Genome-wide analyses of member identification, expression pattern, and protein-protein interaction of EPF/EPFL gene family in Gossypium.

BACKGROUND: Epidermal patterning factor / -like (EPF/EPFL) gene family encodes a class of cysteine-rich secretory peptides, which are widelyfound in terrestrial plants.Multiple studies has indicated that EPF/EPFLs might play significant roles in coordinating plant development and growth, especially as the morphogenesis processes of stoma, awn, stamen, and fruit skin. However, few research on EPF/EPFL gene family was reported in Gossypium. RESULTS: We separately identified 20 G. raimondii, 24 G. arboreum, 44 G. hirsutum, and 44 G. barbadense EPF/EPFL genes in the 4 representative cotton species, which were divided into four clades together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 17 Selaginella moellendorffii ones based on their evolutionary relationships. The similar gene structure and common motifs indicated the high conservation among the EPF/EPFL members, while the uneven distribution in chromosomes implied the variability during the long-term evolutionary process. Hundreds of collinearity relationships were identified from the pairwise comparisons of intraspecifc and interspecific genomes, which illustrated gene duplication might contribute to the expansion of cotton EPF/EPFL gene family. A total of 15 kinds of cis-regulatory elements were predicted in the promoter regions, and divided into three major categories relevant to the biological processes of development and growth, plant hormone response, and abiotic stress response. Having performing the expression pattern analyses with the basic of the published RNA-seq data, we found most of GhEPF/EPFL and GbEPF/EPFL genes presented the relatively low expression levels among the 9 tissues or organs, while showed more dramatically different responses to high/low temperature and salt or drought stresses. Combined with transcriptome data of developing ovules and fibers and quantitative Real-time PCR results (qRT-PCR) of 15 highly expressed GhEPF/EPFL genes, it could be deduced that the cotton EPF/EPFL genes were closely related with fiber development. Additionally, the networks of protein-protein interacting among EPF/EPFLs concentrated on the cores of GhEPF1 and GhEPF7, and thosefunctional enrichment analyses indicated that most of EPF/EPFLs participate in the GO (Gene Ontology) terms of stomatal development and plant epidermis development, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of DNA or base excision repair. CONCLUSION: Totally, 132 EPF/EPFL genes were identified for the first time in cotton, whose bioinformatic analyses of cis-regulatory elements and expression patterns combined with qRT-PCR experiments to prove the potential functions in the biological processes of plant growth and responding to abiotic stresses, specifically in the fiber development. These results not only provide comprehensive and valuable information for cotton EPF/EPFL gene family, but also lay solid foundation for screening candidate EPF/EPFL genes in further cotton breeding.

Quantitative Trait Locus Mapping for Plant Height and Branch Number in CCRI70 Recombinant Inbred Line Population of Upland Cotton (Gossypium hirsutum).

Upland cotton accounts for a high percentage (95%) of the world's cotton production. Plant height (PH) and branch number (BN) are two important agronomic traits that have an impact on improving the level of cotton mechanical harvesting and cotton yield. In this research, a recombinant inbred line (RIL) population with 250 lines developed from the variety CCRI70 was used for constructing a high-density genetic map and identification of quantitative trait locus (QTL). The results showed that the map harbored 8298 single nucleotide polymorphism (SNP) markers, spanning a total distance of 4876.70 centimorgans (cMs). A total of 69 QTLs for PH (9 stable) and 63 for BN (11 stable) were identified and only one for PH was reported in previous studies. The QTLs for PH and BN harbored 495 and 446 genes, respectively. Combining the annotation information, expression patterns and previous studies of these genes, six genes could be considered as potential candidate genes for PH and BN. The results could be helpful for cotton researchers to better understand the genetic mechanism of PH and BN development, as well as provide valuable genetic resources for cotton breeders to manipulate cotton plant architecture to meet future demands.

Genome-wide identification and expression-pattern analysis of sulfate transporter (SULTR) gene family in cotton under multiple abiotic stresses and fiber development.

Sulfate transporter (SULTR) proteins are in charge of the transport and absorption on sulfate substances, and have been reported to play vital roles in the biological processes of plant growth and stress response. However, there were few reports of genome-wide identification and expression-pattern analysis of SULTRs in Hibiscus mutabilis. Gossypium genus is a ideal model for studying the allopolyploidy, therefore two diploid species (G. raimondii and G. arboreum) and two tetraploid species (G. hirsutum and G. barbadense) were chosen in this study to perform bioinformatic analyses, identifying 18, 18, 35, and 35 SULTR members, respectively. All the 106 cotton SULTR genes were utilized to construct the phylogenetic tree together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 8 Zea mays ones, which was divided into Group1-Group4. The clustering analyses of gene structures and 10 conserved motifs among the cotton SULTR genes showed the consistent evolutionary relationship with the phylogenetic tree, and the results of gene-duplication identification among the four representative Gossypium species indicated that genome-wide or segment duplication might make main contributions to the expansion of SULTR gene family in cotton. Having conducted the cis-regulatory element analysis in promoter region, we noticed that the existing salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) elements could have influences with expression levels of cotton SULTR genes. The expression patterns of GhSULTR genes were also investigated on the 7 different tissues or organs and the developing ovules and fibers, most of which were highly expressed in root, stem, sepal, receptacel, ovule at 10 DPA, and fiber at 20 and 25 DPA. In addition, more active regulatory were observed in GhSULTR genes responding to multiple abiotic stresses, and 12 highly expressed genes showed the similar expression patterns in the quantitative Real-time PCR experiments under cold, heat, salt, and drought treatments. These findings broaden our insight into the evolutionary relationships and expression patterns of the SULTR gene family in cotton, and provide the valuable information for further screening the vital candidate genes on trait improvement.

Biotechnology and Solutions: Insect-Pest-Resistance Management for Improvement and Development of Bt Cotton (Gossypium hirsutum L.).

Cotton (Gossypium spp. L.) is a major origin of natural fiber, and is projected at 117 million bales worldwide for 2021/22. A variety of biotic and abiotic stresses have considerable negative impacts on cotton. The significantly decreased applications of chemical insecticidal sprays in the agro-ecosystem have greatly affected the biodiversity and dynamics of primary and secondary insects. Various control measures were taken around the globe to increase production costs. Temperature, drought, and salinity, and biotic stresses such as bacteria, viruses, fungi, nematodes, insects, and mites cause substantial losses to cotton crops. Here, we summarize a number of biotic and abiotic stresses upsetting Bt cotton crop with present and future biotechnology solution strategies that include a refuge strategy, multi-gene pyramiding, the release of sterile insects, seed mixing, RNAi, CRISPR/Cas9, biotic signaling, and the use of bioagents. Surveillance of insect resistance, monitoring of grower compliance, and implementation of remedial actions can lead to the sustainable use of cotton across the globe.

Hub Genes in Stable QTLs Orchestrate the Accumulation of Cottonseed Oil in Upland Cotton via Catalyzing Key Steps of Lipid-Related Pathways.

Upland cotton is the fifth-largest oil crop in the world, with an average supply of nearly 20% of vegetable oil production. Cottonseed oil is also an ideal alternative raw material to be efficiently converted into biodiesel. However, the improvement in kernel oil content (KOC) of cottonseed has not received sufficient attention from researchers for a long time, due to the fact that the main product of cotton planting is fiber. Previous studies have tagged QTLs and identified individual candidate genes that regulate KOC of cottonseed. The regulatory mechanism of oil metabolism and accumulation of cottonseed are still elusive. In the current study, two high-density genetic maps (HDGMs), which were constructed based on a recombinant inbred line (RIL) population consisting of 231 individuals, were used to identify KOC QTLs. A total of forty-three stable QTLs were detected via these two HDGM strategies. Bioinformatic analysis of all the genes harbored in the marker intervals of the stable QTLs revealed that a total of fifty-one genes were involved in the pathways related to lipid biosynthesis. Functional analysis via coexpression network and RNA-seq revealed that the hub genes in the co-expression network that also catalyze the key steps of fatty acid synthesis, lipid metabolism and oil body formation pathways (ACX4, LACS4, KCR1, and SQD1) could jointly orchestrate oil accumulation in cottonseed. This study will strengthen our understanding of oil metabolism and accumulation in cottonseed and contribute to KOC improvement in cottonseed in the future, enhancing the security and stability of worldwide food supply.

QTL Verification and Candidate Gene Screening of Fiber Quality and Lint Percentage in the Secondary Segregating Population of Gossypium hirsutum.

Fiber quality traits, especially fiber strength, length, and micronaire (FS, FL, and FM), have been recognized as critical fiber attributes in the textile industry, while the lint percentage (LP) was an important indicator to evaluate the cotton lint yield. So far, the genetic mechanism behind the formation of these traits is still unclear. Quantitative trait loci (QTL) identification and candidate gene validation provide an effective methodology to uncover the genetic and molecular basis of FL, FS, FM, and LP. A previous study identified three important QTL/QTL cluster loci, harboring at least one of the above traits on chromosomes A01, A07, and D12 via a recombinant inbred line (RIL) population derived from a cross of Lumianyan28 (L28) × Xinluzao24 (X24). A secondary segregating population (F2) was developed from a cross between L28 and an RIL, RIL40 (L28 × RIL40). Based on the population, genetic linkage maps of the previous QTL cluster intervals on A01 (6.70-10.15 Mb), A07 (85.48-93.43 Mb), and D12 (0.40-1.43 Mb) were constructed, which span 12.25, 15.90, and 5.56 cM, with 2, 14, and 4 simple sequence repeat (SSR) and insertion/deletion (Indel) markers, respectively. QTLs of FL, FS, FM, and LP on these three intervals were verified by composite interval mapping (CIM) using WinQTL Cartographer 2.5 software via phenotyping of F2 and its derived F2:3 populations. The results validated the previous primary QTL identification of FL, FS, FM, and LP. Analysis of the RNA-seq data of the developing fibers of L28 and RIL40 at 10, 20, and 30 days post anthesis (DPA) identified seven differentially expressed genes (DEGs) as potential candidate genes. qRT-PCR verified that five of them were consistent with the RNA-seq result. These genes may be involved in regulating fiber development, leading to the formation of FL, FS, FM, and LP. This study provides an experimental foundation for further exploration of these functional genes to dissect the genetic mechanism of cotton fiber development.

Comparative transcriptional and co-expression network analysis of two upland cotton accessions with extreme phenotypic differences reveals molecular mechanisms of fiber development.

Introduction: Upland cotton (Gossypium hirsutum) is the main source of natural fiber in the global textile industry, and thus its fiber quality and yield are important parameters. In this study, comparative transcriptomics was used to analyze differentially expressed genes (DEGs) due to its ability to effectively screen candidate genes during the developmental stages of cotton fiber. However, research using this method is limited, particularly on fiber development. The aim of this study was to uncover the molecular mechanisms underlying the whole period of fiber development and the differences in transcriptional levels. Methods: Comparative transcriptomes are used to analyze transcriptome data and to screen for differentially expressed genes. STEM and WGCNA were used to screen for key genes involved in fiber development. qRT-PCR was performed to verify gene expression of selected DEGs and hub genes. Results: Two accessions of upland cotton with extreme phenotypic differences, namely EZ60 and ZR014121, were used to carry out RNA sequencing (RNA-seq) on fiber samples from different fiber development stages. The results identified 704, 376, 141, 269, 761, and 586 genes that were upregulated, and 1,052, 476, 355, 259, 702, and 847 genes that were downregulated at 0, 5, 10, 15, 20, and 25 days post anthesis, respectively. Similar expression patterns of DEGs were monitored using short time-series expression miner (STEM) analysis, and associated pathways of DEGs within profiles were investigated. In addition, weighted gene co-expression network analysis (WGCNA) identified five key modules in fiber development and screened 20 hub genes involved in the development of fibers. Discussion: Through the annotation of the genes, it was found that the excessive expression of resistance-related genes in the early fiber development stages affects the fiber yield, whereas the sustained expression of cell elongation-related genes is critical for long fibers. This study provides new information that can be used to improve fibers in newly developed upland cotton genotypes.

Genome-Wide Analysis and Functional Characterization of LACS Gene Family Associated with Lipid Synthesis in Cotton (Gossypium spp.).

Cotton (Gossypium spp.) is the fifth largest oil crop in the world, and cottonseed provides abundant vegetable oil resources and industrial bioenergy fuels for people; therefore, it is of practical significance to increase the oil content of cotton seeds for improving the oil yield and economic benefits of planting cotton. Long-chain acyl-coenzyme A (CoA) synthetase (LACS) capable of catalyzing the formation of acyl-CoAs from free fatty acids has been proven to significantly participate in lipid metabolism, of which whole-genome identification and functional characterization of the gene family have not yet been comprehensively analyzed in cotton. In this study, a total of sixty-five LACS genes were confirmed in two diploid and two tetraploid Gossypium species, which were divided into six subgroups based on phylogenetic relationships with twenty-one other plants. An analysis of protein motif and genomic organizations displayed structural and functional conservation within the same group but diverged among the different group. Gene duplication relationship analysis illustrates the LACS gene family in large scale expansion through WGDs/segmental duplications. The overall Ka/Ks ratio indicated the intense purifying selection of LACS genes in four cotton species during evolution. The LACS genes promoter elements contain numerous light response cis-elements associated with fatty acids synthesis and catabolism. In addition, the expression of almost all GhLACS genes in high seed oil were higher compared to those in low seed oil. We proposed LACS gene models and shed light on their functional roles in lipid metabolism, demonstrating their engineering potential for modulating TAG synthesis in cotton, and the genetic engineering of cottonseed oil provides a theoretical basis.

Lint percentage and boll weight QTLs in three excellent upland cotton (Gossypium hirsutum): ZR014121, CCRI60, and EZ60.

BACKGROUND: Upland cotton (Gossypium hirsutum L.) is the most economically important species in the cotton genus (Gossypium spp.). Enhancing the cotton yield is a major goal in cotton breeding programs. Lint percentage (LP) and boll weight (BW) are the two most important components of cotton lint yield. The identification of stable and effective quantitative trait loci (QTLs) will aid the molecular breeding of cotton cultivars with high yield. RESULTS: Genotyping by target sequencing (GBTS) and genome-wide association study (GWAS) with 3VmrMLM were used to identify LP and BW related QTLs from two recombinant inbred line (RIL) populations derived from high lint yield and fiber quality lines (ZR014121, CCRI60 and EZ60). The average call rate of a single locus was 94.35%, and the average call rate of an individual was 92.10% in GBTS. A total of 100 QTLs were identified; 22 of them were overlapping with the reported QTLs, and 78 were novel QTLs. Of the 100 QTLs, 51 QTLs were for LP, and they explained 0.29-9.96% of the phenotypic variation; 49 QTLs were for BW, and they explained 0.41-6.31% of the phenotypic variation. One QTL (qBW-E-A10-1, qBW-C-A10-1) was identified in both populations. Six key QTLs were identified in multiple-environments; three were for LP, and three were for BW. A total of 108 candidate genes were identified in the regions of the six key QTLs. Several candidate genes were positively related to the developments of LP and BW, such as genes involved in gene transcription, protein synthesis, calcium signaling, carbon metabolism, and biosynthesis of secondary metabolites. Seven major candidate genes were predicted to form a co-expression network. Six significantly highly expressed candidate genes of the six QTLs after anthesis were the key genes regulating LP and BW and affecting cotton yield formation. CONCLUSIONS: A total of 100 stable QTLs for LP and BW in upland cotton were identified in this study; these QTLs could be used in cotton molecular breeding programs. Putative candidate genes of the six key QTLs were identified; this result provided clues for future studies on the mechanisms of LP and BW developments.

Genome-wide characterization of trichome birefringence-like genes provides insights into fiber yield improvement.

Cotton is an important fiber crop. The cotton fiber is an extremely long trichome that develops from the epidermis of an ovule. The trichome is a general and multi-function plant organ, and trichome birefringence-like (TBL) genes are related to trichome development. At the genome-wide scale, we identified TBLs in four cotton species, comprising two cultivated tetraploids (Gossypium hirsutum and G. barbadense) and two ancestral diploids (G. arboreum and G. raimondii). Phylogenetic analysis showed that the TBL genes clustered into six groups. We focused on GH_D02G1759 in group IV because it was located in a lint percentage-related quantitative trait locus. In addition, we used transcriptome profiling to characterize the role of TBLs in group IV in fiber development. The overexpression of GH_D02G1759 in Arabidopsis thaliana resulted in more trichomes on the stems, thereby confirming its function in fiber development. Moreover, the potential interaction network was constructed based on the co-expression network, and it was found that GH_D02G1759 may interact with several genes to regulate fiber development. These findings expand our knowledge of TBL family members and provide new insights for cotton molecular breeding.

Fine mapping and candidate gene analysis of qFL-A12-5: a fiber length-related QTL introgressed from Gossypium barbadense into Gossypium hirsutum.

KEY MESSAGE: The fiber length-related qFL-A12-5 identified in CSSLs introgressed from Gossypium barbadense into Gossypium hirsutum was fine-mapped to an 18.8 kb region on chromosome A12, leading to the identification of the GhTPR gene as a potential regulator of cotton fiber length. Fiber length is a key determinant of fiber quality in cotton, and it is a key target of artificial selection for breeding and domestication. Although many fiber length-related quantitative trait loci have been identified, there are few reports on their fine mapping or candidate gene validation, thus hampering efforts to understand the mechanistic basis of cotton fiber development. Our previous study identified the qFL-A12-5 associated with superior fiber quality on chromosome A12 in the chromosome segment substitution line (CSSL) MBI7747 (BC4F3:5). A single segment substitution line (CSSL-106) screened from BC6F2 was backcrossed to construct a larger segregation population with its recurrent parent CCRI45, thus enabling the fine mapping of 2852 BC7F2 individuals using denser simple sequence repeat markers to narrow the qFL-A12-5 to an 18.8 kb region of the genome, in which six annotated genes were identified in Gossypium hirsutum. Quantitative real-time PCR and comparative analyses led to the identification of GH_A12G2192 (GhTPR) encoding a tetratricopeptide repeat-like superfamily protein as a promising candidate gene for qFL-A12-5. A comparative analysis of the protein-coding regions of GhTPR among Hai1, MBI7747, and CCRI45 revealed two non-synonymous mutations. The overexpression of GhTPR resulted in longer roots in Arabidopsis, suggesting that GhTPR may regulate cotton fiber development. These results provide a foundation for future efforts to improve cotton fiber length.

Silver lining to a climate crisis in multiple prospects for alleviating crop waterlogging under future climates.

Extreme weather events threaten food security, yet global assessments of impacts caused by crop waterlogging are rare. Here we first develop a paradigm that distils common stress patterns across environments, genotypes and climate horizons. Second, we embed improved process-based understanding into a farming systems model to discern changes in global crop waterlogging under future climates. Third, we develop avenues for adapting cropping systems to waterlogging contextualised by environment. We find that yield penalties caused by waterlogging increase from 3-11% historically to 10-20% by 2080, with penalties reflecting a trade-off between the duration of waterlogging and the timing of waterlogging relative to crop stage. We document greater potential for waterlogging-tolerant genotypes in environments with longer temperate growing seasons (e.g., UK, France, Russia, China), compared with environments with higher annualised ratios of evapotranspiration to precipitation (e.g., Australia). Under future climates, altering sowing time and adoption of waterlogging-tolerant genotypes reduces yield penalties by 18%, while earlier sowing of winter genotypes alleviates waterlogging by 8%. We highlight the serendipitous outcome wherein waterlogging stress patterns under present conditions are likely to be similar to those in the future, suggesting that adaptations for future climates could be designed using stress patterns realised today.

Genome-wide artificial introgressions of Gossypium barbadense into G. hirsutum reveal superior loci for simultaneous improvement of cotton fiber quality and yield traits.

INTRODUCTION: The simultaneous improvement of fiber quality and yield for cotton is strongly limited by the narrow genetic backgrounds of Gossypium hirsutum (Gh) and the negative genetic correlations among traits. An effective way to overcome the bottlenecks is to introgress the favorable alleles of Gossypium barbadense (Gb) for fiber quality into Gh with high yield. OBJECTIVES: This study was to identify superior loci for the improvement of fiber quality and yield. METHODS: Two sets of chromosome segment substitution lines (CSSLs) were generated by crossing Hai1 (Gb, donor-parent) with cultivar CCRI36 (Gh) and CCRI45 (Gh) as genetic backgrounds, and cultivated in 6 and 8 environments, respectively. The kmer genotyping strategy was improved and applied to the population genetic analysis of 743 genomic sequencing data. A progeny segregating population was constructed to validate genetic effects of the candidate loci. RESULTS: A total of 68,912 and 83,352 genome-wide introgressed kmers were identified in the CCRI36 and CCRI45 populations, respectively. Over 90 % introgressions were homologous exchanges and about 21 % were reverse insertions. In total, 291 major introgressed segments were identified with stable genetic effects, of which 66(22.98 %), 64(21.99 %), 35(12.03 %), 31(10.65 %) and 18(6.19 %) were beneficial for the improvement of fiber length (FL), strength (FS), micronaire, lint-percentage (LP) and boll-weight, respectively. Thirty-nine introgression segments were detected with stable favorable additive effects for simultaneous improvement of 2 or more traits in Gh genetic background, including 6 could increase FL/FS and LP. The pyramiding effects of 3 pleiotropic segments (A07:C45Clu-081, D06:C45Clu-218, D02:C45Clu-193) were further validated in the segregating population. CONCLUSION: The combining of genome-wide introgressions and kmer genotyping strategy showed significant advantages in exploring genetic resources. Through the genome-wide comprehensive mining, a total of 11 clusters (segments) were discovered for the stable simultaneous improvement of FL/FS and LP, which should be paid more attention in the future.

AAQSP increases mapping resolution of stable QTLs through applying NGS-BSA in multiple genetic backgrounds.

KEY MESSAGE: In this study, we present AAQSP as an extension of existing NGS-BSA applications for identifying stable QTLs at high resolution. GhPAP16 and GhIQD14 fine mapped on chromosome D09 of upland cotton are identified as important candidate genes for lint percentage (LP). Bulked segregant analysis combined with next generation sequencing (NGS-BSA) allows rapid identification of genome sequence differences responsible for phenotypic variation. The NGS-BSA approach applied to crops mainly depends on comparing two bulked DNA samples of individuals from an F2 population. Since some F2 individuals still maintain high heterozygosity, heterosis will exert complications in pursuing NGS-BSA in such populations. In addition, the genetic background influences the stability of gene expression in crops, so some QTLs mapped in one segregating population may not be widely applied in crop improvement. The AAQSP (Association Analysis of QTL-seq on Semi-homologous Populations) reported in our study combines the optimized scheme of constructing BSA bulks with NGS-BSA analysis in two (or more) different parental genetic backgrounds for isolating the stable QTLs. With application of AAQSP strategy and construction of a high-density linkage map, we have successfully identified a QTL significantly related to lint percentage (LP) in cultivated upland cotton, followed by map-based cloning to dissect two candidate genes, GhPAP16 and GhIQD14. This study demonstrated that AAQSP can efficiently identify stable QTLs for complex traits of interest, and thus accelerate the genetic improvement of upland cotton and other crop plants.

UDP-glucose pyrophosphorylase: genome-wide identification, expression and functional analyses in Gossypium hirsutum.

In this study, a total of 66 UDP-glucose pyrophosphorylase (UGP) (EC 2.7.7.9) genes were identified from the genomes of four cotton species, which are the members of Pfam glycosyltransferase family (PF01702) and catalyze the reaction between glucose-1-phosphate and UTP to produce UDPG. The analysis of evolutionary relationship, gene structure, and expression provides the basis for studies on function of UGP genes in cotton. The evolutionary tree and gene structure analysis revealed that the UGP gene family is evolutionarily conserved. Collinearity and Ka/Ks analysis indicated that amplification of UGP genes is due to repetitive crosstalk generating between new family genes, while being under strong selection pressure. The analysis of cis-acting elements exhibited that UGP genes play important role in cotton growth, development, abiotic and hormonal stresses. Six UGP genes that were highly expressed in cotton fiber at 15 DPA were screened by transcriptome data and qRT-PCR analysis. The addition of low concentrations of IAA and GA3 to ovule cultures revealed that energy efficiency promoted the development of ovules and fiber clusters, and qRT-PCR showed that expression of these six UGP genes was differentially increased. These results suggest that the UGP gene may play an important role in fiber development, and provides the opportunity to plant researchers to explore the mechanisms involve in fiber development in cotton.

Linkage and association analyses reveal that hub genes in energy-flow and lipid biosynthesis pathways form a cluster in upland cotton.

Upland cotton is an important allotetraploid crop that provides both natural fiber for the textile industry and edible vegetable oil for the food or feed industry. To better understand the genetic mechanism that regulates the biosynthesis of storage oil in cottonseed, we identified the genes harbored in the major quantitative trait loci/nucleotides (QTLs/QTNs) of kernel oil content (KOC) in cottonseed via both multiple linkage analyses and genome-wide association studies (GWAS). In 'CCRI70' RILs, six stable QTLs were simultaneously identified by linkage analysis of CHIP and SLAF-seq strategies. In '0-153' RILs, eight stable QTLs were detected by consensus linkage analysis integrating multiple strategies. In the natural panel, thirteen and eight loci were associated across multiple environments with two algorithms of GWAS. Within the confidence interval of a major common QTL on chromosome 3, six genes were identified as participating in the interaction network highly correlated with cottonseed KOC. Further observations of gene differential expression showed that four of the genes, LtnD, PGK, LPLAT1, and PAH2, formed hub genes and two of them, FER and RAV1, formed the key genes in the interaction network. Sequence variations in the coding regions of LtnD, FER, PGK, LPLAT1, and PAH2 genes may support their regulatory effects on oil accumulation in mature cottonseed. Taken together, clustering of the hub genes in the lipid biosynthesis interaction network provides new insights to understanding the mechanism of fatty acid biosynthesis and TAG assembly and to further genetic improvement projects for the KOC in cottonseeds.

Inheritance, QTLs, and Candidate Genes of Lint Percentage in Upland Cotton.

Cotton (Gossypium spp.) is an important natural fiber plant. Lint percentage (LP) is one of the most important determinants of cotton yield and is a typical quantitative trait with high variation and heritability. Many cotton LP genetic linkages and association maps have been reported. This work summarizes the inheritance, quantitative trait loci (QTLs), and candidate genes of LP to facilitate LP genetic study and molecular breeding. More than 1439 QTLs controlling LP have been reported. Excluding replicate QTLs, 417 unique QTLs have been identified on 26 chromosomes, including 243 QTLs identified at LOD >3. More than 60 are stable, major effective QTLs that can be used in marker-assisted selection (MAS). More than 90 candidate genes for LP have been reported. These genes encode MYB, HOX, NET, and other proteins, and most are preferentially expressed during fiber initiation and elongation. A putative molecular regulatory model of LP was constructed and provides the foundation for the genetic study and molecular breeding of LP.

lncRNA7 and lncRNA2 modulate cell wall defense genes to regulate cotton resistance to Verticillium wilt.

In plants, long noncoding RNAs (lncRNAs) regulate disease resistance against fungi and other pathogens. However, the specific mechanism behind this regulation remains unclear. In this study, we identified disease resistance-related lncRNAs as well as their regulating genes and assessed their functions by infection of cotton (Gossypium) chromosome segment substitution lines with Verticillium dahliae. Our results demonstrated that lncRNA7 and its regulating gene Pectin methylesterase inhibitor 13 (GbPMEI13) positively regulated disease resistance via the silencing approach, while ectopic overexpression of GbPMEI13 in Arabidopsis (Arabidopsis thaliana) promoted growth and enhanced resistance to V. dahliae. In contrast, lncRNA2 and its regulating gene Polygalacturonase 12 (GbPG12) negatively regulated resistance to V. dahliae. We further found that fungal disease-related agents, including the pectin-derived oligogalacturonide (OG), could downregulate the expression of lncRNA2 and GbPG12, leading to pectin accumulation. Conversely, OG upregulated the expression of lncRNA7, which encodes a plant peptide phytosulfokine (PSK-α), which was confirmed by lncRNA7 overexpression and Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS) experiments. We showed that PSK-α promoted 3-Indoleacetic acid (IAA) accumulation and activated GbPMEI13 expression through Auxin Response Factor 5. Since it is an inhibitor of pectin methylesterase (PME), GbPMEI13 promotes pectin methylation and therefore increases the resistance to V. dahliae. Consistently, we also demonstrated that GbPMEI13 inhibits the mycelial growth and spore germination of V. dahliae in vitro. In this study, we demonstrated that lncRNA7, lncRNA2, and their regulating genes modulate cell wall defense against V. dahliae via auxin-mediated signaling, providing a strategy for cotton breeding.

Multi-environment Evaluations Across Ecological Regions Reveal That the Kernel Oil Content of Cottonseed Is Equally Determined by Genotype and Environment.

Cotton is the fifth-largest oil crop in the world. A high kernel oil content (KOC) and high stability are important cottonseed attributes for food security. In this study, the phenotype of KOC and the genotype-by-environment interaction factors were collectively dissected using 250 recombinant inbred lines, their parental cultivars sGK156 and 901-001, and CCRI70 across multi-environments. ANOVA and correlation analysis showed that both genotype and environment contributed significantly to KOC accumulation. Analyses of additive main effect multiplicative interaction and genotype-by-environment interaction biplot models presented the effects of genotype, environment, and genotype by environment on KOC performance and the stability of the experimental materials. Interaction network analysis revealed that meteorological and geographical factors explained 38% of the total KOC variance, with average daily rainfall contributing the largest positive impact and cumulative rainfall having the largest negative impact on KOC accumulation. This study provides insight into KOC accumulation and could direct selection strategies for improved KOC and field management of cottonseed in the future.

QTL mapping and candidate gene prediction for fiber yield and quality traits in a high-generation cotton chromosome substitution line with Gossypium barbadense segments.

Gossypium provides the foremost natural fiber for supporting the rapid development of the textile industry. Quantitative trait locus (QTL) mapping of fiber yield and quality traits is, thus, of great significance for providing a foundation for the genetic improvement of key target traits in cotton production. In this study, a superior chromosome segment substitution line (CSSL), MBI8255, with high yield and premium fiber quality characteristics was cultivated from the BC5F3:5 lineage derived from G. barbadense Hai1 and G. hirsutum CCRI36, and was chosen to construct a segregation population containing 123 F2 individuals with CCRI36. A total of 71 polymorphic SSR (simple sequence repeat) markers were identified based on a previous high-density linkage map, and 17 QTLs distributed on five chromosomes were detected, of which 10 QTLs for cotton yield explained 0.26-15.41% of phenotypic variations, while 7 QTLs for fiber quality explained 0.84-9.38% of phenotypic variations, separately containing four and one stable QTLs detected from over two environments. Among three identified QTL clusters, only the Chr19 QTL cluster harbored two stable and one unstable QTL for three different traits, and hence this significant region, which included 1546 genes, was subjected to functional enrichment and transcriptome expression analyses, ultimately screening eight candidate genes relevant to fiber development. This study not only provides useful information for the further fine-mapping and functional verification of candidate genes, but also offers a solid foundation for revealing the molecular mechanisms of fiber formation.