Mauricio Ulloa

Toward Sequencing Cotton (Gossypium) Genomes

Despite rapidly decreasing costs and innovative technologies, sequencing of angiosperm genomes is not yet undertaken lightly. Generating larger amounts of sequence data more quickly does not address the difficulties of sequencing and assembling complex genomes de novo. The cotton ( Gossypium spp.)

CMD: a Cotton Microsatellite Database resource for Gossypium genomics

BackgroundThe 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.DescriptionAt 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.ConclusionThe 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.

RFLP genetic linkage maps from four F2.3 populations and a joinmap of Gossypium hirsutum L.

Abstract An RFLP genetic linkage joinmap was constructed from four different mapping populations of cotton (Gossypium hirsutum L.). Genetic maps from two of the four populations have been previously reported. The third genetic map was constructed from 199 bulk-sampled plots of an F2.3 (HQ95–6×’MD51ne’) population. The map comprises 83 loci mapped to 24 linkage groups with an average distance between markers of 10.0 centiMorgan (cM), covering 830.1 cM or approximately 18% of the genome. The fourth genetic map was developed from 155 bulk-sampled plots of an F2.3 (119– 5 sub-okra×’MD51ne’) population. This map comprises 56 loci mapped to 16 linkage groups with an average distance between markers of 9.3 cM, covering 520.4 cM or approximately 11% of the cotton genome. A core of 104 cDNA probes was shared between populations, yielding 111 RFLP loci. The constructed genetic linkage joinmap from the above four populations comprises 284 loci mapped to 47 linkage groups with the average distance between markers of 5.3 cM, covering 1,502.6 cM or approximately 31% of the total recombinational length of the cotton genome. The linkage groups contained from 2 to 54 loci each and ranged in distance from 1.0 to 142.6 cM. The joinmap provided further knowledge of competitive chromosome arrangement, parental relationships, gene order, and increased the potential to map genes for the improvement of the cotton crop. This is the first genetic linkage joinmap assembled in G. hirsutum with a core of RFLP markers assayed on different genetic backgrounds of cotton populations (Acala, Delta, and Texas plain). Research is ongoing for the identification of quantitative trait loci for agronomic, physiological and fiber quality traits on these maps, and the identification of RFLP loci lineage for G. hirsutum from its diploid progenitors (the A and D genomes).

A High-Density Simple Sequence Repeat and Single Nucleotide Polymorphism Genetic Map of the Tetraploid Cotton Genome

Genetic linkage maps play fundamental roles in understanding genome structure, explaining genome formation events during evolution, and discovering the genetic bases of important traits. A high-density cotton (Gossypium spp.) genetic map was developed using representative sets of simple sequence repeat (SSR) and the first public set of single nucleotide polymorphism (SNP) markers to genotype 186 recombinant inbred lines (RILs) derived from an interspecific cross between Gossypium hirsutum L. (TM-1) and G. barbadense L. (3-79). The genetic map comprised 2072 loci (1825 SSRs and 247 SNPs) and covered 3380 centiMorgan (cM) of the cotton genome (AD) with an average marker interval of 1.63 cM. The allotetraploid cotton genome produced equivalent recombination frequencies in its two subgenomes (At and Dt). Of the 2072 loci, 1138 (54.9%) were mapped to 13 At-subgenome chromosomes, covering 1726.8 cM (51.1%), and 934 (45.1%) mapped to 13 Dt-subgenome chromosomes, covering 1653.1 cM (48.9%). The genetically smallest homeologous chromosome pair was Chr. 04 (A04) and 22 (D04), and the largest was Chr. 05 (A05) and 19 (D05). Duplicate loci between and within homeologous chromosomes were identified that facilitate investigations of chromosome translocations. The map augments evidence of reciprocal rearrangement between ancestral forms of Chr. 02 and 03 versus segmental homeologs 14 and 17 as centromeric regions show homeologous between Chr. 02 (A02) and 17 (D02), as well as between Chr. 03 (A03) and 14 (D03). This research represents an important foundation for studies on polyploid cottons, including germplasm characterization, gene discovery, and genome sequence assembly.

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.

Cotton genetic resources in the western states of Mexico: in situ conservation status and germplasm collection for ex situ preservation.

The Gossypium hirsutum gene pool from Mexico is one of the primary sources for improvement of most of the Acala and Upland cotton growing in the world today. Mexico is also the center of diversity of the Gossypium genus with 11 of the 13 known diploid Gossypium species of the Western Hemisphere endemic to its boundaries. In 2002 and 2003, the current status of these important genetic resources was surveyed, and germplasm was collected in the western states of Mexico. Information was collected to verify current status and circumscription of the endemic Gossypium species. Sixty years after the first in-depth studies of Gossypium in Mexico, increasing human population, modernization and urbanization have severely reduced the survival of G. hirsutum landraces. Representatives of cotton landraces evidently survive only as curiosities in garden plots or dooryards, or as occasional feral plants. Populations of seven known species, G. aridum, G. barbadense, G. gossypioides, G. hirsutum, G. laxum, G. lobatum, and G. schwendimanii, and one undescribed wild diploid Gossypium taxon were located during the survey. In situ conservation of some of these species is threatened. Samples of the collected germplasm were deposited in the Cotton Collection of the US National Plant Germplasm System where they will be available to scientists worldwide for research, breeding, and education. Additionally, a Gossypium species nursery is being established by the Mexican government for the preservation, and as a working legacy, of this resource. Knowledge of the diversity and, consequently, utilization of the genetic resources in these species cannot be fully realized in situ under current conditions.

QTL analysis for transgressive resistance to root-knot nematode in interspecific cotton (Gossypium spp.) progeny derived from susceptible parents.

The southern root-knot nematode (RKN, Meloidogyne incognita) is a major soil-inhabiting plant parasite that causes significant yield losses in cotton (Gossypium spp.). Progeny from crosses between cotton genotypes susceptible to RKN produced segregants in subsequent populations which were highly resistant to this parasite. A recombinant inbred line (RIL) population of 138 lines developed from a cross between Upland cotton TM-1 (G. hirsutum L.) and Pima 3–79 (G. barbadense L.), both susceptible to RKN, was used to identify quantitative trait loci (QTLs) determining responses to RKN in greenhouse infection assays with simple sequence repeat (SSR) markers. Compared to both parents, 53.6% and 52.1% of RILs showed less (P<0.05) root-galling index (GI) and had lower (P<0.05) nematode egg production (eggs per gram root, EGR). Highly resistant lines (transgressive segregants) were identified in this RIL population for GI and/or EGR in two greenhouse experiments. QTLs were identified using the single-marker analysis nonparametric mapping Kruskal-Wallis test. Four major QTLs located on chromosomes 3, 4, 11, and 17 were identified to account for 8.0 to 12.3% of the phenotypic variance (R2) in root-galling. Two major QTLs accounting for 9.7% and 10.6% of EGR variance were identified on chromosomes 14 and 23 (P<0.005), respectively. In addition, 19 putative QTLs (P<0.05) accounted for 4.5–7.7% of phenotypic variance (R2) in GI, and 15 QTLs accounted for 4.2–7.3% of phenotypic variance in EGR. In lines with alleles positive for resistance contributed by both parents in combinations of two to four QTLs, dramatic reductions of >50% in both GI and EGR were observed. The transgressive segregants with epistatic effects derived from susceptible parents indicate that high levels of nematode resistance in cotton may be attained by pyramiding positive alleles using a QTL mapping approach.

Development of a core set of SSR markers for the characterization of Gossypium germplasm

Molecular markers such as simple sequence repeats (SSR) are a useful tool for characterizing genetic diversity of Gossypium germplasm. Genetic profiles by DNA fingerprinting of cotton accessions can only be compared among different collections if a common set of molecular markers are used by different laboratories and/or research projects. Herein, we propose and report a core set of 105 SSR markers with wide genome coverage of at least four evenly distributed markers per chromosome for the 26 tetraploid cotton chromosomes. The core marker set represents the efforts of ten research groups involved in marker development, and have been systematically evaluated for DNA polymorphism on the 12 genotypes belonging to six Gossypium species [known collectively as the cotton marker database (CMD) panel]. A total of 35 marker bins in triplex sets were arranged from the 105 markers that were each labeled with one of the three fluorescent dyes (FAM, HEX, and NED). Results from this study indicated that the core marker set was robust in revealing DNA polymorphism either between and within species. Average value of polymorphism information content (PIC) among the CMD panel was 0.65, and that within the cultivated cotton species Gossypium hirsutum was 0.29. Based on the similarity matrix and phylogenetic analysis of the CMD panel, the core marker set appeared to be sufficient in characterizing the diversity within G. hirsutum and other Gossypium species. The portability of this core marker set would facilitate the systematic characterization and the simultaneous comparison among various research efforts involved in genetic diversity analysis and germplasm resource preservation.

Status of the USA cotton germplasm collection and crop vulnerability.

The National Plant Germplasm System (NPGS) is a cooperative effort among State, Federal and Private organizations aimed at preserving one of agriculture’s greatest assets: plant genetic diversity. The NPGS serves the scientific community by collecting, storing, and distributing germplasm as well as maintaining a searchable database of trait descriptors. Serving the NPGS, a Crop Germplasm Committee (CGC) is elected for each crop and is comprised of a group of scientists concerned with development, maintenance, characterization, and utilization of germplasm collections. Each CGC serves in an advisory role and provides a status report every seven years to determine scientific efforts, adequacy of germplasm base representation, and progress in breeding through utilization of germplasm. In addition, each committee can call attention to areas of concerns regarding facilities and staffing associated with the maintenance, collection, and taxonomic activities for a specific crop within the system. The following report was developed by the CGC for cotton and provides a record of collections, activities, concerns, crop vulnerabilities, and recommendations associated with the cotton collection for the period 1997–2005. Information provided within this document is a much expanded and detailed description of a report provided to the NPGS and includes the most exhaustive citation of germplasm depositions and research activity descriptions available anywhere in the USA for this time period. This documentation will be a valuable resource to breeders, geneticists, and taxonomists with an interest in this important food and fiber crop.

Mapping resistance gene analogs (RGAs) in cultivated tetraploid cotton using RGA-AFLP analysis

Diseases cause significant losses in cotton production throughout the US Cotton Belt. Growing resistant cultivars can significantly improve cotton yields and effectively reduce production inputs. Disease resistance (R) genes have been isolated in numerous plant species and the R genes with domains of nucleotide binding sites (NB) and leucine rich repeats (LRR) represent the largest R gene family. Degenerate primers designed based on conserved motifs of plant disease resistance genes were used alone or in combination with AFLP primers to analyze disease resistance gene analogs (RGAs) in a recombinant inbred line (RIL) population of Pima (Gossypium barbadense) 3–79 and Upland cotton (G. hirsutum) line NM 24016. Eighty-eight polymorphic RGA markers were amplified by 8 pairs of RGA degenerate primers, while 131 polymorphic RGA-AFLP markers were produced from six pairs of RGA-AFLP primer combinations. Of the 219 polymorphic RGA and RGA-AFLP markers that were identified, 212 were assigned to 18 chromosomes and linkage groups based on existing SSR markers that are on known chromosomes. However, the RGA and RGA-AFLP markers are not evenly distributed among chromosomes in that 189 RGA and RGA-AFLP markers (88%) are assigned onto three “giant” chromosomes, i.e., C6, C12, and C15, suggesting RGA clusters in the cotton genome. Several RGA and RGA-AFLP markers were mapped to the same linkage group carrying a root-knot nematode resistance gene. The identification and mapping of RGA and RGA-AFLP markers provide a framework to facilitate marker-assisted selection of disease resistance in cotton breeding and to understand the physical relationship of cotton resistance genes.