Regulatory controls of duplicated gene expression during fiber development in allotetraploid cotton.

Polyploidy complicates transcriptional regulation and increases phenotypic diversity in organisms. The dynamics of genetic regulation of gene expression between coresident subgenomes in polyploids remains to be understood. Here we document the genetic regulation of fiber development in allotetraploid cotton Gossypium hirsutum by sequencing 376 genomes and 2,215 time-series transcriptomes. We characterize 1,258 genes comprising 36 genetic modules that control staged fiber development and uncover genetic components governing their partitioned expression relative to subgenomic duplicated genes (homoeologs). Only about 30% of fiber quality-related homoeologs show phenotypically favorable allele aggregation in cultivars, highlighting the potential for subgenome additivity in fiber improvement. We envision a genome-enabled breeding strategy, with particular attention to 48 favorable alleles related to fiber phenotypes that have been subjected to purifying selection during domestication. Our work delineates the dynamics of gene regulation during fiber development and highlights the potential of subgenomic coordination underpinning phenotypes in polyploid plants.

Flame resistant cotton lines generated by synergistic epistasis in a MAGIC population.

Textiles made from cotton fibers are flammable and thus often include flame retardant additives for consumer safety. Transgressive segregation in multi-parent populations facilitates new combinations of alleles of genes and can result in traits that are superior to those of any of the parents. A screen of 257 recombinant inbred lines from a multi-parent advanced generation intercross (MAGIC) population for naturally enhance flame retardance (FR) was conducted. All eleven parents, like all conventional white fiber cotton cultivars produce flammable fabric. MAGIC recombinant inbred lines (RILs) that produced fibers with significantly lower heat release capacities (HRC) as measured by microscale combustion calorimetry (MCC) were identified and the stability of the phenotypes of the outliers were confirmed when the RILs were grown at an additional location. Of the textiles fabricated from the five superior RILs, four exhibited the novel characteristic of inherent flame resistance. When exposed to open flame by standard 45° incline flammability testing, these four fabrics self-extinguished. To determine the genetic architecture of this novel trait, linkage, epistatic and multi-locus genome wide association studies (GWAS) were conducted with 473k SNPs identified by whole genome sequencing (WGS). Transcriptomes of developing fiber cells from select RILs were sequenced (RNAseq). Together, these data provide insight into the genetic mechanism of the unexpected emergence of flame-resistant cotton by transgressive segregation in a breeding program. The incorporation of this trait into global cotton germplasm by breeding has the potential to greatly reduce the costs and impacts of flame-retardant chemicals.

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.

Genomic innovation and regulatory rewiring during evolution of the cotton genus Gossypium.

Phenotypic diversity and evolutionary innovation ultimately trace to variation in genomic sequence and rewiring of regulatory networks. Here, we constructed a pan-genome of the Gossypium genus using ten representative diploid genomes. We document the genomic evolutionary history and the impact of lineage-specific transposon amplification on differential genome composition. The pan-3D genome reveals evolutionary connections between transposon-driven genome size variation and both higher-order chromatin structure reorganization and the rewiring of chromatin interactome. We linked changes in chromatin structures to phenotypic differences in cotton fiber and identified regulatory variations that decode the genetic basis of fiber length, the latter enabled by sequencing 1,005 transcriptomes during fiber development. We showcase how pan-genomic, pan-3D genomic and genetic regulatory data serve as a resource for delineating the evolutionary basis of spinnable cotton fiber. Our work provides insights into the evolution of genome organization and regulation and will inform cotton improvement by enabling regulome-based approaches.

A deletion/duplication in the Ligon lintless-2 locus induces siRNAs that inhibit cotton fiber cell elongation.

Most cultivated cotton (Gossypium hirsutum L.) varieties have two types of seed fibers: short fuzz fiber strongly adhered to the seed coat, and long lint fiber used in the textile industry. The Ligon lintless-2 (Li2) cotton mutant has a normal vegetative phenotype but produces very short lint fiber on the seeds. The Li2 mutation is controlled by a single dominant gene. We discovered a large structural rearrangement at the end of chromosome D13 in the Li2 mutant based on whole-genome sequencing and genetic mapping of segregating populations. The rearrangement contains a 177-kb deletion and a 221-kb duplication positioned as a tandem inverted repeat. The gene Gh_D13G2437 is located at the junction of the inverted repeat in the duplicated region. During transcription such structure spontaneously forms self-complementary hairpin RNA of Gh_D13G2437 followed by production of small interfering RNA (siRNA). Gh_D13G2437 encodes a Ran-Binding Protein 1 (RanBP1) that preferentially expresses during cotton fiber elongation. The abundance of siRNA produced from Gh_D13G2437 reciprocally corresponds with the abundance of highly homologous (68%-98% amino acid sequence identity) RanBP1 family transcripts during fiber elongation, resulting in a shorter fiber phenotype in the Li2. Overexpression of Gh_D13G2437 in the Li2 mutant recovered the long lint fiber phenotype. Taken together, our findings revealed that siRNA-induced silencing of a family of RanBP1s inhibit elongation of cotton fiber cells in the Li2 mutant.

A GWAS identified a major QTL for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in a MAGIC population of Upland cotton and a meta-analysis of QTLs for Fusarium wilt resistance.

KEY MESSAGE: A major QTL conferring resistance to Fusarium wilt race 4 in a narrow region of chromosome D02 was identified in a MAGIC population of 550 RILs of Upland cotton. Numerous studies have been conducted to investigate the genetic basis of Fusarium wilt (FW, caused by Fusarium oxysporum f. sp. vasinfectum, FOV) resistance using bi-parental and association mapping populations in cotton. In this study, a multi-parent advanced generation inter-cross (MAGIC) population of 550 recombinant inbred lines (RILs), together with their 11 Upland cotton (Gossypium hirsutum) parents, was used to identify QTLs for FOV race 4 (FOV4) resistance. Among the parents, Acala Ultima, M-240 RNR, and Stoneville 474 were the most resistant, while Deltapine Acala 90, Coker 315, and Stoneville 825 were the most susceptible. Twenty-two MAGIC lines were consistently resistant to FOV4. Through a genome-wide association study (GWAS) based on 473,516 polymorphic SNPs, a major FOV4 resistance QTL within a narrow region on chromosomes D02 was detected, allowing identification of 14 candidate genes. Additionally, a meta-analysis of 133 published FW resistance QTLs showed a D subgenome and individual chromosome bias and no correlation between homeologous chromosome pairs. This study represents the first GWAS study using a largest genetic population and the most comprehensive meta-analysis for FW resistance in cotton. The results illustrated that 550 lines were not enough for high resolution mapping to pinpoint a candidate gene, and experimental errors in phenotyping cotton for FW resistance further compromised the accuracy and precision in QTL localization and identification of candidate genes. This study identified FOV4-resistant parents and MAGIC lines, and the first major QTL for FOV4 resistance in Upland cotton, providing useful information for developing FOV4-resistant cultivars and further genomic studies towards identification of causal genes for FOV4 resistance in cotton.

Dynamic 3D genome architecture of cotton fiber reveals subgenome-coordinated chromatin topology for 4-staged single-cell differentiation.

BACKGROUND: Despite remarkable advances in our knowledge of epigenetically mediated transcriptional programming of cell differentiation in plants, little is known about chromatin topology and its functional implications in this process. RESULTS: To interrogate its significance, we establish the dynamic three-dimensional (3D) genome architecture of the allotetraploid cotton fiber, representing a typical single cell undergoing staged development in plants. We show that the subgenome-relayed switching of the chromatin compartment from active to inactive is coupled with the silencing of developmentally repressed genes, pinpointing subgenome-coordinated contribution to fiber development. We identify 10,571 topologically associating domain-like (TAD-like) structures, of which 25.6% are specifically organized in different stages and 75.23% are subject to partition or fusion between two subgenomes. Notably, dissolution of intricate TAD-like structure cliques showing long-range interactions represents a prominent characteristic at the later developmental stage. Dynamic chromatin loops are found to mediate the rewiring of gene regulatory networks that exhibit a significant difference between the two subgenomes, implicating expression bias of homologous genes. CONCLUSIONS: This study sheds light on the spatial-temporal asymmetric chromatin structures of two subgenomes in the cotton fiber and offers a new insight into the regulatory orchestration of cell differentiation in plants.

Genomic interrogation of a MAGIC population highlights genetic factors controlling fiber quality traits in cotton.

Cotton (Gossypium hirsutum L.) fiber is the most important resource of natural and renewable fiber for the textile industry. However, the understanding of genetic components and their genome-wide interactions controlling fiber quality remains fragmentary. Here, we sequenced a multiple-parent advanced-generation inter-cross (MAGIC) population, consisting of 550 individuals created by inter-crossing 11 founders, and established a mosaic genome map through tracing the origin of haplotypes that share identity-by-descent (IBD). We performed two complementary GWAS methods-SNP-based GWAS (sGWAS) and IBD-based haplotype GWAS (hGWAS). A total of 25 sQTLs and 14 hQTLs related to cotton fiber quality were identified, of which 26 were novel QTLs. Two major QTLs detected by both GWAS methods were responsible for fiber strength and length. The gene Ghir_D11G020400 (GhZF14) encoding the MATE efflux family protein was identified as a novel candidate gene for fiber length. Beyond the additive QTLs, we detected prevalent epistatic interactions that contributed to the genetics of fiber quality, pinpointing another layer for trait variance. This study provides new targets for future molecular design breeding of superior fiber quality.

Mapping-by-sequencing the locus of EMS-induced mutation responsible for tufted-fuzzless seed phenotype in cotton.

Cotton fiber mutants are valuable resources for studying functions of altered genes and their roles in fiber development. The n4t is a recessive tufted-fuzzless seed mutant created through chemical mutagenesis with ethyl methanesulfonate. Genetic analysis indicated that the tufted-fuzzless phenotype is controlled by a single recessive locus. In this study, we developed an F2 population of 602 progeny plants and sequenced the genomes of the parents and two DNA bulks from F2 progenies showing the mutant phenotype. We identified DNA sequence variants between the tufted-fuzzless mutant and wild type by aligning the sequence reads to the reference TM-1 genome and designed subgenome-specific SNP markers. We mapped the n4t locus on chromosome D04 within a genomic interval of about 411 kb. In this region, seven genes showed significant differential expression between the tufted-fuzzless mutant and wild type. Possible candidate genes are discussed in this study. The utilization of the n4t mutant along with other fiber mutants will facilitate our understanding of the molecular mechanisms of cotton fiber cell growth and development.

Surface and Thermal Characterization of Cotton Fibers of Phenotypes Differing in Fiber Length.

Cotton is one of the most important and widely grown crops in the world. Understanding the synthesis mechanism of cotton fiber elongation can provide valuable tools to the cotton industry for improving cotton fiber yield and quality at the molecular level. In this work, the surface and thermal characteristics of cotton fiber samples collected from a wild type (WT) and three mutant lines (Li1, Li2-short, Li2-long, Li2-mix, and liy) were comparatively investigated. Microimaging revealed a general similarity trend of WT ≥ Li2-long ≈ Li2-mix > Li1 > Li2 short ≈ liy with Ca detected on the surface of the last two. Attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and thermogravimetric measurements also showed that Li2-short and liy were more similar to each other, and Li2-long and Li2-mix closer to WT while Li1 was quite independent. FT-IR results further demonstrated that wax and amorphous cellulose were co-present in fiber structures during the fiber formation processes. The correlation analysis found that the FT-IR-based maturity parameter was well correlated (p ≤ 0.05) to the onset decomposition temperature and all three weight-loss parameters at onset, peak, and end decomposition stages, suggesting that the maturity degree is a better parameter than crystallinity index (CI) and other FT-IR parameters that reflect the thermal stability of the cotton fiber. In summary, this work demonstrated that genetic mutation altered the surface and thermal characteristics in the same way for Li2-short and liy, but with different mechanisms for the other three mutant cotton fiber samples.

GWAS reveals consistent QTL for drought and salt tolerance in a MAGIC population of 550 lines derived from intermating of 11 Upland cotton (Gossypium hirsutum) parents.

Cotton is grown in arid and semi-arid regions where abiotic stresses such as drought and salt are prevalent. There is a lack of studies that simultaneously address the genetic and genomic basis of tolerance to drought and salt stress. In this study, a multi-parent advanced generation inter-cross (MAGIC) population of 550 recombinant inbred lines (RILs) together with their 11 Upland cotton parents with a total of 473,516 polymorphic SNP markers was used to identify quantitative trait loci (QTL) for drought tolerance (DT) and salt tolerance (ST) at the seedling stage based on two replicated greenhouse tests. Transgressive segregation occurred in the MAGIC-RILs, indicating that tolerant and sensitive alleles recombined for tolerance to the abiotic stress during the intermating process for the population development. A total of 20 QTL were detected for DT including 13 and 7 QTL based on plant height (PH) and dry shoot weight (DSW), respectively; and 23 QTL were detected for ST including 12 and 11 QTL for PH and DSW, respectively. There were several chromosomes with QTL clusters for abiotic stress tolerance including four QTL on chromosome A13 and three QTL on A01 for DT, and four QTL on D08 and three QTL on A11 for ST. Nine QTL (21% of the 43 QTL) detected were in common between DT and ST, indicating a common genetic basis for DT and ST. The narrow chromosomal regions for most of the QTL detected in this study allowed identification of 53 candidate genes associated with responses to salt and drought stress and abiotic stimulus. The QTL identified for both DT and ST have significantly augmented the repertoire of QTL for abiotic stress tolerance that can be used for marker-assisted selection to develop cultivars with resilience to drought and/or salt and further genomic studies towards the identification of drought and salt tolerance genes in cotton.

Elucidation of sequence polymorphism in fuzzless-seed cotton lines.

Most commercially produced cotton cultivars have two types of fibers on the seed coat, short fuzz and long lint. Lint fiber is used in the textile industry, while fuzz is considered an undesirable trait. Both types of fibers are believed to be controlled by the same regulators; however, their mechanisms of actions are still obscure. Cotton fiber mutants provide an excellent system to study the genes that regulate fiber development. Here we described four uncharacterized and three previously reported cotton mutants with fuzzless seed phenotypes. To evaluate whether or not the genes previously associated with fuzzless seed phenotypes have mutations we sequenced whole genomic DNA of seven mutants and wild type varieties. We identified multiple polymorphic changes among the tested genes. Non-synonymous SNPs in the coding region of the MML3-A gene was common in the six mutant lines tested in this study, showing both dominant and recessive fuzzless phenotypes. We have mapped the locus of the causative mutation for one of the uncharacterized fuzzless lines using an F2 population that originated from a cross between the dominant fuzzless mutant and a wild type. Further, we have clarified the current knowledge about the causative n2 mutations by analyzing the sequence data and previously reported mapping data. The key genes and possible mechanisms of fiber differentiation are discussed in this study.

Combined GWAS and eQTL analysis uncovers a genetic regulatory network orchestrating the initiation of secondary cell wall development in cotton.

The cotton fibre serves as a valuable experimental system to study cell wall synthesis in plants, but our understanding of the genetic regulation of this process during fibre development remains limited. We performed a genome-wide association study (GWAS) and identified 28 genetic loci associated with fibre quality in allotetraploid cotton. To investigate the regulatory roles of these loci, we sequenced fibre transcriptomes of 251 cotton accessions and identified 15 330 expression quantitative trait loci (eQTL). Analysis of local eQTL and GWAS data prioritised 13 likely causal genes for differential fibre quality in a transcriptome-wide association study (TWAS). Characterisation of distal eQTL revealed unequal genetic regulation patterns between two subgenomes, highlighted by an eQTL hotspot (Hot216) that established a genome-wide genetic network regulating the expression of 962 genes. The primary regulatory role of Hot216, and specifically the gene encoding a KIP-related protein, was found to be the transcriptional regulation of genes responsible for cell wall synthesis, which contributes to fibre length by modulating the developmental transition from rapid cell elongation to secondary cell wall synthesis. This study uncovered the genetic regulation of fibre-cell development and revealed the molecular basis of the temporal modulation of secondary cell wall synthesis during plant cell elongation.

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.

An EMS-induced mutation in a tetratricopeptide repeat-like superfamily protein gene (Ghir_A12G008870) on chromosome A12 is responsible for the liy short fiber phenotype in cotton.

KEY MESSAGE: The EMS-induced threonine/isoleucine substitution in a tetratricopeptide repeat-like superfamily protein encoded by gene Ghir_A12G008870 is responsible for the Ligon-lintless-y (liy) short fiber phenotype in cotton. A short fiber mutant Ligon-lintless-y was created through treating the seeds of the cotton line MD15 with ethyl methanesulfonate. Genetic analysis indicated that the short fiber phenotype is controlled by a single recessive locus designated liy. From F2 populations derived from crosses between the mutant and its wild type (WT), we selected 132 short fiber progeny (liy/liy) and made two DNA bulks. We sequenced these DNA bulks along with the two parents of the population. The liy locus was located on chromosome A12. Using multiple F2 populations and F3 progeny plants, we mapped the liy locus within a genomic region of 1.18 Mb. In this region, there is only one gene, i.e., Ghir_A12G008870 encoding a tetratricopeptide repeat-like superfamily protein that has a non-synonymous mutation between the liy mutant and its WT. Analysis of a SNP marker representing this gene in the F2 and F3 progeny plants demonstrated its complete linkage with the liy short fiber phenotype. We further analyzed this SNP marker in a panel of 384 cotton varieties. The mutant allele is absent in all varieties analyzed. RNAseq and RT-qPCR analysis of the gene Ghir_A12G008870 during fiber development showed a significant expression difference between the liy mutant and its WT in developing fiber cells beginning at 12 days post-anthesis. Virus-induced gene silencing of the gene Ghir_A12G008870 significantly reduced the fiber length of the WT cotton line MD15. Taken together, our results suggest that the gene Ghir_A12G008870 is involved in the cotton fiber cell elongation process and is a promising candidate gene responsible for the liy short fiber phenotype.

Genetic and transcriptomic dissection of the fiber length trait from a cotton (Gossypium hirsutum L.) MAGIC population.

BACKGROUND: Improving cotton fiber length without reducing yield is one of the major goals of cotton breeding. However, genetic improvement of cotton fiber length by breeding has been a challenge due to the narrow genetic diversity of modern cotton cultivars and negative correlations between fiber quality and yield traits. A multi-parent advanced generation inter-cross (MAGIC) population developed through random mating provides an excellent genetic resource that allows quantitative trait loci (QTL) and causal genes to be identified. RESULTS: An Upland cotton MAGIC population, consisting of 550 recombinant inbred lines (RILs) derived from eleven different cultivars, was used to identify fiber length QTLs and potential genes that contribute to longer fibers. A genome wide association study (GWAS) identified a cluster of single nucleotide polymorphisms (SNPs) on chromosome (Chr.) D11 that is significantly associated with fiber length. Further evaluation of the Chr. D11 genomic region among lines of the MAGIC population detected that 90% of RILs have a D11 haplotype similar to the reference TM-1 genome (D11-ref), whereas 10% of RILs inherited an alternative haplotype from one of the parents (D11-alt). The average length of fibers of D11-alt RILs was significantly shorter compared to D11-ref RILs, suggesting that alleles in the D11-alt haplotype contributed to the inferior fiber quality. RNAseq analysis of the longest and shortest fiber length RILs from D11-ref and D11-alt populations identified 949 significantly differentially expressed genes (DEGs). Gene set enrichment analysis revealed that different functional categories of genes were over-represented during fiber elongation between the four selected RILs. We found 12 genes possessing non-synonymous SNPs (nsSNPs) significantly associated with the fiber length, and three that were highly significant and were clustered at D11:24-Mb, including D11G1928, D11G1929 and D11G1931. CONCLUSION: The results of this study provide insights into molecular aspects of genetic variation in fiber length and suggests candidate genes for genetic manipulation for cotton improvement.

A novel variant of Gh_D02G0276 is required for root-knot nematode resistance on chromosome 14 (D02) in Upland cotton.

KEY MESSAGE: MAGIC population sequencing and virus-induced gene silencing identify Gh_D02G0276 as a novel root-knot nematode resistance gene on chromosome 14 in Upland cotton. The southern root-knot nematode [RKN; Meloidogyne incognita (Kofoid & White)] remains the primary yield-limiting biotic stress to Upland cotton (Gossypium hirsutum L.) throughout the southeastern USA. While useful genetic markers have been developed for two major RKN resistance loci on chromosomes 11 (A11) and 14 (D02), these markers are not completely effective because the causative genes have not been identified. Here, we sequenced 550 recombinant inbred lines (RILs) from a multi-parent advanced generation intercross (MAGIC) population to identify five RILs that had informative recombinations near the D02-RKN resistance locus. The RKN resistance phenotypes of these five RILs narrowed the D02-RKN locus to a 30-kb region with four candidate genes. We conducted virus-induced gene silencing (VIGS) on each of these genes and found that Gh_D02G0276 was required for suppression of RKN egg production conferred by the Chr. D02 resistance gene. The resistant lines all possessed an allele of Gh_D02G0276 that showed non-synonymous mutations and was prematurely truncated. Furthermore, a Gh_D02G0276-specific marker for the resistance allele variant was able to identify RKN-resistant germplasm from a collection of 367 cotton accessions. The Gh_D02G0276 peptide shares similarity with domesticated hAT-like transposases with additional novel N- and C-terminal domains that resemble the target of known RKN effector molecules and a prokaryotic motif, respectively. The truncation in the resistance allele results in a loss of a plant nuclear gene-specific C-terminal motif, potentially rendering this domain antigenic due to its high homology with bacterial proteins. The conclusive identification of this RKN resistance gene opens new avenues for understanding plant resistance mechanisms to RKN as well as opportunities to develop more efficient marker-assisted selection in cotton breeding programs.

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.

The P450 gene CYP749A16 is required for tolerance to the sulfonylurea herbicide trifloxysulfuron sodium in cotton (Gossypium hirsutum L.).

BACKGROUND: Weed management is critical to global crop production and is complicated by rapidly evolving herbicide resistance in weeds. New sources of herbicide resistance are needed for crop plants so that applied herbicides can be rotated or combined to thwart the evolution of resistant weeds. The diverse family of cytochrome P450 proteins has been suggested to be a source of detoxifying herbicide metabolism in both weed and crop plants, and greater understanding of these genes will offer avenues for crop improvement and novel weed management practices. RESULTS: Here, we report the identification of CYP749A16 (Gh_D10G1401) which is responsible for the natural tolerance exhibited by most cotton, Gossypium hirsutum L., cultivars to the herbicide trifloxysulfuron sodium (TFS, CGA 362622, commercial formulation Envoke). A 1-bp frameshift insertion in the third exon of CYP749A16 results in the loss of tolerance to TFS. The DNA marker designed from this insertion perfectly co-segregated with the phenotype in 2145 F2 progeny of a cross between the sensitive cultivar Paymaster HS26 and tolerant cultivar Stoneville 474, and in 550 recombinant inbred lines of a multi-parent advanced generation inter-cross population. Marker analysis of 382 additional cotton cultivars identified twelve cultivars containing the 1-bp frameshift insertion. The marker genotypes matched perfectly with phenotypes in 188 plants from the selected twelve cultivars. Virus-induced gene silencing of CYP749A16 generated sensitivity in the tolerant cotton cultivar Stoneville 474. CONCLUSIONS: CYP749A16 located on chromosome D10 is required for TFS herbicide tolerance in cotton. This finding should add to the repertoire of tools available to farmers and breeders for the advancement of agricultural productivity.

Genome-wide analysis of gene expression of EMS-induced short fiber mutant Ligon lintless-y (liy) in cotton (Gossypium hirsutum L.).

In this work we describe a chemically-induced short fiber mutant cotton line, Ligon-lintless-y (liy), which is controlled by a single recessive locus and affects multiple traits, including height of the plant, and length and maturity of fiber. An RNAseq analysis was used to evaluate global transcriptional changes during cotton fiber development at 3, 8 and 16days post anthesis. We found that 613, 2629 and 3397 genes were significantly down-regulated, while 2700, 477 and 3260 were significantly up-regulated in liy at 3, 8 and 16 DPA. Gene set enrichment analysis revealed that many metabolic pathways, including carbohydrate, cell wall, hormone metabolism and transport were substantially altered in liy developing fibers. We discuss perturbed expression of genes involved in signal transduction and biosynthesis of phytohormones, such as auxin, abscisic acid, gibberellin and ethylene. The results of this study provide new insights into transcriptional regulation of cotton fiber development.