Nematode-resistance loci in Upland cotton genomes are associated with structural differences.

Reniform and root-knot nematode are two of the most destructive pests of conventional upland cotton, Gossypium hirsutum, L. and continue to be a major threat to cotton fiber production in semi-arid regions of the southern United States and Central America. Fortunately, naturally occurring tolerance to these nematodes has been identified in the Pima cotton species (G. barbadense) and several upland cotton varieties (G. hirsutum), which has led to a robust breeding program that has successfully introgressed and stacked these independent resistant traits into several upland cotton lineages with superior agronomic traits, e.g. BAR 32-30 and BARBREN-713. This work identifies the genomic variations of these nematode tolerant accessions by comparing their respective genomes to the susceptible, high-quality fiber producing parental line of this lineage: Phytogen 355 (PSC355). We discover several large genomic differences within marker regions that harbor putative resistance genes as well as expression mechanisms shared by the two resistant lines, with respect to the susceptible PSC355 parental line. This work emphasizes the utility of whole genome comparisons as a means of elucidating large and small nuclear differences by lineage and phenotype.  .

Origin and diversity of the wild cottons (Gossypium hirsutum) of Mound Key, Florida.

Elucidating genetic diversity within wild forms of modern crops is essential for understanding domestication and the possibilities of wild germplasm utilization. Gossypium hirsutum is a predominant source of natural plant fibers and the most widely cultivated cotton species. Wild forms of G. hirsutum are challenging to distinguish from feral derivatives, and truly wild populations are uncommon. Here we characterize a population from Mound Key Archaeological State Park, Florida using genome-wide SNPs extracted from 25 individuals over three sites. Our results reveal that this population is genetically dissimilar from other known wild, landrace, and domesticated cottons, and likely represents a pocket of previously unrecognized wild genetic diversity. The unexpected level of divergence between the Mound Key population and other wild cotton populations suggests that the species may harbor other remnant and genetically distinct populations that are geographically scattered in suitable habitats throughout the Caribbean. Our work thus has broader conservation genetic implications and suggests that further exploration of natural diversity in this species is warranted.

Genome resources for three modern cotton lines guide future breeding efforts.

Cotton (Gossypium hirsutum L.) is the key renewable fibre crop worldwide, yet its yield and fibre quality show high variability due to genotype-specific traits and complex interactions among cultivars, management practices and environmental factors. Modern breeding practices may limit future yield gains due to a narrow founding gene pool. Precision breeding and biotechnological approaches offer potential solutions, contingent on accurate cultivar-specific data. Here we address this need by generating high-quality reference genomes for three modern cotton cultivars ('UGA230', 'UA48' and 'CSX8308') and updating the 'TM-1' cotton genetic standard reference. Despite hypothesized genetic uniformity, considerable sequence and structural variation was observed among the four genomes, which overlap with ancient and ongoing genomic introgressions from 'Pima' cotton, gene regulatory mechanisms and phenotypic trait divergence. Differentially expressed genes across fibre development correlate with fibre production, potentially contributing to the distinctive fibre quality traits observed in modern cotton cultivars. These genomes and comparative analyses provide a valuable foundation for future genetic endeavours to enhance global cotton yield and sustainability.

Evolutionary Dynamics of Chromatin Structure and Duplicate Gene Expression in Diploid and Allopolyploid Cotton.

Polyploidy is a prominent mechanism of plant speciation and adaptation, yet the mechanistic understandings of duplicated gene regulation remain elusive. Chromatin structure dynamics are suggested to govern gene regulatory control. Here, we characterized genome-wide nucleosome organization and chromatin accessibility in allotetraploid cotton, Gossypium hirsutum (AADD, 2n = 4X = 52), relative to its two diploid parents (AA or DD genome) and their synthetic diploid hybrid (AD), using DNS-seq. The larger A-genome exhibited wider average nucleosome spacing in diploids, and this intergenomic difference diminished in the allopolyploid but not hybrid. Allopolyploidization also exhibited increased accessibility at promoters genome-wide and synchronized cis-regulatory motifs between subgenomes. A prominent cis-acting control was inferred for chromatin dynamics and demonstrated by transposable element removal from promoters. Linking accessibility to gene expression patterns, we found distinct regulatory effects for hybridization and later allopolyploid stages, including nuanced establishment of homoeolog expression bias and expression level dominance. Histone gene expression and nucleosome organization are coordinated through chromatin accessibility. Our study demonstrates the capability to track high-resolution chromatin structure dynamics and reveals their role in the evolution of cis-regulatory landscapes and duplicate gene expression in polyploids, illuminating regulatory ties to subgenomic asymmetry and dominance.

Genomic and Cytogenetic Analysis of Synthetic Polyploids between Diploid and Tetraploid Cotton (Gossypium) Species.

Cotton (Gossypium spp.) is the most important natural fiber source in the world. The genetic potential of cotton can be successfully and efficiently exploited by identifying and solving the complex fundamental problems of systematics, evolution, and phylogeny, based on interspecific hybridization of cotton. This study describes the results of interspecific hybridization of G. herbaceum L. (A1-genome) and G. mustelinum Miers ex Watt (AD4-genome) species, obtaining fertile hybrids through synthetic polyploidization of otherwise sterile triploid forms with colchicine (C22H25NO6) treatment. The fertile F1C hybrids were produced from five different cross combinations: (1) G. herbaceum subsp. frutescens × G. mustelinum; (2) G. herbaceum subsp. pseudoarboreum × G. mustelinum; (3) G. herbaceum subsp. pseudoarboreum f. harga × G. mustelinum; (4) G. herbaceum subsp. africanum × G. mustelinum; (5) G. herbaceum subsp. euherbaceum (variety A-833) × G. mustelinum. Cytogenetic analysis discovered normal conjugation of bivalent chromosomes in addition to univalent, open, and closed ring-shaped quadrivalent chromosomes at the stage of metaphase I in the F1C and F2C hybrids. The setting of hybrid bolls obtained as a result of these crosses ranged from 13.8-92.2%, the fertility of seeds in hybrid bolls from 9.7-16.3%, and the pollen viability rates from 36.6-63.8%. Two transgressive plants with long fiber of 35.1-37.0 mm and one plant with extra-long fiber of 39.1-41.0 mm were identified in the F2C progeny of G. herbaceum subsp. frutescens × G. mustelinum cross. Phylogenetic analysis with 72 SSR markers that detect genomic changes showed that tetraploid hybrids derived from the G. herbaceum × G. mustelinum were closer to the species G. mustelinum. The G. herbaceum subsp. frutescens was closer to the cultivated form, and its subsp. africanum was closer to the wild form. New knowledge of the interspecific hybridization and synthetic polyploidization was developed for understanding the genetic mechanisms of the evolution of tetraploid cotton during speciation. The synthetic polyploids of cotton obtained in this study would provide beneficial genes for developing new cotton varieties of the G. hirsutum species, with high-quality cotton fiber and strong tolerance to biotic or abiotic stress. In particular, the introduction of these polyploids to conventional and molecular breeding can serve as a bridge of transferring valuable genes related to high-quality fiber and stress tolerance from different cotton species to the new cultivars.

The Gossypium herbaceum L. Wagad genome as a resource for understanding cotton domestication.

Gossypium herbaceum is a species of cotton native to Africa and Asia that is one of the 2 domesticated diploids. Together with its sister-species G. arboreum, these A-genome taxa represent models of the extinct A-genome donor of modern polyploid cotton, which provide about 95% of cotton grown worldwide. As part of a larger effort to characterize variation and improve resources among diverse diploid and polyploid cotton genomes, we sequenced and assembled the genome of G. herbaceum cultivar (cv.) Wagad, representing the first domesticated accession for this species. This chromosome-level genome was generated using a combination of PacBio long-read technology, HiC, and Bionano optical mapping and compared to existing genome sequences in cotton. We compare the genome of this cultivar to the existing genome of wild G. herbaceum subspecies africanum to elucidate changes in the G. herbaceum genome concomitant with domestication and extend these analyses to gene expression using available RNA-seq. Our results demonstrate the utility of the G. herbaceum cv. Wagad genome in understanding domestication in the diploid species, which could inform modern breeding programs.

Dual Domestication, Diversity, and Differential Introgression in Old World Cotton Diploids.

Domestication in the cotton genus is remarkable in that it has occurred independently four different times at two different ploidy levels. Relatively little is known about genome evolution and domestication in the cultivated diploid species Gossypium herbaceum and Gossypium arboreum, due to the absence of wild representatives for the latter species, their ancient domestication, and their joint history of human-mediated dispersal and interspecific gene flow. Using in-depth resequencing of a broad sampling from both species, we provide support for their independent domestication, as opposed to a progenitor-derivative relationship, showing that diversity (mean π = 6 × 10-3) within species is similar, and that divergence between species is modest (FST = 0.413). Individual accessions were homozygous for ancestral single-nucleotide polymorphisms at over half of variable sites, while fixed, derived sites were at modest frequencies. Notably, two chromosomes with a paucity of fixed, derived sites (i.e., chromosomes 7 and 10) were also strongly implicated as having experienced high levels of introgression. Collectively, these data demonstrate variable permeability to introgression among chromosomes, which we propose is due to divergent selection under domestication and/or the phenomenon of F2 breakdown in interspecific crosses. Our analyses provide insight into the evolutionary forces that shape diversity and divergence in the diploid cultivated species and establish a foundation for understanding the contribution of introgression and/or strong parallel selection to the extensive morphological similarities shared between species.

An In Vitro Co-Culture System for Rapid Differential Response to Fusarium oxysporum f. sp. vasinfectum Race 4 in Three Cotton Cultivars.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a devastating fungus pathogen that causes Fusarium wilt in both domesticated cotton species, Gossypium hirsutum (Upland) and G. barbadense (Pima). Greenhouse and field-based pathogenicity assays can be a challenge because of nonuniform inoculum levels, the presence of endophytes, and varying environmental factors. Therefore, an in vitro coculture system was designed to support the growth of both domesticated cotton species and FOV4 via an inert polyphenolic foam substrate with a liquid medium. A Fusarium wilt-susceptible Pima cotton cultivar, G. barbadense 'GB1031'; a highly resistant Pima cotton cultivar, G. barbadense 'DP348RF'; and a susceptible Upland cotton cultivar, G. hirsutum 'TM-1', were evaluated for 30 days during coculture with FOV4 in this foam-based system. Thirty days after inoculation, disease symptoms were more severe in both susceptible cultivars, which displayed higher percentages of foliar damage, and greater plant mortality than observed in 'DP348RF', the resistant Pima cotton cultivar. This foam-based in vitro system may be useful for screening cotton germplasm for resistance to a variety of fungus pathogens and may facilitate the study of biotic interactions in domesticated cotton species under controlled environmental conditions.

Homology-based identification of candidate genes for male sterility editing in upland cotton (Gossypium hirsutum L.).

Upland cotton (Gossypium hirsutum L.) accounts for more than 90% of the world's cotton production, providing natural material for the textile and oilseed industries worldwide. One strategy for improving upland cotton yields is through increased adoption of hybrids; however, emasculation of cotton flowers is incredibly time-consuming and genetic sources of cotton male sterility are limited. Here we review the known biochemical modes of plant nuclear male sterility (NMS), often known as plant genetic male sterility (GMS), and characterized them into four groups: transcriptional regulation, splicing, fatty acid transport and processing, and sugar transport and processing. We have explored protein sequence homology from 30 GMS genes of three monocots (maize, rice, and wheat) and three dicots (Arabidopsis, soybean, and tomato). We have analyzed evolutionary relationships between monocot and dicot GMS genes to describe the relative similarity and relatedness of these genes identified. Five were lowly conserved to their source species, four unique to monocots, five unique to dicots, 14 highly conserved among all species, and two in the other category. Using this source, we have identified 23 potential candidate genes within the upland cotton genome for the development of new male sterile germplasm to be used in hybrid cotton breeding. Combining homology-based studies with genome editing may allow for the discovery and validation of GMS genes that previously had no diversity observed in cotton and may allow for development of a desirable male sterile mutant to be used in hybrid cotton production.

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.

The Gossypium anomalum genome as a resource for cotton improvement and evolutionary analysis of hybrid incompatibility.

Cotton is an important crop that has been the beneficiary of multiple genome sequencing efforts, including diverse representatives of wild species for germplasm development. Gossypium anomalum is a wild African diploid species that harbors stress-resistance and fiber-related traits with potential application to modern breeding efforts. In addition, this species is a natural source of cytoplasmic male sterility and a resource for understanding hybrid lethality in the genus. Here, we report a high-quality de novo genome assembly for G. anomalum and characterize this genome relative to existing genome sequences in cotton. In addition, we use the synthetic allopolyploids 2(A2D1) and 2(A2D3) to discover regions in the G. anomalum genome potentially involved in hybrid lethality, a possibility enabled by introgression of regions homologous to the D3 (Gossypium davidsonii) lethality loci into the synthetic 2(A2D3) allopolyploid.

Two gap-free reference genomes and a global view of the centromere architecture in rice.

Rice (Oryza sativa), a major staple throughout the world and a model system for plant genomics and breeding, was the first crop genome sequenced almost two decades ago. However, reference genomes for all higher organisms to date contain gaps and missing sequences. Here, we report the assembly and analysis of gap-free reference genome sequences for two elite O. sativa xian/indica rice varieties, Zhenshan 97 and Minghui 63, which are being used as a model system for studying heterosis and yield. Gap-free reference genomes provide the opportunity for a global view of the structure and function of centromeres. We show that all rice centromeric regions share conserved centromere-specific satellite motifs with different copy numbers and structures. In addition, the similarity of CentO repeats in the same chromosome is higher than across chromosomes, supporting a model of local expansion and homogenization. Both genomes have over 395 non-TE genes located in centromere regions, of which ∼41% are actively transcribed. Two large structural variants at the end of chromosome 11 affect the copy number of resistance genes between the two genomes. The availability of the two gap-free genomes lays a solid foundation for further understanding genome structure and function in plants and breeding climate-resilient varieties.

Parallel and Intertwining Threads of Domestication in Allopolyploid Cotton.

The two cultivated allopolyploid cottons, Gossypium hirsutum and Gossypium barbadense, represent a remarkable example of parallel independent domestication, both involving dramatic morphological transformations under selection from wild perennial plants to annualized row crops. Deep resequencing of 643 newly sampled accessions spanning the wild-to-domesticated continuum of both species, and their allopolyploid relatives, are combined with existing data to resolve species relationships and elucidate multiple aspects of their parallel domestication. It is confirmed that wild G. hirsutum and G. barbadense were initially domesticated in the Yucatan Peninsula and NW South America, respectively, and subsequently spread under domestication over 4000-8000 years to encompass most of the American tropics. A robust phylogenomic analysis of infraspecific relationships in each species is presented, quantify genetic diversity in both, and describe genetic bottlenecks associated with domestication and subsequent diffusion. As these species became sympatric over the last several millennia, pervasive genome-wide bidirectional introgression occurred, often with striking asymmetries involving the two co-resident genomes of these allopolyploids. Diversity scans revealed genomic regions and genes unknowingly targeted during domestication and additional subgenomic asymmetries. These analyses provide a comprehensive depiction of the origin, divergence, and adaptation of cotton, and serve as a rich resource for cotton improvement.

The Gossypium stocksii genome as a novel resource for cotton improvement.

Cotton is an important textile crop whose gains in production over the last century have been challenged by various diseases. Because many modern cultivars are susceptible to several pests and pathogens, breeding efforts have included attempts to introgress wild, naturally resistant germplasm into elite lines. Gossypium stocksii is a wild cotton species native to Africa, which is part of a clade of vastly understudied species. Most of what is known about this species comes from pest resistance surveys and/or breeding efforts, which suggests that G. stocksii could be a valuable reservoir of natural pest resistance. Here, we present a high-quality de novo genome sequence for G. stocksii. We compare the G. stocksii genome with resequencing data from a closely related, understudied species (Gossypium somalense) to generate insight into the relatedness of these cotton species. Finally, we discuss the utility of the G. stocksii genome for understanding pest resistance in cotton, particularly resistance to cotton leaf curl virus.

The Gossypium longicalyx Genome as a Resource for Cotton Breeding and Evolution.

Cotton is an important crop that has made significant gains in production over the last century. Emerging pests such as the reniform nematode have threatened cotton production. The rare African diploid species Gossypium longicalyx is a wild species that has been used as an important source of reniform nematode immunity. While mapping and breeding efforts have made some strides in transferring this immunity to the cultivated polyploid species, the complexities of interploidal transfer combined with substantial linkage drag have inhibited progress in this area. Moreover, this species shares its most recent common ancestor with the cultivated A-genome diploid cottons, thereby providing insight into the evolution of long, spinnable fiber. Here we report a newly generated de novo genome assembly of G. longicalyx This high-quality genome leveraged a combination of PacBio long-read technology, Hi-C chromatin conformation capture, and BioNano optical mapping to achieve a chromosome level assembly. The utility of the G. longicalyx genome for understanding reniform immunity and fiber evolution is discussed.

Genetic Analysis of the Transition from Wild to Domesticated Cotton (Gossypium hirsutum L.).

The evolution and domestication of cotton is of great interest from both economic and evolutionary standpoints. Although many genetic and genomic resources have been generated for cotton, the genetic underpinnings of the transition from wild to domesticated cotton remain poorly known. Here we generated an intraspecific QTL mapping population specifically targeting domesticated cotton phenotypes. We used 466 F2 individuals derived from an intraspecific cross between the wild Gossypium hirsutum var. yucatanense (TX2094) and the elite cultivar G. hirsutum cv. Acala Maxxa, in two environments, to identify 120 QTL associated with phenotypic changes under domestication. While the number of QTL recovered in each subpopulation was similar, only 22 QTL were considered coincident (i.e., shared) between the two locations, eight of which shared peak markers. Although approximately half of QTL were located in the A-subgenome, many key fiber QTL were detected in the D-subgenome, which was derived from a species with unspinnable fiber. We found that many QTL are environment-specific, with few shared between the two environments, indicating that QTL associated with G. hirsutum domestication are genomically clustered but environmentally labile. Possible candidate genes were recovered and are discussed in the context of the phenotype. We conclude that the evolutionary forces that shape intraspecific divergence and domestication in cotton are complex, and that phenotypic transformations likely involved multiple interacting and environmentally responsive factors.

The Genome Sequence of Gossypioides kirkii Illustrates a Descending Dysploidy in Plants.

One of the extraordinary aspects of plant genome evolution is variation in chromosome number, particularly that among closely related species. This is exemplified by the cotton genus (Gossypium) and its relatives, where most species and genera have a base chromosome number of 13. The two exceptions are sister genera that have n = 12 (the Hawaiian Kokia and the East African and Madagascan Gossypioides). We generated a high-quality genome sequence of Gossypioides kirkii (n = 12) using PacBio, Bionano, and Hi-C technologies, and compared this assembly to genome sequences of Kokia (n = 12) and Gossypium diploids (n = 13). Previous analysis demonstrated that the directionality of their reduced chromosome number was through large structural rearrangements. A series of structural rearrangements were identified comparing the de novo G. kirkii genome sequence to genome sequences of Gossypium, including chromosome fusions and inversions. Genome comparison between G. kirkii and Gossypium suggests that multiple steps are required to generate the extant structural differences.

De Novo Genome Sequence Assemblies of Gossypium raimondii and Gossypium turneri.

Cotton is an agriculturally important crop. Because of its importance, a genome sequence of a diploid cotton species (Gossypium raimondii, D-genome) was first assembled using Sanger sequencing data in 2012. Improvements to DNA sequencing technology have improved accuracy and correctness of assembled genome sequences. Here we report a new de novo genome assembly of G. raimondii and its close relative G. turneri The two genomes were assembled to a chromosome level using PacBio long-read technology, HiC, and Bionano optical mapping. This report corrects some minor assembly errors found in the Sanger assembly of G. raimondii We also compare the genome sequences of these two species for gene composition, repetitive element composition, and collinearity. Most of the identified structural rearrangements between these two species are due to intra-chromosomal inversions. More inversions were found in the G. turneri genome sequence than the G. raimondii genome sequence. These findings and updates to the D-genome sequence will improve accuracy and translation of genomics to cotton breeding and genetics.

The first genetic map for yellow lupin enables genetic dissection of adaptation traits in an orphan grain legume crop.

BACKGROUND: Yellow lupin (Lupinus luteus L.) is a promising grain legume for productive and sustainable crop rotations. It has the advantages of high tolerance to soil acidity and excellent seed quality, but its current yield potential is poor, especially in low rainfall environments. Key adaptation traits such as phenology and enhanced stress tolerance are often complex and controlled by several genes. Genomic-enabled technologies may help to improve our basic understanding of these traits and to provide selective markers in breeding. However, in yellow lupin there are very limited genomic resources to support research and no published information is available on the genetic control of adaptation traits. RESULTS: We aimed to address these deficiencies by developing the first linkage map for yellow lupin and conducting quantitative trait locus (QTL) analysis of yield under well-watered (WW) and water-deficit (WT) conditions. Two next-generation sequencing marker approaches - genotyping-by-sequencing (GBS) and Diversity Array Technology (DArT) sequencing - were employed to genotype a recombinant inbred line (RIL) population developed from a bi-parental cross between wild and domesticated parents. A total of 2,458 filtered single nucleotide polymorphism (SNP) and presence / absence variation (PAV) markers were used to develop a genetic map comprising 40 linkage groups, the first reported for this species. A number of significant QTLs controlling total biomass and 100-seed weight under two water (WW and WD) regimes were found on linkage groups YL-03, YL-09 and YL-26 that together explained 9 and 28% of total phenotypic variability. QTLs associated with length of the reproductive phase and time to flower were found on YL-01, YL-21, YL-35 and YL-40 that together explained a total of 12 and 44% of total phenotypic variation. CONCLUSION: These genomic resources and the QTL information offer significant potential for use in marker-assisted selection in yellow lupin.

Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense.

Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1-4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.