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Abstract Background Many elite genes have been identified from the available cotton genomic data, providing various genetic resources for gene-driven breeding. However, backbone cultivar-driven breeding is the most widely applied strategy. Revealing the genetic basis of cultivar-driven strategy’s restriction is crucial for transition of cotton breeding strategy. Result CRI12 is a backbone cultivar in cultivar-driven breeding. Here we sequence the pedigree of CRI12 using Nanopore long-read sequencing. We construct a graphical pedigree genome using the high-quality CRI12 genome and 13,138 structural variations within 20 different pedigree members. We find that low hereditary stability of elite segments in backbone cultivars is a drawback of cultivar-driven strategy. We also identify 623 functional segments in CRI12 for multiple agronomic traits in presence and absence variation-based genome-wide association study on three cohorts. We demonstrate that 25 deleterious segments are responsible for the geographical divergence of cotton in pathogen resistance. We also characterize an elite pathogen-resistant gene (GhKHCP) utilized in modern cotton breeding. In addition, we identify 386 pedigree fingerprint segments by comparing the segments of the CRI12 pedigree with those of a large cotton population. Conclusion We characterize the genetic patterns of functional segments in the pedigree of CRI12 using graphical genome method, revealing restrictions of cultivar-driven strategies in cotton breeding. These findings provide theoretical support for transitioning from cultivar-driven to gene-driven strategy in cotton breeding.

Abstract Background The glyoxalase system includes glyoxalase I (GLXI), glyoxalase II (GLXII) and glyoxalase III (GLXIII), which are responsible for methylglyoxal (MG) detoxification and involved in abiotic stress responses such as drought, salinity and heavy metal. Results In this study, a total of 620 GLX family genes were identified from 21 different plant species. The results of evolutionary analysis showed that GLX genes exist in all species from lower plants to higher plants, inferring that GLX genes might be important for plants, and GLXI and GLXII account for the majority. In addition, motif showed an expanding trend in the process of evolution. The analysis of cis-acting elements in 21 different plant species showed that the promoter region of the GLX genes were rich in phytohormones and biotic and abiotic stress-related elements, indicating that GLX genes can participate in a variety of life processes. In cotton, GLXs could be divided into two groups and most GLXIs distributed in group I, GLXIIs and GLXIIIs mainly belonged to group II, indicating that there are more similarities between GLXII and GLXIII in cotton evolution. The transcriptome data analysis and quantitative real-time PCR analysis (qRT-PCR) show that some members of GLX family would respond to high temperature treatment in G.hirsutum. The protein interaction network of GLXs in G.hirsutum implied that most members can participate in various life processes through protein interactions. Conclusions The results elucidated the evolutionary history of GLX family genes in plants and lay the foundation for their functions analysis in cotton.

Abstract Background Cotton is the source of natural fibers globally, fulfilling 90% of the textile industry's requirements. However, fiber development is a complex biological process comprising four stages. Fiber develops from a single cell, and cell elongation is a vital process in fiber development. Therefore, it is pertinent to understand and exploit mechanisms underlying cell elongation during fiber development. A previous report about cell division control protein 42 (CDC-42) with its key role in cell elongation in eukaryotes inspired us to explore its homologs Rho GTPases for understanding of cell elongation during cotton fiber development. Result We classified 2 066 Rho proteins from 8 Gossypium species into 5 and 8 groups within A and D sub-genomes, respectively. Asymmetric evolution of Rho members was observed among five tetraploids. Population fixation statistics between two short and long fiber genotypes identified highly diverged regions encompassing 34 Rho genes in G. hirustum, and 31 of them were retained through further validation by genome wide association analysis (GWAS). Moreover, a weighted gene co-expression network characterized genome-wide expression patteren of Rho genes based on previously published transcriptome data. Twenty Rho genes from five modules were identified as hub genes which were potentially related to fiber development. Interaction networks of 5 Rho genes based on transcriptional abundance and gene ontology (GO) enrichment emphasized the involvement of Rho in cell wall biosynthesis, fatty acid elongation, and other biological processes. Conclusion Our study characterized the Rho proteins in cotton, provided insights into the cell elongation of cotton fiber and potential application in cotton fiber improvement.

Abstract Background Upland cotton provides the most natural fiber in the world. During fiber development, the quality and yield of fiber were influenced by gene transcription. Revealing sequence features related to transcription has a profound impact on cotton molecular breeding. We applied convolutional neural networks to predict gene expression status based on the sequences of gene transcription start regions. After that, a gradient-based interpretation and an N-adjusted kernel transformation were implemented to extract sequence features contributing to transcription. Results Our models had approximate 80% accuracies, and the area under the receiver operating characteristic curve reached over 0.85. Gradient-based interpretation revealed 5' untranslated region contributed to gene transcription. Furthermore, 6 DOF binding motifs and 4 transcription activator binding motifs were obtained by N-adjusted kernel-motif transformation from models in three developmental stages. Apart from 10 general motifs, 3 DOF5.1 genes were also detected. In silico analysis about these motifs’ binding proteins implied their potential functions in fiber formation. Besides, we also found some novel motifs in plants as important sequence features for transcription. Conclusions In conclusion, the N-adjusted kernel transformation method could interpret convolutional neural networks and reveal important sequence features related to transcription during fiber development. Potential functions of motifs interpreted from convolutional neural networks could be validated by further wet-lab experiments and applied in cotton molecular breeding.

Abstract Introduction Genome sequence plays an important role in both basic and applied studies. Gossypium raimondii, the putative contributor of the D subgenome of upland cotton (G. hirsutum), highlights the need to improve the genome quality rapidly and efficiently. Methods We performed Hi-C sequencing of G. raimondii and reassembled its genome based on a set of new Hi-C data and previously published scaffolds. We also compared the reassembled genome sequence with the previously published G. raimondii genomes for gene and genome sequence collinearity. Result A total of 98.42% of scaffold sequences were clustered successfully, among which 99.72% of the clustered sequences were ordered and 99.92% of the ordered sequences were oriented with high-quality. Further evaluation of results by heat-map and collinearity analysis revealed that the current reassembled genome is significantly improved than the previous one (Nat Genet 44:98–1103, 2012). Conclusion This improvement in G. raimondii genome not only provides a better reference to increase study efficiency but also offers a new way to assemble cotton genomes. Furthermore, Hi-C data of G. raimondii may be used for 3D structure research or regulating analysis.

Abstract Background Gossypium hirsutum L. (cotton) is one of the most economically important crops in the world due to its significant source of fiber, feed, foodstuff, oil and biofuel products. However, the utilization of cottonseed was limited due to the presence of small and darkly pigmented glands that contain large amounts of gossypol, which is toxic to human beings and non-ruminant animals. To date, some progress has been made in the pigment gland formation, but the underlying molecular mechanism of its formation was still unclear. Results In this study, we identified an AP2/ERF transcription factor named GhERF105 (GH_A12G2166), which was involved in the regulation of gland pigmentation by the comparative transcriptome analysis of the leaf of glanded and glandless plants. It encoded an ERF protein containing a converved AP2 domain which was localized in the nucleus with transcriptional activity, and showed the high expression in glanded cotton accessions that contained much gossypol. Virus-induced gene silencing (VIGS) against GhERF105 caused the dramatic reduction in the number of glands and significantly lowered levels of gossypol in cotton leaves. GhERF105 showed the patterns of spatiotemporal and inducible expression in the glanded plants. Conclusions These results suggest that GhERF105 contributes to the pigment gland formation and gossypol biosynthesis in partial organs of glanded plant. It also provides a potential molecular basis to generate ‘glandless-seed’ and ‘glanded-plant’ cotton cultivar.

Abstract Background Genome sequencing technologies have been improved at an exponential pace but precise chromosome-scale genome assembly still remains a great challenge. The draft genome of cultivated G. arboreum was sequenced and assembled with shotgun sequencing approach, however, it contains several misassemblies. To address this issue, we generated an improved reassembly of G. arboreum chromosome 12 using genetic mapping and reference-assisted approaches and evaluated this reconstruction by comparing with homologous chromosomes of G. raimondii and G. hirsutum. Results In this study, we generated a high quality assembly of the 94.64 Mb length of G. arboreum chromosome 12 (A_A12) which comprised of 144 scaffolds and contained 3361 protein coding genes. Evaluation of results using syntenic and collinear analysis of reconstructed G. arboreum chromosome A_A12 with its homologous chromosomes of G. raimondii (D_D08) and G. hirsutum (AD_A12 and AD_D12) confirmed the significant improved quality of current reassembly as compared to previous one. We found major misassemblies in previously assembled chromosome 12 (A_Ca9) of G. arboreum particularly in anchoring and orienting of scaffolds into a pseudo-chromosome. Further, homologous chromosomes 12 of G. raimondii (D_D08) and G. arboreum (A_A12) contained almost equal number of transcription factor (TF) related genes, and showed good collinear relationship with each other. As well, a higher rate of gene loss was found in corresponding homologous chromosomes of tetraploid (AD_A12 and AD_D12) than diploid (A_A12 and D_D08) cotton, signifying that gene loss is likely a continuing process in chromosomal evolution of tetraploid cotton. Conclusion This study offers a more accurate strategy to correct misassemblies in sequenced draft genomes of cotton which will provide further insights towards its genome organization.