N6-methyladenosine RNA modification regulates photoperiod sensitivity in cotton.

The methylation of N6-methyladenosine (m6A) involves writers, erasers, and readers, acting synergistically in posttranscriptional regulation. These processes influence various biological processes, including plant floral transition. However, the specific role of m6A modifications in photoperiod sensitivity in cotton (Gossypium hirsutum) remains obscure. To elucidate this, in this study, we conducted transcriptome-wide m6A sequencing during critical flowering transition stages in the photoperiod-sensitive wild G. hirsutum var. yucatanense (yucatanense) and the photoperiod-insensitive cultivated cotton G. hirsutum acc. TM-1 (TM-1). Our results revealed significant variations in m6A methylation of 2 cotton varieties, with yucatanense exhibiting elevated m6A modification levels compared with TM-1 under long-day conditions. Notably, distinct m6A peaks between TM-1 and yucatanense correlated significantly with photoperiod sensitivity. Moreover, our study highlighted the role of the demethylase G. hirsutum ALKB homolog 5 (GhALKBH5) in modulating m6A modification levels. Silencing GhALKBH5 led to a decreased mRNA level of key photoperiodic flowering genes (GhADO3, GhAGL24, and GhFT1), resulting in delayed bud emergence and flowering. Reverse transcription quantitative PCR analyses confirmed that silencing GhADO3 and GhAGL24 significantly downregulated the expression of the floral integrator GhFT1. Collectively, our findings unveiled a transcriptional regulatory mechanism in which GhALKBH5-mediated m6A demethylation of crucial photoperiodic flowering transcripts modulated photoperiod sensitivity in cotton.

Dynamic patterns of gene expressional and regulatory variations in cotton heterosis.

Purpose: Although the application of heterosis has significantly increased crop yield over the past century, the mechanisms underlying this phenomenon still remain obscure. Here, we applied transcriptome sequencing to unravel the impacts of parental expression differences and transcriptomic reprogramming in cotton heterosis. Methods: A high-quality transcriptomic atlas covering 15 developmental stages and tissues was constructed for XZM2, an elite hybrid of upland cotton (Gossypium hirsutum L.), and its parental lines, CRI12 and J8891. This atlas allowed us to identify gene expression differences between the parents and to characterize the transcriptomic reprogramming that occurs in the hybrid. Results: Our analysis revealed abundant gene expression differences between the parents, with pronounced tissue specificity; a total of 1,112 genes exhibited single-parent expression in at least one tissue. It also illuminated transcriptomic reprogramming in the hybrid XZM2, which included both additive and non-additive expression patterns. Coexpression networks between parents and hybrid constructed via weighted gene coexpression network analysis identified modules closely associated with fiber development. In particular, key regulatory hub genes involved in fiber development showed high-parent dominant or over dominant patterns in the hybrid, potentially driving the emergence of heterosis. Finally, high-depth resequencing data was generated and allele-specific expression patterns examined in the hybrid, enabling the dissection of cis- and trans-regulation contributions to the observed expression differences. Conclusion: Parental transcriptional differences and transcriptomic reprogramming in the hybrid, especially the non-additive upregulation of key genes, play an important role in shaping heterosis. Collectively, these findings provide new insights into the molecular basis of heterosis in cotton.

Impacts of parental genomic divergence in non-syntenic regions on cotton heterosis.

INTRODUCTION: Heterosis has revolutionized crop breeding, enhancing global agricultural production. However, the mechanisms underlying heterosis remain obscure. Xiangzamian 2# (XZM2), a super hybrid upland cotton (Gossypium hirsutum L.) characterized by high-yield heterosis, has been developed and extensively planted in China. OBJECTIVES: We conducted a systematic analysis of CRI12 and J8891, two parents of XZM2. We aimed to reveal the precise genetic information and the role of non-syntenic divergence in shaping heterosis, laying a foundation for advancing understanding of heterosis. METHODS: We de novo assembled high-quality genomes of CRI12 and J8891, and further uncovered abundant genetic variations and non-syntenic regions between the parents. Whole-genome comparison, association analysis, transcriptomic analysis and relative identity-by-descent (rIBD) estimation were conducted to identify structural variations (SVs) and introgressions within non-syntenic blocks and to analyze their impacts on promoting heterosis. RESULTS: Parental genetic divergence increased in non-syntenic regions. Furthermore, these regions, accounting for only 16.71% of the total genome, contained more loci with significantly higher heterotic effects, far exceeding the syntenic background. SVs covered 97.26% of non-syntenic sequences and caused widespread gene expression differences in these regions, driving dynamic complementation of gene expression in the hybrid. A set of SVs were responsible for trait improvement and had positive effects on heterosis, contributing larger heritability than short variations. We characterized numerous parental-specific introgressions from G. barbadense. Specifically, a functional introgression segment within non-syntenic blocks introduced an elite haplotype, which significantly increased lint yield and enhanced heterosis. CONCLUSION: Our study clarified non-syntenic regions to harbor more loci with higher heterotic effects, revealed their importance in promoting heterosis and supported the crucial role of genetic complementation in heterosis. SVs and introgressions were identified as key factors responsible for non-syntenic divergence between the parents. They had important effects on gene expression and trait improvement, positively contributing to heterosis.

Unravelling the genetic basis and regulation networks related to fibre quality improvement using chromosome segment substitution lines in cotton.

The elucidation of genetic architecture and molecular regulatory networks underlying complex traits remains a significant challenge in life science, largely due to the substantial background effects that arise from epistasis and gene-environment interactions. The chromosome segment substitution line (CSSL) is an ideal material for genetic and molecular dissection of complex traits due to its near-isogenic properties; yet a comprehensive analysis, from the basic identification of substitution segments to advanced regulatory network, is still insufficient. Here, we developed two cotton CSSL populations on the Gossypium hirsutum background, representing wide adaptation and high lint yield, with introgression from G. barbadense, representing superior fibre quality. We sequenced 99 CSSLs that demonstrated significant differences from G. hirsutum in fibre, and characterized 836 dynamic fibre transcriptomes in three crucial developmental stages. We developed a workflow for precise resolution of chromosomal substitution segments; the genome sequencing revealed substitutions collectively representing 87.25% of the G. barbadense genome. Together, the genomic and transcriptomic survey identified 18 novel fibre-quality-related quantitative trait loci with high genetic contributions and the comprehensive landscape of fibre development regulation. Furthermore, analysis determined unique cis-expression patterns in CSSLs to be the driving force for fibre quality alteration; building upon this, the co-expression regulatory network revealed biological relationships among the noted pathways and accurately described the molecular interactions of GhHOX3, GhRDL1 and GhEXPA1 during fibre elongation, along with reliable predictions for their interactions with GhTBA8A5. Our study will enhance more strategic employment of CSSL in crop molecular biology and breeding programmes.

UDP-glucosyltransferase 71C4 controls the flux of phenylpropanoid metabolism to shape cotton seed development.

Seeds play a crucial role in plant reproduction, making it essential to identify genes that affect seed development. In this study, we focused on UDP-glucosyltransferase 71C4 (UGT71C4) in cotton, a member of the glycosyltransferase family that shapes seed width and length, thereby influencing seed index and seed cotton yield. Overexpression of UGT71C4 results in seed enlargement owing to its glycosyltransferase activity on flavonoids, which redirects metabolic flux from lignin to flavonoid metabolism. This shift promotes cell proliferation in the ovule via accumulation of flavonoid glycosides, significantly enhancing seed cotton yield and increasing the seed index from 10.66 g to 11.91 g. By contrast, knockout of UGT71C4 leads to smaller seeds through activation of the lignin metabolism pathway and redirection of metabolic flux back to lignin synthesis. This redirection leads to increased ectopic lignin deposition in the ovule, inhibiting ovule growth and development, and alters yield components, increasing the lint percentage from 41.42% to 43.40% and reducing the seed index from 10.66 g to 8.60 g. Our research sheds new light on seed size development and reveals potential pathways for enhancing seed yield.

Genomic insights into local adaptation of upland cotton in China and Pakistan.

KEY MESSAGE: Different kinship and resistance to cotton leaf curl disease (CLCuD) and heat were found between upland cotton cultivars from China and Pakistan. 175 SNPs and 82 InDels loci related to yield, fiber quality, CLCuD, and heat resistance were identified. Elite alleles found in Pakistani accessions aided local adaptation to climatic condition of two countries. Adaptation of upland cotton (Gossypium hirsutum) beyond its center of origin is expected to be driven by tailoring of the genome and genes to enhance yield and quality in new ecological niches. Here, resequencing of 456 upland cotton accessions revealed two distinct kinships according to the associated country. Fiber quality and lint percentage were consistent across kinships, but resistance to cotton leaf curl disease (CLCuD) and heat was distinctly exhibited by accessions from Pakistan, illustrating highly local adaption. A total of 175 SNP and 82 InDel loci related to yield, fiber quality, CLCuD and heat resistance were identified; among them, only two overlapped between Pakistani and Chinese accessions underscoring the divergent domestication and improvement targets in each country. Loci associated with resistance alleles to leaf curl disease and high temperature were largely found in Pakistani accessions to counter these stresses prevalent in Pakistan. These results revealed that breeding activities led to the accumulation of unique alleles and helped upland cotton become adapted to the respective climatic conditions, which will contribute to elucidating the genetic mechanisms that underlie resilience traits and help develop climate-resilient cotton cultivars for use worldwide.

GHCU, a Molecular Chaperone, Regulates Leaf Curling by Modulating the Distribution of KNGH1 in Cotton.

Leaf shape is considered to be one of the most significant agronomic traits in crop breeding. However, the molecular basis underlying leaf morphogenesis in cotton is still largely unknown. In this study, through genetic mapping and molecular investigation using a natural cotton mutant cu with leaves curling upward, the causal gene GHCU is successfully identified as the key regulator of leaf flattening. Knockout of GHCU or its homolog in cotton and tobacco using CRISPR results in abnormal leaf shape. It is further discovered that GHCU facilitates the transport of the HD protein KNOTTED1-like (KNGH1) from the adaxial to the abaxial domain. Loss of GHCU function restricts KNGH1 to the adaxial epidermal region, leading to lower auxin response levels in the adaxial boundary compared to the abaxial. This spatial asymmetry in auxin distribution produces the upward-curled leaf phenotype of the cu mutant. By analysis of single-cell RNA sequencing and spatiotemporal transcriptomic data, auxin biosynthesis genes are confirmed to be expressed asymmetrically in the adaxial-abaxial epidermal cells. Overall, these findings suggest that GHCU plays a crucial role in the regulation of leaf flattening through facilitating cell-to-cell trafficking of KNGH1 and hence influencing the auxin response level.

Gossypium purpurascens genome provides insight into the origin and domestication of upland cotton.

INTRODUCTION: Allotetraploid upland cotton (Gossypium hirsutum L.) is native to the Mesoamerican and Caribbean regions, had been improved in the southern United States by the mid-eighteenth century, was then dispersed worldwide. However, a Hainan Island Native Cotton (HIC) has long been grown extensively on Hainan Island, China. OBJECTIVES: Explore HIC's evolutionary relationship and genomic diversity with other tetraploid cottons, its origin and whether it was used for YAZHOUBU (Yazhou cloth, World Intangible Cultural Heritage) weaving, and the role of structural variations (SVs) in upland cotton domestication. METHODS: We assembled a high-quality genome of one HIC plant. We performed phylogenetic analysis, divergence time estimation, principal component analysis and population differentiation estimation using cotton assemblies and/or resequencing data. SVs were detected by whole-genome comparison. A F2 population was used for linkage analysis and to study effects of SVs. Buoyancy and salt water tolerance tests for seeds were conducted. RESULTS: We found that the HIC belongs to G. purpurascens. G. purpurascens is best classified as a primitive race of G. hirsutum. The potential for long range transoceanic dispersal of G. purpurascens seeds was proved. A set of SVs, selective sweep regions between G. hirsutum races and cultivars, and quantitative trait loci (QTLs) of eleven agronomic traits were obtained. SVs, especially large-scale SVs, were found to have important effects on cotton domestication and improvement. Of them, eight large-scale inversions strongly associated with yield and fiber quality have probably undergone artificial selection in domestication. CONCLUSION: G. purpurascens including HIC is a primitive race of G. hirsutum, probably disperse to Hainan from Central America by floating on ocean currents, may have been partly domesticated, planted and was likely used for YAZHOUBU weaving in Hainan much earlier than the Pre-Columbian period. SV plays an important role in cotton domestication and improvement.

GoSTR, a negative modulator of stem trichome formation in cotton.

Trichomes, the outward projection of plant epidermal tissue, provide an effective defense against stress and insect pests. Although numerous genes have been identified to be involved in trichome development, the molecular mechanism for trichome cell fate determination is not well enunciated. Here, we reported GoSTR functions as a master repressor for stem trichome formation, which was isolated by map-based cloning based on a large F2 segregating population derived from a cross between TM-1 (pubescent stem) and J220 (smooth stem). Sequence alignment revealed a critical G-to-T point mutation in GoSTR's coding region that converted codon 2 from GCA (Alanine) to TCA (Serine). This mutation occurred between the majority of Gossypium hirsutum with pubescent stem (GG-haplotype) and G. barbadense with glabrous stem (TT-haplotype). Silencing of GoSTR in J220 and Hai7124 via virus-induced gene silencing resulted in the pubescent stems but no visible change in leaf trichomes, suggesting stem trichomes and leaf trichomes are genetically distinct. Yeast two-hybrid assay and luciferase complementation imaging assay showed GoSTR interacts with GoHD1 and GoHOX3, two key regulators of trichome development. Comparative transcriptomic analysis further indicated that many transcription factors such as GhMYB109, GhTTG1, and GhMYC1/GhDEL65 which function as positive regulators of trichomes were significantly upregulated in the stem from the GoSTR-silencing plant. Taken together, these results indicate that GoSTR functions as an essential negative modulator of stem trichomes and its transcripts will greatly repress trichome cell differentiation and growth. This study provided valuable insights for plant epidermal hair initiation and differentiation research.

Genomic insights into the genetic basis of cotton breeding in China.

The excellent Upland cotton (Gossypium hirsutum) cultivars developed since 1949 have made a huge contribution to cotton production in China, the world's largest producer and consumer of cotton. However, the genetic and genomic basis for the improvements of these cotton cultivars remains largely unclear. In this study, we selected 16 Upland cotton cultivars with important historical status in Chinese cotton breeding and constructed a multiparent, advanced generation, intercross (MAGIC) population comprising 920 recombinant inbred lines. A genome-wide association study using the MAGIC population identified 54 genomic loci associated with lint yield and fiber quality. Of them, 25 (46.30%) pleiotropic genomic loci cause simultaneous changes of lint yield and/or fiber quality traits, revealing complex trade-offs and linkage drags in Upland cotton agronomic traits. Deep sequencing data of 11 introduced ancestor cultivars and publicly available resequencing datasets of 839 cultivars developed in China during the past 70 years were integrated to explore the historical distribution and origin of the elite or selected alleles. Interestingly, 85% of these elite alleles were selected and fixed from different American ancestors, consistent with cotton breeding practices in China. However, seven elite alleles of native origin that are responsible for Fusarium wilt resistance, early maturing, good-quality fiber, and other characteristics were not found in American ancestors but have greatly contributed to Chinese cotton breeding and wide cultivation. Taken together, these results provide a genetic basis for further improving cotton cultivars and reveal that the genetic composition of Chinese cotton cultivars is narrow and mainly derived from early introduced American varieties.

Structural variation (SV)-based pan-genome and GWAS reveal the impacts of SVs on the speciation and diversification of allotetraploid cottons.

Structural variations (SVs) have long been described as being involved in the origin, adaption, and domestication of species. However, the underlying genetic and genomic mechanisms are poorly understood. Here, we report a high-quality genome assembly of Gossypium barbadense acc. Tanguis, a landrace that is closely related to formation of extra-long-staple (ELS) cultivated cotton. An SV-based pan-genome (Pan-SV) was then constructed using a total of 182 593 non-redundant SVs, including 2236 inversions, 97 398 insertions, and 82 959 deletions from 11 assembled genomes of allopolyploid cotton. The utility of this Pan-SV was then demonstrated through population structure analysis and genome-wide association studies (GWASs). Using segregation mapping populations produced through crossing ELS cotton and the landrace along with an SV-based GWAS, certain SVs responsible for speciation, domestication, and improvement in tetraploid cottons were identified. Importantly, some of the SVs presently identified as associated with the yield and fiber quality improvement had not been identified in previous SNP-based GWAS. In particular, a 9-bp insertion or deletion was found to associate with elimination of the interspecific reproductive isolation between Gossypium hirsutum and G. barbadense. Collectively, this study provides new insights into genome-wide, gene-scale SVs linked to important agronomic traits in a major crop species and highlights the importance of SVs during the speciation, domestication, and improvement of cultivated crop species.

Duplicate mutations of GhCYP450 lead to the production of ms5m6 male sterile line in cotton.

KEY MESSAGE: The duplicated male sterile genes ms5m6 in cotton were map-based cloned and validated by the virus-induced gene silencing assays. Duplicate mutations of the GhCYP450 gene encoding a cytochrome P450 protein are responsible for the male sterility in cotton. The utilization of male sterility in cotton plays a vital role in improving yield and fiber quality. A complete male sterile line (ms5ms6) has been extensively used to develop hybrid cotton worldwide. Using Zhongkang-A (ZK-A) developed by transferring Bt and ms5ms6 genes into the commercial cultivar Zhongmiansuo 12, the duplicate genes were map-based cloned and confirmed via the virus-induced gene silencing (VIGS) assays. The duplicate mutations of GhCYP450 genes encoding a cytochrome P450 protein were responsible for producing male sterility in ms5ms6 in cotton. Sequence alignment showed that GhCYP450-Dt in ZK-A differed in two critical aspects from the fertile wild-type TM-1: GhCYP450-Dt has three amino acid (D98E, E168K, G198R) changes in the coding region and a 7-bp (GGAAAAA) insertion in the promoter domain; GhCYP450-At appears to be premature termination of GhCYP450 translation. Further morphological observation and cytological examination of GhCYP450-silenced plants induced by VIGS exhibited shorter filaments and no mature pollen grains. These results indicate that GhCYP450 is essential for pollen exine formation and pollen development for male fertility. Investigating the mechanisms of ms5ms6 male sterility will deepen our understanding of the development and utilization of heterosis.

Identification of elite fiber quality loci in upland cotton based on the genotyping-by-target-sequencing technology.

Genome-wide association studies (GWAS) of fiber quality traits of upland cotton were conducted to identify the single-nucleotide polymorphic (SNP) loci associated with cotton fiber quality, which lays the foundation for the mining of elite] cotton fiber gene resources and its application in molecular breeding. A total of 612 upland cotton accessions were genotyped using the ZJU Cotton Chip No. 1 40K chip array via the liquid-phase probe hybridization-based genotyping-by-target-sequencing (GBTS) technology. In the present study, five fiber quality traits, namely fiber length, fiber strength, micronaire, uniformity and elongation, showed different degrees of variation in different environments. The average coefficient of variation of fiber strength was the greatest, whereas the average coefficient of variation of uniformity was the least. Significant or extremely significant correlations existed among the five fiber quality traits, especially fiber length, strength, uniformity and elongation all being significantly negative correlated with micronaire. Population cluster analysis divided the 612 accessions into four groups: 73 assigned to group I, 226 to group II, 220 to group III and 93 to group IV. Genome-wide association studies of five fiber quality traits in five environments was performed and a total of 42 SNP loci associated with target traits was detected, distributed on 19 chromosomes, with eight loci associated with fiber length, five loci associated with fiber strength, four loci associated with micronaire, twelve loci associated with fiber uniformity and thirteen loci associated with fiber elongation. Of them, seven loci were detected in more than two environments. Nine SNP loci related to fiber length, fiber strength, uniformity and elongation were found on chromosome A07, seven loci related to fiber length, fiber strength, micronaire and elongation were detected on chromosome D01, and five loci associated with fiber length, uniformity and micronaire were detected on chromosome D11. The results from this study could provide more precise molecular markers and genetic resources for cotton breeding for better fiber quality in the future.

Visible gland constantly traces virus-induced gene silencing in cotton.

A virus-induced gene silencing (VIGS) system was established to induce endogenous target gene silencing by post-transcriptional gene silencing (PTGS), which is a powerful tool for gene function analysis in plants. Compared with stable transgenic plant via Agrobacterium-mediated gene transformation, phenotypes after gene knockdown can be obtained rapidly, effectively, and high-throughput through VIGS system. This approach has been successfully applied to explore unknown gene functions involved in plant growth and development, physiological metabolism, and biotic and abiotic stresses in various plants. In this system, GhCLA1 was used as a general control, however, silencing of this gene leads to leaf albino, wilting, and plant death ultimately. As such, it cannot indicate the efficiency of target gene silencing throughout the whole plant growth period. To address this question, in this study, we developed a novel marker gene, Gossypium PIGMENT GLAND FORMATION GENE (GoPGF), as the control to trace the efficiency of gene silencing in the infected tissues. GoPGF has been proved a key gene in gland forming. Suppression of GoPGF does not affect the normal growth and development of cotton. The number of gland altered related to the expression level of GoPGF gene. So it is a good marker that be used to trace the whole growth stages of plant. Moreover, we further developed a method of friction inoculation to enhance and extend the efficiency of VIGS, which facilitates the analysis of gene function in both the vegetative stage and reproductive stage. This improved VIGS technology will be a powerful tool for the rapid functional identification of unknown genes in genomes.

Identification of candidate genes in cotton associated with specific seed traits and their initial functional characterization in Arabidopsis.

Oilseed crops are used to produce vegetable oil to satisfy the requirements of humans and livestock. Cotton (Gossypium spp.) is of great economic value because it is used as both an important textile commodity and a nutrient-rich resource. Cottonseed oil is rich in polyunsaturated fatty acids and does not contain trans fatty acids; hence, it is considered a healthy vegetable oil. However, research on the genetic basis for cottonseed protein content, oil production, and fatty acid composition is lacking. Here, we investigated the protein content, oil content, and fatty acid composition in terms of oleic acid (C18:1) and linoleic acid (C18:2) in mature cottonseeds from 318 Gossypium hirsutum accessions. Moreover, we examined the dynamic change of protein content and lipid composition including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) in developing seeds from 258 accessions at 10 and 20 days post-anthesis. Then, we conducted a genome-wide association study and identified 152 trait-associated loci and 64 candidate genes responsible for protein and oil-related contents in mature cottonseeds and ovules. Finally, six candidate genes were experimentally validated to be involved in the regulation of fatty acid biosynthesis through heterologous expression in Arabidopsis. These results comprise a solid foundation for expanding our understanding of lipid biosynthesis in cotton, which will help breeders manipulate protein and oil contents to make it a fully developed 'fiber, food, and oil crop'.

Development of the engineered "glanded plant and glandless seed" cotton.

After fiber, cottonseed is the second most important by-product of cotton production. However, high concentrations of toxic free gossypol deposited in the glands of the cottonseed greatly hamper its effective usage as food or feed. Here, we developed a cotton line with edible cottonseed by specifically silencing the endogenous expression of GoPGF in the seeds, which led to a glandless phenotype with an ultra-low gossypol content in the seeds and nearly normal gossypol in other parts of the plants. This engineered cotton maintains normal resistance to insect pests, but the gossypol content in the seeds dropped by 98%, and thus, it can be consumed directly as food. The trait of a low gossypol content in the cottonseeds was stable and heritable, while the protein, oil content, and fiber yield or quality were nearly unchanged compared to the transgenic receptor W0. In addition, comparative transcriptome analysis showed that down-regulated genes in the ovules of the glandless cotton were enriched in terpenoid biosynthesis, indicating the underlying relationship between gland formation and gossypol biosynthesis. These results pave the way for the comprehensive utilization of cotton as a fiber, oil, and feed crop in the future.

Advanced genes expression pattern greatly contributes to divergence in Verticillium wilt resistance between Gossypium barbadense and Gossupium hirsutum.

Verticillium, representing one of the world's major pathogens, causes Verticillium wilt in important woody species, ornamentals, agricultural, etc., consequently resulting in a serious decline in production and quality, especially in cotton. Gossupium hirutum and Gossypium barbadense are two kinds of widely cultivated cotton species that suffer from Verticillium wilt, while G. barbadense has much higher resistance toward it than G. hirsutum. However, the molecular mechanism regarding their divergence in Verticillium wilt resistance remains largely unknown. In the current study, G. barbadense cv. Hai7124 and G. hirsutum acc. TM-1 were compared at 0, 12, 24, 48, 72, 96, 120, and 144 h post-inoculation (hpi) utilizing high throughput RNA-Sequencing. As a result, a total of 3,549 and 4,725 differentially expressed genes (DEGs) were identified, respectively. In particular, the resistant type Hai7124 displayed an earlier and faster detection and signaling response to the Verticillium dahliae infection and demonstrated higher expression levels of defense-related genes over TM-1 with respect to transcription factors, plant hormone signal transduction, plant-pathogen interaction, and nucleotide-binding leucine-rich repeat (NLR) genes. This study provides new insights into the molecular mechanisms of divergence in Verticillium wilt resistance between G. barbadense and G. hirsutum and important candidate genes for breeding V. dahliae resistant cotton cultivars.

Retrieving a disrupted gene encoding phospholipase A for fibre enhancement in allotetraploid cultivated cotton.

After polyploidization originated from one interspecific hybridization event in Gossypium, Gossypium barbadense evolved to produce extra-long staple fibres than Gossypium hirsutum (Upland cotton), which produces a higher fibre yield. The genomic diversity between G. barbadense and G. hirsutum thus provides a genetic basis for fibre trait variation. Recently, rapid accumulation of gene disruption or deleterious mutation was reported in allotetraploid cotton genomes, with unknown impacts on fibre traits. Here, we identified gene disruptions in allotetraploid G. hirsutum (18.14%) and G. barbadense (17.38%) through comparison with their presumed diploid progenitors. Relative to conserved genes, these disrupted genes exhibited faster evolution rate, lower expression level and altered gene co-expression networks. Within a module regulating fibre elongation, a hub gene experienced gene disruption in G. hirsutum after polyploidization, with a 2-bp deletion in the coding region of GhNPLA1D introducing early termination of translation. This deletion was observed in all of the 34 G. hirsutum landraces and 36 G. hirsutum cultivars, but not in 96% of 57 G. barbadense accessions. Retrieving the disrupted gene GhNPLA1D using its homoeolog GhNPLA1A achieved longer fibre length in G. hirsutum. Further enzyme activity and lipids analysis confirmed that GhNPLA1A encodes a typical phospholipase A and promotes cotton fibre elongation via elevating intracellular levels of linolenic acid and 34:3 phosphatidylinositol. Our work opens a strategy for identifying disrupted genes and retrieving their functions in ways that can provide valuable resources for accelerating fibre trait enhancement in cotton breeding.

Construction of a high-density genetic map and identification of QTLs related to agronomic and physiological traits in an interspecific (Gossypium hirsutum × Gossypium barbadense) F2 population.

BACKGROUND: Advances in genome sequencing technology, particularly restriction-site associated DNA sequence (RAD-seq) and whole-genome resequencing, have greatly aided the construction of cotton interspecific genetic maps based on single nucleotide polymorphism (SNPs), Indels, and other types of markers. High-density genetic maps can improve accuracy of quantitative trait locus (QTL) mapping, narrow down location intervals, and facilitate identification of the candidate genes. RESULT: In this study, 249 individuals from an interspecific F2 population (TM-1 and Hai7124) were re-sequenced, yielding 6303 high-confidence bin markers spanning 5057.13 cM across 26 cotton chromosomes. A total of 3380 recombination hot regions RHRs were identified which unevenly distributed on the 26 chromosomes. Based on this map, 112 QTLs relating to agronomic and physiological traits from seedling to boll opening stage were identified, including 15 loci associated with 14 traits that contained genes harboring nonsynonymous SNPs. We analyzed the sequence and expression of these ten candidate genes and discovered that GhRHD3 (GH_D10G0500) may affect fiber yield while GhGPAT6 (GH_D04G1426) may affect photosynthesis efficiency. CONCLUSION: Our research illustrates the efficiency of constructing a genetic map using binmap and QTL mapping on the basis of a certain size of the early-generation population. High-density genetic map features high recombination exchanges in number and distribution. The QTLs and the candidate genes identified based on this high-density genetic map may provide important gene resources for the genetic improvement of cotton.