Impact of salinity stress on cotton and opportunities for improvement through conventional and biotechnological approaches.

Excess salinity can affect the growth and development of all plants. Salinization jeopardizes agroecosystems, induces oxidative reactions in most cultivated plants and reduces biomass which affects crop yield. Some plants are affected more than others, depending upon their ability to endure the effects of salt stress. Cotton is moderately tolerant to salt stress among cultivated crops. The fundamental tenet of plant breeding is genetic heterogeneity in available germplasm for acquired characteristics. Variation for salinity tolerance enhancing parameters (morphological, physiological and biochemical) is a pre-requisite for the development of salt tolerant cotton germplasm followed by indirect selection or hybridization programs. There has been a limited success in the development of salt tolerant genotypes because this trait depends on several factors, and these factors as well as their interactions are not completely understood. However, advances in biochemical and molecular techniques have made it possible to explore the complexity of salt tolerance through transcriptomic profiling. The focus of this article is to discuss the issue of salt stress in crop plants, how it alters the physiology and morphology of the cotton crop, and breeding strategies for the development of salinity tolerance in cotton germplasm.

Leveraging National Germplasm Collections to Determine Significantly Associated Categorical Traits in Crops: Upland and Pima Cotton as a Case Study.

Observable qualitative traits are relatively stable across environments and are commonly used to evaluate crop genetic diversity. Recently, molecular markers have largely superseded describing phenotypes in diversity surveys. However, qualitative descriptors are useful in cataloging germplasm collections and for describing new germplasm in patents, publications, and/or the Plant Variety Protection (PVP) system. This research focused on the comparative analysis of standardized cotton traits as represented within the National Cotton Germplasm Collection (NCGC). The cotton traits are named by 'descriptors' that have non-numerical sub-categories (descriptor states) reflecting the details of how each trait manifests or is absent in the plant. We statistically assessed selected accessions from three major groups of Gossypium as defined by the NCGC curator: (1) "Stoneville accessions (SA)," containing mainly Upland cotton (Gossypium hirsutum) cultivars; (2) "Texas accessions (TEX)," containing mainly G. hirsutum landraces; and (3) Gossypium barbadense (Gb), containing cultivars or landraces of Pima cotton (Gossypium barbadense). For 33 cotton descriptors we: (a) revealed distributions of character states for each descriptor within each group; (b) analyzed bivariate associations between paired descriptors; and (c) clustered accessions based on their descriptors. The fewest significant associations between descriptors occurred in the SA dataset, likely reflecting extensive breeding for cultivar development. In contrast, the TEX and Gb datasets showed a higher number of significant associations between descriptors, likely correlating with less impact from breeding efforts. Three significant bivariate associations were identified for all three groups, bract nectaries:boll nectaries, leaf hair:stem hair, and lint color:seed fuzz color. Unsupervised clustering analysis recapitulated the species labels for about 97% of the accessions. Unexpected clustering results indicated accessions that may benefit from potential further investigation. In the future, the significant associations between standardized descriptors can be used by curators to determine whether new exotic/unusual accessions most closely resemble Upland or Pima cotton. In addition, the study shows how existing descriptors for large germplasm datasets can be useful to inform downstream goals in breeding and research, such as identifying rare individuals with specific trait combinations and targeting breakdown of remaining trait associations through breeding, thus demonstrating the utility of the analytical methods employed in categorizing germplasm diversity within the collection.

Genome assembly of two nematode-resistant cotton lines (Gossypium hirsutum L.).

Upland cotton (Gossypium hirsutum L.) is susceptible to damage by the root-knot and the reniform nematodes, causing yield losses greater than 4% annually in the United States. In addition, these nematodes are synergistic with seeding disease and root rot pathogens that exacerbate diseases and subsequent yield losses. Production practices to minimize nematode damage include crop rotation and nematicides, but these techniques need to be repeated and are expensive. The use of resistant cultivars is deemed the most effective and economical approach for managing nematodes in cotton. Here, we describe the genomes of two nematode-resistant lines of cotton, BARBREN-713 and BAR 32-30. These genomes may expedite the development of DNA markers that can be used to efficiently introduce nematode resistance into commercially valuable Upland lines.

Assessing Drought and Heat Stress-Induced Changes in the Cotton Leaf Metabolome and Their Relationship With Hyperspectral Reflectance.

The study of phenotypes that reveal mechanisms of adaptation to drought and heat stress is crucial for the development of climate resilient crops in the face of climate uncertainty. The leaf metabolome effectively summarizes stress-driven perturbations of the plant physiological status and represents an intermediate phenotype that bridges the plant genome and phenome. The objective of this study was to analyze the effect of water deficit and heat stress on the leaf metabolome of 22 genetically diverse accessions of upland cotton grown in the Arizona low desert over two consecutive years. Results revealed that membrane lipid remodeling was the main leaf mechanism of adaptation to drought. The magnitude of metabolic adaptations to drought, which had an impact on fiber traits, was found to be quantitatively and qualitatively associated with different stress severity levels during the two years of the field trial. Leaf-level hyperspectral reflectance data were also used to predict the leaf metabolite profiles of the cotton accessions. Multivariate statistical models using hyperspectral data accurately estimated (R 2 > 0.7 in ∼34% of the metabolites) and predicted (Q 2 > 0.5 in 15-25% of the metabolites) many leaf metabolites. Predicted values of metabolites could efficiently discriminate stressed and non-stressed samples and reveal which regions of the reflectance spectrum were the most informative for predictions. Combined together, these findings suggest that hyperspectral sensors can be used for the rapid, non-destructive estimation of leaf metabolites, which can summarize the plant physiological status.

Evaluation and genome-wide association study of resistance to bacterial blight race 18 in U.S. Upland cotton germplasm.

Bacterial blight (BB), caused by Xanthomonas citri pv. malvacearum (Xcm), is a destructive disease to cotton production in many countries. In the U.S., Xcm race 18 is the most virulent and widespread race and can cause serious yield losses. Planting BB-resistant cotton cultivars is the most effective method of controlling this disease. In this study, 335 U.S. Upland cotton accessions were evaluated for resistance to race 18 using artificial inoculations by scratching cotyledons on an individual plant basis in a greenhouse. The analysis of variance detected significant genotypic variation in disease incidence, and 50 accessions were resistant including 38 lines with no symptoms on either cotyledons or true leaves. Many of the resistant lines were developed in the MAR (multi-adversity resistance) breeding program at Texas A&M University, whereas others were developed before race 18 was first reported in the U.S. in 1973, suggesting a broad base of resistance to race 18. A genome-wide association study (GWAS) based on 26,301 single nucleotide polymorphic (SNP) markers detected 11 quantitative trait loci (QTL) anchored by 79 SNPs, including three QTL on each of the three chromosomes A01, A05 and D02, and one QTL on each of D08 and D10. This study has identified a set of obsolete Upland germplasm with resistance to race 18 and specific chromosomal regions delineated by SNPs for resistance. The results will assist in breeding cotton for BB resistance and facilitate further genomic studies in fine mapping resistance genes to enhance the understanding of the genetic basis of BB resistance in cotton.

Networks of Physiological Adjustments and Defenses, and Their Synergy With Sodium (Na+) Homeostasis Explain the Hidden Variation for Salinity Tolerance Across the Cultivated Gossypium hirsutum Germplasm.

The abilities to mobilize and/or sequester excess ions within and outside the plant cell are important components of salt-tolerance mechanisms. Mobilization and sequestration of Na+ involves three transport systems facilitated by the plasma membrane H+/Na+ antiporter (SOS1), vacuolar H+/Na+ antiporter (NHX1), and Na+/K+ transporter in vascular tissues (HKT1). Many of these mechanisms are conserved across the plant kingdom. While Gossypium hirsutum (upland cotton) is significantly more salt-tolerant relative to other crops, the critical factors contributing to the phenotypic variation hidden across the germplasm have not been fully unraveled. In this study, the spatio-temporal patterns of Na+ accumulation along with other physiological and biochemical interactions were investigated at different severities of salinity across a meaningful genetic diversity panel across cultivated upland Gossypium. The aim was to define the importance of holistic or integrated effects relative to the direct effects of Na+ homeostasis mechanisms mediated by GhHKT1, GhSOS1, and GhNHX1. Multi-dimensional physio-morphometric attributes were investigated in a systems-level context using univariate and multivariate statistics, randomForest, and path analysis. Results showed that mobilized or sequestered Na+ contributes significantly to the baseline tolerance mechanisms. However, the observed variance in overall tolerance potential across a meaningful diversity panel were more significantly attributed to antioxidant capacity, maintenance of stomatal conductance, chlorophyll content, and divalent cation (Mg2+) contents other than Ca2+ through a complex interaction with Na+ homeostasis. The multi-tier macro-physiological, biochemical and molecular data generated in this study, and the networks of interactions uncovered strongly suggest that a complex physiological and biochemical synergy beyond the first-line-of defense (Na+ sequestration and mobilization) accounts for the total phenotypic variance across the primary germplasm of Gossypium hirsutum. These findings are consistent with the recently proposed Omnigenic Theory for quantitative traits and should contribute to a modern look at phenotypic selection for salt tolerance in cotton breeding.

Genome-wide identification and characterization of HSP70 gene family in four species of cotton.

Heat shock proteins (HSPs) are important elements of the cellular group of molecular chaperones. Specifically, HSP70 proteins protect cells from being damaged when plants are exposed to environmental stresses. These proteins are catalysts that manage the correct folding of other proteins, and they play a key role in the development of tolerance against biotic and abiotic stresses. In the present study, 113 HSP70 genes were retrieved from the available genome assemblies of four cotton species, including Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii. The HSP70 genes were clustered into 11 subfamilies based on phylogeny. One hundred and nine (109) gene duplications were found across these four species. Localization of genes revealed that several HSP70 genes reside in the cytoplasm. Synonymous and non-synonymous substitution rates revealed that functional segregation of HSP70 genes in cotton is due to purifying selection. Furthermore, HSP70 genes in cotton are expressed constitutively during developmental stages. These findings are valuable to understand the complex mechanism of HSP70 gene regulation that occurs in signaling pathways in response to plant stress.

A genome-wide association study uncovers consistent quantitative trait loci for resistance to Verticillium wilt and Fusarium wilt race 4 in the US Upland cotton.

KEY MESSAGE: A high-resolution GWAS detected consistent QTL for resistance to Verticillium wilt and Fusarium wilt race 4 in 376 U.S. Upland cotton accessions based on six independent replicated greenhouse tests. Verticillium wilt (VW, caused by Verticillium dahliae Kleb.) and Fusarium wilt (FOV, caused by Fusarium oxysporum f.sp. vasinfectum Atk. Sny & Hans) are the most important soil-borne fungal diseases in cotton. To augment and refine resistance quantitative trait loci (QTL), we conducted a genome-wide association study (GWAS) using high-density genotyping with the CottonSNP63K array. Resistance of 376 US Upland cotton accessions to a defoliating VW and virulent FOV4 was evaluated in four and two independent replicated greenhouse tests, respectively. A total of 15 and 13 QTL for VW and FOV4 resistances were anchored by 30 (on five chromosomes) and 56 (on six chromosomes) significant single nucleotide polymorphic (SNPs) markers, respectively. QTL on c8, c10, c16, and c21 were consistent in two or more tests for VW resistance, while two QTL on c8 and c14 were consistent for FOV4 resistance in two tests. Two QTL clusters on c16 and c19 were observed for both VW and FOV4 resistance, suggesting that these genomic regions may harbor genes in response to both diseases. Using BLAST search against the sequenced TM-1 genome, 30 and 35 candidate genes were identified on four QTL for VW resistance and on three QTL for FOV4 resistance, respectively. These genomic regions were rich in NBS-LRR genes presented in clusters. The results create opportunities for further studies to determine the correlations of field resistance with these QTL, molecular examinations of VW and FOV4 resistances, marker-assisted selection (MAS) and eventual cloning of QTL for disease resistance in cotton.

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.

Lipid metabolites in seeds of diverse Gossypium accessions: molecular identification of a high oleic mutant allele.

MAIN CONCLUSION: Genetically diverse cottonseeds show altered compositions and spatial distributions of phosphatidylcholines and triacylglycerols. Lipidomics profiling led to the discovery of a novel FAD2 - 1 allele, fad2 - 1D - 1 , resulting in a high oleic phenotype. The domestication and breeding of cotton for elite, high-fiber cultivars have led to reduced variation of seed constituents within currently cultivated upland cotton genotypes. However, a recent screen of the genetically diverse U.S. National Cotton Germplasm Collection identified Gossypium accessions with marked differences in seed oil and protein content. Here, several of these accessions representing substantial variation in seed oil content were analyzed for quantitative and spatial differences in lipid compositions by mass spectrometric approaches. Results indicate considerable variation in amount and spatial distribution of pathway metabolites for triacylglycerol biosynthesis in embryos across Gossypium accessions, suggesting that this variation might be exploited by breeders for seed composition traits. By way of example, these lipid metabolite differences led to the identification of a mutant allele of the D-subgenome homolog of the delta-12 desaturase (fad2-1D-1) in a wild accession of G. barbadense that has a high oil and high oleic seed phenotype. This mutation is a 90-bp insertion in the 3' end of the FAD2-1D coding sequence and a modification of the 3' end of the gene beyond the coding sequence leading to the introduction of a premature stop codon. Given the large amounts of cottonseed produced around the world that is currently not processed into higher value products, these efforts might be one avenue to raise the overall value of the cotton crop for producers.

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.

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.

Molecular characterization of the Gossypium Diversity Reference Set of the US National Cotton Germplasm Collection.

KEY MESSAGE: A core marker set containing markers developed to be informative within a single commercial cotton species can elucidate diversity structure within a multi-species subset of the Gossypium germplasm collection. An understanding of the genetic diversity of cotton (Gossypium spp.) as represented in the US National Cotton Germplasm Collection is essential to develop strategies for collecting, conserving, and utilizing these germplasm resources. The US collection is one of the largest world collections and includes not only accessions with improved yield and fiber quality within cultivated species, but also accessions possessing sources of abiotic and biotic stress resistance often found in wild species. We evaluated the genetic diversity of a subset of 272 diploid and 1,984 tetraploid accessions in the collection (designated the Gossypium Diversity Reference Set) using a core set of 105 microsatellite markers. Utility of the core set of markers in differentiating intra-genome variation was much greater in commercial tetraploid genomes (99.7 % polymorphic bands) than in wild diploid genomes (72.7 % polymorphic bands), and may have been influenced by pre-selection of markers for effectiveness in the commercial species. Principal coordinate analyses revealed that the marker set differentiated interspecific variation among tetraploid species, but was only capable of partially differentiating among species and genomes of the wild diploids. Putative species-specific marker bands in G. hirsutum (73) and G. barbadense (81) were identified that could be used for qualitative identification of misclassifications, redundancies, and introgression within commercial tetraploid species. The results of this broad-scale molecular characterization are essential to the management and conservation of the collection and provide insight and guidance in the use of the collection by the cotton research community in their cotton improvement efforts.

RNAi-mediated Ultra-low gossypol cottonseed trait: performance of transgenic lines under field conditions.

Cottonseed remains a low-value by-product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra-low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ-cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild-type levels of gossypol and related terpenoids. Although it was a relatively small-scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild-type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%-8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi-based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever-increasing needs of humanity.