fiber ESTs and the gene-based markers complement existing available resources useful in basic and applied research for crop improvement in cotton.

A high density consensus genetic map of tetraploid cotton that integrates multiple component maps through molecular marker redundancy check.

A consensus genetic map of tetraploid cotton was constructed using six high-density maps and after the integration of a sequence-based marker redundancy check. Public cotton SSR libraries (17,343 markers) were curated for sequence redundancy using 90% as a similarity cutoff. As a result, 20% of the markers (3,410) could be considered as redundant with some other markers. The marker redundancy information had been a crucial part of the map integration process, in which the six most informative interspecific Gossypium hirsutum×G. barbadense genetic maps were used for assembling a high density consensus (HDC) map for tetraploid cotton. With redundant markers being removed, the HDC map could be constructed thanks to the sufficient number of collinear non-redundant markers in common between the component maps. The HDC map consists of 8,254 loci, originating from 6,669 markers, and spans 4,070 cM, with an average of 2 loci per cM. The HDC map presents a high rate of locus duplications, as 1,292 markers among the 6,669 were mapped in more than one locus. Two thirds of the duplications are bridging homoeologous A(T) and D(T) chromosomes constitutive of allopolyploid cotton genome, with an average of 64 duplications per A(T)/D(T) chromosome pair. Sequences of 4,744 mapped markers were used for a mutual blast alignment (BBMH) with the 13 major scaffolds of the recently released Gossypium raimondii genome indicating high level of homology between the diploid D genome and the tetraploid cotton genetic map, with only a few minor possible structural rearrangements. Overall, the HDC map will serve as a valuable resource for trait QTL comparative mapping, map-based cloning of important genes, and better understanding of the genome structure and evolution of tetraploid cotton.

cDNA-AFLP-based genetical genomics in cotton fibers.

Genetical genomics, or genetic analysis applied to gene expression data, has not been widely used in plants. We used quantitative cDNA-AFLP to monitor the variation in the expression level of cotton fiber transcripts among a population of inter-specific Gossypium hirsutum × G. barbadense recombinant inbred lines (RILs). Two key fiber developmental stages, elongation (10 days post anthesis, dpa), and secondary cell wall thickening (22 dpa), were studied. Normalized intensity ratios of 3,263 and 1,201 transcript-derived fragments (TDFs) segregating over 88 RILs were analyzed for quantitative trait loci (QTL) mapping for the 10 and 22 dpa fibers, respectively. Two-thirds of all TDFs mapped between 1 and 6 eQTLs (LOD > 3.5). Chromosome 21 had a higher density of eQTLs than other chromosomes in both data sets and, within chromosomes, hotspots of presumably trans-acting eQTLs were identified. The eQTL hotspots were compared to the location of phenotypic QTLs for fiber characteristics among the RILs, and several cases of co-localization were detected. Quantitative RT-PCR for 15 sequenced TDFs showed that 3 TDFs had at least one eQTL at a similar location to those identified by cDNA-AFLP, while 3 other TDFs mapped an eQTL at a similar location but with opposite additive effect. In conclusion, cDNA-AFLP proved to be a cost-effective and highly transferable platform for genome-wide and population-wide gene expression profiling. Because TDFs are anonymous, further validation and interpretation (in silico analysis, qPCR gene profiling) of the eQTL and eQTL hotspots will be facilitated by the increasing availability of cDNA and genomic sequence resources in cotton.

Meta-analysis of cotton fiber quality QTLs across diverse environments in a Gossypium hirsutum x G. barbadense RIL population.

BACKGROUND: Cotton fibers (produced by Gossypium species) are the premier natural fibers for textile production. The two tetraploid species, G. barbadense (Gb) and G. hirsutum (Gh), differ significantly in their fiber properties, the former having much longer, finer and stronger fibers that are highly prized. A better understanding of the genetics and underlying biological causes of these differences will aid further improvement of cotton quality through breeding and biotechnology. We evaluated an inter-specific Gh x Gb recombinant inbred line (RIL) population for fiber characteristics in 11 independent experiments under field and glasshouse conditions. Sites were located on 4 continents and 5 countries and some locations were analyzed over multiple years. RESULTS: The RIL population displayed a large variability for all major fiber traits. QTL analyses were performed on a per-site basis by composite interval mapping. Among the 651 putative QTLs (LOD > 2), 167 had a LOD exceeding permutation based thresholds. Coincidence in QTL location across data sets was assessed for the fiber trait categories strength, elongation, length, length uniformity, fineness/maturity, and color. A meta-analysis of more than a thousand putative QTLs was conducted with MetaQTL software to integrate QTL data from the RIL and 3 backcross populations (from the same parents) and to compare them with the literature. Although the global level of congruence across experiments and populations was generally moderate, the QTL clustering was possible for 30 trait x chromosome combinations (5 traits in 19 different chromosomes) where an effective co-localization of unidirectional (similar sign of additivity) QTLs from at least 5 different data sets was observed. Most consistent meta-clusters were identified for fiber color on chromosomes c6, c8 and c25, fineness on c15, and fiber length on c3. CONCLUSIONS: Meta-analysis provided a reliable means of integrating phenotypic and genetic mapping data across multiple populations and environments for complex fiber traits. The consistent chromosomal regions contributing to fiber quality traits constitute good candidates for the further dissection of the genetic and genomic factors underlying important fiber characteristics, and for marker-assisted selection.

A new interspecific, Gossypium hirsutum x G. barbadense, RIL population: towards a unified consensus linkage map of tetraploid cotton.

We report the development of a new interspecific cotton recombinant inbred line (RIL) population of 140 lines deriving from an interspecific cross between Gossypium hirsutum (Gh) and G. barbadense (Gb), using the same two parents that have served for the construction of a BC(1) map and for the marker-assisted backcross selection program underway at CIRAD. Two marker systems, microsatellites and AFLPs, were used. An important feature of the RIL population was its marked segregation distortion with a genome-wide bias to Gh alleles (parental genome ratio is 71/29). The RIL map displays an excellent colinearity with the BC(1) map, although it is severely contracted in terms of map size. Existence of 255 loci in common (between 6 and 14 per chromosome) allowed the integration of the two data sets. A consensus BC(1)-RIL map based upon 215 individuals (75 BC1 + 140 RIL) was built. It consisted of 1,745 loci, spanned 3,637 cM, intermediate between the sizes of the two component maps, and constituted a solid framework to cross align cotton maps using common markers. The new RIL population will be further exploited for fiber property QTL mapping and eQTL mapping.

CMD: a Cotton Microsatellite Database resource for Gossypium genomics.

BACKGROUND: The Cotton Microsatellite Database (CMD) http://www.cottonssr.org is a curated and integrated web-based relational database providing centralized access to publicly available cotton microsatellites, an invaluable resource for basic and applied research in cotton breeding. DESCRIPTION: At present CMD contains publication, sequence, primer, mapping and homology data for nine major cotton microsatellite projects, collectively representing 5,484 microsatellites. In addition, CMD displays data for three of the microsatellite projects that have been screened against a panel of core germplasm. The standardized panel consists of 12 diverse genotypes including genetic standards, mapping parents, BAC donors, subgenome representatives, unique breeding lines, exotic introgression sources, and contemporary Upland cottons with significant acreage. A suite of online microsatellite data mining tools are accessible at CMD. These include an SSR server which identifies microsatellites, primers, open reading frames, and GC-content of uploaded sequences; BLAST and FASTA servers providing sequence similarity searches against the existing cotton SSR sequences and primers, a CAP3 server to assemble EST sequences into longer transcripts prior to mining for SSRs, and CMap, a viewer for comparing cotton SSR maps. CONCLUSION: The collection of publicly available cotton SSR markers in a centralized, readily accessible and curated web-enabled database provides a more efficient utilization of microsatellite resources and will help accelerate basic and applied research in molecular breeding and genetic mapping in Gossypium spp.