Genomic confirmation of Gossypium barbadense introgression into G. hirsutum and a subsequent MAGIC population.

Introgression of superior fiber traits from Pima cotton (Gossypium barbadense, GB) into high yield Upland cotton (G. hirsutum) has been a breeding objective for many years in a few breeding programs in the world. However, progress has been very slow due to introgression barriers resulting from whole genome hybridization between the two species. To minimize such barriers, chromosome substitution lines (CS-B) from Pima cotton 3-79 in an Upland cotton cultivar TM-1 were developed. A multiparent advanced generation inter-cross (MAGIC) population consisting of 180 recombinant inbred lines (RILs) was subsequently made using the 18 CS-B lines and three Upland cotton cultivars as parents. In this research, we sequenced the whole genomes of the 21 parents and 180 RILs to examine the G. barbadense introgression. Of the 18 CS-B lines, 11 contained the target GB chromosome or chromosome segment, two contained more than two GB chromosomes, and five did not have the expected introgression. Residual introgression in non-target chromosomes was prevalent in all CS-B lines. A clear structure existed in the MAGIC population and the 180 RILs were distributed into three groups, i.e., high, moderate, and low GB introgression. Large blocks of GB chromosome introgression were still present in some RILs after five cycles of random-mating, an indication of recombination suppression or other unknown reasons present in the population. Identity by descent analysis revealed that the MAGIC RILs contained less introgression than expected. This research presents an insight on understanding the complex problems of introgression between cotton species.

Genetic diversity and population structure analyses and genome-wide association studies of photoperiod sensitivity in cotton (Gossypium hirsutum L.).

KEY MESSAGE: Genetic diversity and population structure analyses showed progressively narrowed diversity in US Upland cotton compared to land races. GWAS identified genomic regions and candidate genes for photoperiod sensitivity in cotton. Six hundred fifty-seven accessions that included elite cotton germplasm (DIV panel), lines of a public cotton breeding program (FB panel), and tropical landrace accessions (TLA panel) of Gossypium hirsutum L. were genotyped with cottonSNP63K array and phenotyped for photoperiod sensitivity under long day-length conditions. The genetic diversity analysis using 26,952 polymorphic SNPs indicated a progressively narrowed diversity from the landraces (0.230) to the DIV panel accessions (0.195) and FB panel (0.116). Structure analysis in the US germplasm identified seven subpopulations representing all four major regions of the US cotton belt. Three subpopulations were identified within the landrace accessions. The highest fixation index (FST) of 0.65 was found between landrace accessions of Guatemala and the Plains-type cultivars from Southwest cotton region while the lowest FST values were between the germplasms of Mid-South and Southeastern regions. Genome wide association studies (GWAS) of photoperiod response using 600 phenotyped accessions identified 14 marker trait associations spread across eight Upland cotton chromosomes. Six of these marker trait associations, on four chromosomes (A10, D04, D05, and D06), showed significant epistatic interactions. Targeted genomic analysis identified regions with 19 candidate genes including Transcription factor Vascular Plant One-Zinc Finger 1 (VOZ1) and Protein Photoperiod-Independent Early Flowering 1 (PIE1) genes. Genetic diversity and genome wide analyses of photoperiod sensitivity in diverse cotton germplasms will enable the use of genomic tools to systematically utilize the tropical germplasm and its beneficial alleles for broadening the genetic base in Upland cotton.

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.

LCM and RNA-seq analyses revealed roles of cell cycle and translational regulation and homoeolog expression bias in cotton fiber cell initiation.

BACKGROUND: Cotton fibers provide a powerful model for studying cell differentiation and elongation. Each cotton fiber is a singular and elongated cell derived from epidermal-layer cells of a cotton seed. Efforts to understand this dramatic developmental shift have been impeded by the difficulty of separation between fiber and epidermal cells. RESULTS: Here we employed laser-capture microdissection (LCM) to separate these cell types. RNA-seq analysis revealed transitional differences between fiber and epidermal-layer cells at 0 or 2 days post anthesis. Specifically, down-regulation of putative cell cycle genes was coupled with upregulation of ribosome biosynthesis and translation-related genes, which may suggest their respective roles in fiber cell initiation. Indeed, the amount of fibers in cultured ovules was increased by cell cycle progression inhibitor, Roscovitine, and decreased by ribosome biosynthesis inhibitor, Rbin-1. Moreover, subfunctionalization of homoeologs was pervasive in fiber and epidermal cells, with expression bias towards 10% more D than A homoeologs of cell cycle related genes and 40-50% more D than A homoeologs of ribosomal protein subunit genes. Key cell cycle regulators were predicted to be epialleles in allotetraploid cotton. MYB-transcription factor genes displayed expression divergence between fibers and ovules. Notably, many phytohormone-related genes were upregulated in ovules and down-regulated in fibers, suggesting spatial-temporal effects on fiber cell development. CONCLUSIONS: Fiber cell initiation is accompanied by cell cycle arrest coupled with active ribosome biosynthesis, spatial-temporal regulation of phytohormones and MYB transcription factors, and homoeolog expression bias of cell cycle and ribosome biosynthesis genes. These valuable genomic resources and molecular insights will help develop breeding and biotechnological tools to improve cotton fiber production.

Evaluation of genomic selection methods for predicting fiber quality traits in Upland cotton.

The use of genomic selection (GS) has stimulated a new way to utilize molecular markers in breeding for complex traits in the absence of phenotypic data. GS can potentially decrease breeding cycle by selecting the progeny in the early stages. The objective of this study was to experimentally evaluate the potential value of genomic selection in Upland cotton breeding. Six fiber quality traits were obtained in 3 years of replicated field trials in Starkville, MS. Genotyping-by-sequencing-based genotyping was performed using 550 recombinant inbred lines of the multi-parent advanced generation inter-cross population, and 6292 molecular markers were used for the GS analysis. Several methods were compared including genomic BLUP (GBLUP), ridge regression BLUP (rrBLUP), BayesB, Bayesian LASSO, and reproducing kernel hilbert spaces (RKHS). The average heritability (h2) ranged from 0.38 to 0.88 for all tested traits across the 3 years evaluated. BayesB predicted the highest accuracies among the five GS methods tested. The prediction ability (PA) and prediction accuracy (PACC) varied widely across 3 years for all tested traits and the highest PA and PACC were 0.65, and 0.69, respectively, in 2010 for fiber elongation. Marker density and training population size appeared to be very important factors for PA and PACC in GS. Results indicated that BayesB-based GS method could predict genomic estimated breeding value efficiently in Upland cotton fiber quality attributes and has great potential utility in breeding by reducing cost and time.

Modifications to a LATE MERISTEM IDENTITY1 gene are responsible for the major leaf shapes of Upland cotton (Gossypium hirsutum L.).

Leaf shape varies spectacularly among plants. Leaves are the primary source of photoassimilate in crop plants, and understanding the genetic basis of variation in leaf morphology is critical to improving agricultural productivity. Leaf shape played a unique role in cotton improvement, as breeders have selected for entire and lobed leaf morphs resulting from a single locus, okra (l-D1), which is responsible for the major leaf shapes in cotton. The l-D1 locus is not only of agricultural importance in cotton, but through pioneering chimeric and morphometric studies, it has contributed to fundamental knowledge about leaf development. Here we show that an HD-Zip transcription factor homologous to the LATE MERISTEM IDENTITY1 (LMI1) gene of Arabidopsis is the causal gene underlying the l-D1 locus. The classical okra leaf shape allele has a 133-bp tandem duplication in the promoter, correlated with elevated expression, whereas an 8-bp deletion in the third exon of the presumed wild-type normal allele causes a frame-shifted and truncated coding sequence. Our results indicate that subokra is the ancestral leaf shape of tetraploid cotton that gave rise to the okra allele and that normal is a derived mutant allele that came to predominate and define the leaf shape of cultivated cotton. Virus-induced gene silencing (VIGS) of the LMI1-like gene in an okra variety was sufficient to induce normal leaf formation. The developmental changes in leaves conferred by this gene are associated with a photosynthetic transcriptomic signature, substantiating its use by breeders to produce a superior cotton ideotype.

High-density linkage map construction and QTL analyses for fiber quality, yield and morphological traits using CottonSNP63K array in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Improving fiber quality and yield are the primary research objectives in cotton breeding for enhancing the economic viability and sustainability of Upland cotton production. Identifying the quantitative trait loci (QTL) for fiber quality and yield traits using the high-density SNP-based genetic maps allows for bridging genomics with cotton breeding through marker assisted and genomic selection. In this study, a recombinant inbred line (RIL) population, derived from cross between two parental accessions, which represent broad allele diversity in Upland cotton, was used to construct high-density SNP-based linkage maps and to map the QTLs controlling important cotton traits. RESULTS: Molecular genetic mapping using RIL population produced a genetic map of 3129 SNPs, mapped at a density of 1.41 cM. Genetic maps of the individual chromosomes showed good collinearity with the sequence based physical map. A total of 106 QTLs were identified which included 59 QTLs for six fiber quality traits, 38 QTLs for four yield traits and 9 QTLs for two morphological traits. Sub-genome wide, 57 QTLs were mapped in A sub-genome and 49 were mapped in D sub-genome. More than 75% of the QTLs with favorable alleles were contributed by the parental accession NC05AZ06. Forty-six mapped QTLs each explained more than 10% of the phenotypic variation. Further, we identified 21 QTL clusters where 12 QTL clusters were mapped in the A sub-genome and 9 were mapped in the D sub-genome. Candidate gene analyses of the 11 stable QTL harboring genomic regions identified 19 putative genes which had functional role in cotton fiber development. CONCLUSION: We constructed a high-density genetic map of SNPs in Upland cotton. Collinearity between genetic and physical maps indicated no major structural changes in the genetic mapping populations. Most traits showed high broad-sense heritability. One hundred and six QTLs were identified for the fiber quality, yield and morphological traits. Majority of the QTLs with favorable alleles were contributed by improved parental accession. More than 70% of the mapped QTLs shared the similar map position with previously reported QTLs which suggest the genetic relatedness of Upland cotton germplasm. Identification of QTL clusters could explain the correlation among some fiber quality traits in cotton. Stable and major QTLs and QTL clusters of traits identified in the current study could be the targets for map-based cloning and marker assisted selection (MAS) in cotton breeding. The genomic region on D12 containing the major stable QTLs for micronaire, fiber strength and lint percentage could be potential targets for MAS and gene cloning of fiber quality traits in cotton.

Whole genome sequencing of a MAGIC population identified genomic loci and candidate genes for major fiber quality traits in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Significant associations between candidate genes and six major cotton fiber quality traits were identified in a MAGIC population using GWAS and whole genome sequencing. Upland cotton (Gossypium hirsutum L.) is the world's major renewable source of fibers for textiles. To identify causative genetic variants that influence the major agronomic measures of cotton fiber quality, which are used to set discount or premium prices on each bale of cotton in the USA, we measured six fiber phenotypes from twelve environments, across three locations and 7 years. Our 550 recombinant inbred lines were derived from a multi-parent advanced generation intercross population and were whole-genome-sequenced at 3× coverage, along with the eleven parental cultivars at 20× coverage. The segregation of 473,517 single nucleotide polymorphisms (SNPs) in this population, including 7506 non-synonymous mutations, was combined with phenotypic data to identify seven highly significant fiber quality loci. At these loci, we found fourteen genes with non-synonymous SNPs. Among these loci, some had simple additive effects, while others were only important in a subset of the population. We observed additive effects for elongation and micronaire, when the three most significant loci for each trait were examined. In an informative subset where the major multi-trait locus on chromosome A07:72-Mb was fixed, we unmasked the identity of another significant fiber strength locus in gene Gh_D13G1792 on chromosome D13. The micronaire phenotype only revealed one highly significant genetic locus at one environmental location, demonstrating a significant genetic by environment component. These loci and candidate causative variant alleles will be useful to cotton breeders for marker-assisted selection with minimal linkage drag and potential biotechnological applications.

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.

A MAGIC population-based genome-wide association study reveals functional association of GhRBB1_A07 gene with superior fiber quality in cotton.

BACKGROUND: Cotton supplies a great majority of natural fiber for the global textile industry. The negative correlation between yield and fiber quality has hindered breeders' ability to improve these traits simultaneously. A multi-parent advanced generation inter-cross (MAGIC) population developed through random-mating of multiple diverse parents has the ability to break this negative correlation. Genotyping-by-sequencing (GBS) is a method that can rapidly identify and genotype a large number of single nucleotide polymorphisms (SNP). Genotyping a MAGIC population using GBS technologies will enable us to identify marker-trait associations with high resolution. RESULTS: An Upland cotton MAGIC population was developed through random-mating of 11 diverse cultivars for five generations. In this study, fiber quality data obtained from four environments and 6071 SNP markers generated via GBS and 223 microsatellite markers of 547 recombinant inbred lines (RILs) of the MAGIC population were used to conduct a genome wide association study (GWAS). By employing a mixed linear model, GWAS enabled us to identify markers significantly associated with fiber quantitative trait loci (QTL). We identified and validated one QTL cluster associated with four fiber quality traits [short fiber content (SFC), strength (STR), length (UHM) and uniformity (UI)] on chromosome A07. We further identified candidate genes related to fiber quality attributes in this region. Gene expression and amino acid substitution analysis suggested that a regeneration of bulb biogenesis 1 (GhRBB1_A07) gene is a candidate for superior fiber quality in Upland cotton. The DNA marker CFBid0004 designed from an 18 bp deletion in the coding sequence of GhRBB1_A07 in Acala Ultima is associated with the improved fiber quality in the MAGIC RILs and 105 additional commercial Upland cotton cultivars. CONCLUSION: Using GBS and a MAGIC population enabled more precise fiber QTL mapping in Upland cotton. The fiber QTL and associated markers identified in this study can be used to improve fiber quality through marker assisted selection or genomic selection in a cotton breeding program. Target manipulation of the GhRBB1_A07 gene through biotechnology or gene editing may potentially improve cotton fiber quality.

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.

Genome-wide identification of auxin response factor (ARF) genes and its tissue-specific prominent expression in Gossypium raimondii.

Auxin response factors (ARFs) are recently discovered transcription factors that bind with auxin response elements (AuxRE, TGTCTC) to regulate the expression of early auxin-responsive genes. To our knowledge, the ARF gene family has never been characterized in cotton, the most important fiber crop in the world. In this study, a total of 35 ARF genes, named as GrARFs, were identified in a diploid cotton species Gossypium raimondii. The 35 ARF genes were located in 12 of the 13 cotton chromosomes; the intron/exon distribution of the GrARF genes was similar among sister pairs, whereas the divergence of some GrARF genes suggests the possibility of functional diversification. Our results show that the middle domains of nine GrARF proteins rich in glutamine (Q) are activators, while 26 other GrARF proteins rich in proline (P), serine (S), and threonine (T) are repressors. Our results also show that the expression of GrARF genes is diverse in different tissues. The expression of GrARF1 was significantly higher in leaves, whereas GrARF2a had higher expression level in shoots, which implicates different roles in the tested tissues. The GrARF11 has a higher expression level in buds than that in leaves, while GrARF19.2 shows contrasting expression patterns, having higher expression in leaves than that in buds. This suggests that they play different roles in leaves and buds. During long-term evolution of G. raimondii, some ARF genes were lost and some arose. The identification and characterization of the ARF genes in G. raimondii elucidate its important role in cotton that ARF genes regulate the development of flower buds, sepals, shoots, and leaves.

Small RNA sequencing identifies miRNA roles in ovule and fibre development.

MicroRNAs (miRNAs) have been found to be differentially expressed during cotton fibre development. However, which specific miRNAs and how they are involved in fibre development is unclear. Here, using deep sequencing, 65 conserved miRNA families were identified and 32 families were differentially expressed between leaf and ovule. At least 40 miRNAs were either leaf or ovule specific, whereas 62 miRNAs were shared in both leaf and ovule. qRT-PCR confirmed these miRNAs were differentially expressed during fibre early development. A total of 820 genes were potentially targeted by the identified miRNAs, whose functions are involved in a series of biological processes including fibre development, metabolism and signal transduction. Many predicted miRNA-target pairs were subsequently validated by degradome sequencing analysis. GO and KEGG analyses showed that the identified miRNAs and their targets were classified to 1027 GO terms including 568 biological processes, 324 molecular functions and 135 cellular components and were enriched to 78 KEGG pathways. At least seven unique miRNAs participate in trichome regulatory interaction network. Eleven trans-acting siRNA (tasiRNA) candidate genes were also identified in cotton. One has never been found in other plant species and two of them were derived from MYB and ARF, both of which play important roles in cotton fibre development. Sixteen genes were predicted to be tasiRNA targets, including sucrose synthase and MYB2. Together, this study discovered new miRNAs in cotton and offered evidences that miRNAs play important roles in cotton ovule/fibre development. The identification of tasiRNA genes and their targets broadens our understanding of the complicated regulatory mechanism of miRNAs in cotton.

Regulation of Gene Expression during the Onset of Ligninolytic Oxidation by Phanerochaete chrysosporium on Spruce Wood.

Since uncertainty remains about how white rot fungi oxidize and degrade lignin in wood, it would be useful to monitor changes in fungal gene expression during the onset of ligninolysis on a natural substrate. We grew Phanerochaete chrysosporium on solid spruce wood and included oxidant-sensing beads bearing the fluorometric dye BODIPY 581/591 in the cultures. Confocal fluorescence microscopy of the beads showed that extracellular oxidation commenced 2 to 3 days after inoculation, coincident with cessation of fungal growth. Whole transcriptome shotgun sequencing (RNA-seq) analyses based on the v.2.2 P. chrysosporium genome identified 356 genes whose transcripts accumulated to relatively high levels at 96 h and were at least four times the levels found at 40 h. Transcripts encoding some lignin peroxidases, manganese peroxidases, and auxiliary enzymes thought to support their activity showed marked apparent upregulation. The data were also consistent with the production of ligninolytic extracellular reactive oxygen species by the action of manganese peroxidase-catalyzed lipid peroxidation, cellobiose dehydrogenase-catalyzed Fe(3+) reduction, and oxidase-catalyzed H2O2 production, but the data do not support a role for iron-chelating glycopeptides. In addition, transcripts encoding a variety of proteins with possible roles in lignin fragment uptake and processing, including 27 likely transporters and 18 cytochrome P450s, became more abundant after the onset of extracellular oxidation. Genes encoding cellulases showed little apparent upregulation and thus may be expressed constitutively. Transcripts corresponding to 165 genes of unknown function accumulated more than 4-fold after oxidation commenced, and some of them may merit investigation as possible contributors to ligninolysis.

Development and bin mapping of gene-associated interspecific SNPs for cotton (Gossypium hirsutum L.) introgression breeding efforts.

BACKGROUND: Cotton (Gossypium spp.) is the largest producer of natural fibers for textile and is an important crop worldwide. Crop production is comprised primarily of G. hirsutum L., an allotetraploid. However, elite cultivars express very small amounts of variation due to the species monophyletic origin, domestication and further bottlenecks due to selection. Conversely, wild cotton species harbor extensive genetic diversity of prospective utility to improve many beneficial agronomic traits, fiber characteristics, and resistance to disease and drought. Introgression of traits from wild species can provide a natural way to incorporate advantageous traits through breeding to generate higher-producing cotton cultivars and more sustainable production systems. Interspecific introgression efforts by conventional methods are very time-consuming and costly, but can be expedited using marker-assisted selection. RESULTS: Using transcriptome sequencing we have developed the first gene-associated single nucleotide polymorphism (SNP) markers for wild cotton species G. tomentosum, G. mustelinum, G. armourianum and G. longicalyx. Markers were also developed for a secondary cultivated species G. barbadense cv. 3-79. A total of 62,832 non-redundant SNP markers were developed from the five wild species which can be utilized for interspecific germplasm introgression into cultivated G. hirsutum and are directly associated with genes. Over 500 of the G. barbadense markers have been validated by whole-genome radiation hybrid mapping. Overall 1,060 SNPs from the five different species have been screened and shown to produce acceptable genotyping assays. CONCLUSIONS: This large set of 62,832 SNPs relative to cultivated G. hirsutum will allow for the first high-density mapping of genes from five wild species that affect traits of interest, including beneficial agronomic and fiber characteristics. Upon mapping, the markers can be utilized for marker-assisted introgression of new germplasm into cultivated cotton and in subsequent breeding of agronomically adapted types, including cultivar development.

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.

Spatial mapping of extracellular oxidant production by a white rot basidiomycete on wood reveals details of ligninolytic mechanism.

Oxidative cleavage of the recalcitrant plant polymer lignin is a crucial step in global carbon cycling, and is accomplished most efficiently by fungi that cause white rot of wood. These basidiomycetes secrete many enzymes and metabolites with proposed ligninolytic roles, and it is not clear whether all of these agents are physiologically important during attack on natural lignocellulosic substrates. One new approach to this problem is to infer properties of ligninolytic oxidants from their spatial distribution relative to the fungus on the lignocellulose. We grew Phanerochaete chrysosporium on wood sections in the presence of oxidant-sensing beads based on the ratiometric fluorescent dye BODIPY 581/591. The beads, having fixed locations relative to the fungal hyphae, enabled spatial mapping of cumulative extracellular oxidant distributions by confocal fluorescence microscopy. The results showed that oxidation gradients occurred around the hyphae, and data analysis using a mathematical reaction-diffusion model indicated that the dominant oxidant during incipient white rot had a half-life under 0.1 s. The best available hypothesis is that this oxidant is the cation radical of the secreted P. chrysosporium metabolite veratryl alcohol.

Improved Upland Cotton Germplasm for Multiple Fiber Traits Mediated by Transferring and Pyramiding Novel Alleles From Ethyl Methanesulfonate-Generated Mutant Lines Into Elite Genotypes.

Ethyl methanesulfonate (EMS) mutagenesis offers important advantages for improving crops, such as cotton, with limited diversity in elite gene pools. EMS-induced point mutations are less frequently associated with deleterious traits than alleles from wild or exotic germplasm. From 157 mutant lines that have significantly improved fiber properties, we focused on nine mutant lines here. A total of eight populations were developed by crossing mutant lines in different combinations into GA230 (GA2004230) background. Multiple lines in each population were significantly improved for the fiber trait that distinguished the donor parent(s), demonstrating that an elite breeding line (GA230) could be improved for fiber qualities using the mutant lines. Genotypes improved for multiple fiber traits of interest suggesting that allele pyramiding is possible. Compared to midparent values, individual progeny in the population conferred fiber quality improvements of as much as 31.7% (in population O) for micronaire (MIC), 16.1% (in population P) for length, 22.4% (in population K) for strength, 4.1% (in population Q) for uniformity, 45.8% (in population N) for elongation, and 13.9% (in population O) for lint percentage (lint%). While further testing for stability of the phenotype and estimation of yield potential is necessary, mutation breeding shows promise as an approach to reduce the problem of the genetic bottleneck of upland cotton. The populations developed here may also contribute to identifying candidate genes and causal mutations for fiber quality improvement.

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.

From Sequencing to Genome Editing for Cotton Improvement.

Traditional breeding techniques are proven, but additional knowledge learned from genome sequencing provides vast new data that might help identify gene targets for improving cotton sustainability. CRISPR/Cas9 provides a powerful tool for precision cotton breeding. Here, we discuss the opportunities and challenges of genome sequencing and editing for cotton improvement.