Structural Genomics

TBA

Cotton not only provides the largest renewal source of textile fiber, but also is a model for studying fiber cell differentiation, cellulose biosynthesis, as well as polyploidy and its impact on genome evolution and crop domestication. In this report, we will update the progress of collective efforts on generating reference-grade sequences of the tetraploid cotton genomes and analyzing the structure and function of these genomes using multi-dimensional and integrated approaches.

Host plant resistance is the most practical approach to control the Southern root-knot nematode (Meloidogyne incognita; RKN), which has emerged as one of the most serious economic pest of Upland cotton (Gossypium hirsutum L.). Linkage analysis of an interspecific F2:3 population has identified a resistance locus on chromosome-11 (qMi-C11) affecting galling and another locus on chromosome-14 (qMi-C14) affecting egg production. Although these two QTL regions were fine mapped, there are no expression profiling of genes that may account for this phenomenon. We applied the comparative transcriptomic approach to compare expression profiles of genes between RKN susceptible and resistance genotypes at an early stage of RKN development that coincide with the establishment of a feeding site and at the late stage of RKN development that coincide with RKN egg production. Sequencing of six cDNA libraries produced over 315 million reads of which 240 million reads (76%) were mapped on to the Gossypium hirsutum genome. A total of 1239 differentially expressed genes (DEGs) were identified and clustered according to their expression profiles. A large number of DEGs down regulated in susceptible genotype at the late stage of RKN development while several genes were upregulated in the resistant genotype. Key enriched categories included transcription factor activity, defense response, response to hormones, cell wall organization, and protein serine/threonine kinase activity. Zeatin biosynthesis and biosynthesis of secondary metabolites are among the enriched KEGG pathway. Our results also show that the DEGs in resistant genotype at qMi-C11 and qMi-C14 loci displayed higher expression compared to the DEGs in susceptible genotypes.

When Identifying and responding to a new disease threat, breeders are already behind in the race to find ways to mitigate the effects of the disease and develop cotton cultivars with genetic resistance. A collaborative project between the United States and Pakistan demonstrated the value of international partnerships to identify multiple sources of resistance to cotton leaf curl virus disease in non-commercial cotton and transferring it to elite lines both Pakistani and U.S. farmers. Best management practices to mitigate the effects of the disease, DNA markers improve the efficiency of selection and diagnostic tests to identify the virus were also developed through the project. Lessons learned from this project can be used to create new international partnerships to proactively combat other diseases and several examples will be highlighted.

Cotton functional genomics promise to enhance the understanding of fundamental plant biology to systematically exploit genetic resources for improvement of cotton fibre quality and yield, as well as utilization of genetic information for germplasm improvement. However, determining the cotton gene functions is a much more challenging task which has not progressed at rapid pace. Genome sequencing technologies has been improved with exponent pace,and some species such as G.raimondii, G.arborem, G.hirsutum,G.barbadense have been sequenced. but precise chromosome-scale assembling of genomes remained an important challenge.This paper presents a comprehensive overview on the recent tools and resources available with the major advances of cotton functional genomics to develop elite cotton genotypes ,and we generated significantly correct high quality assembly of G. arboreum single chromosome by genetic map and reference assisted approaches.

Laccase gene, GhLAC15, enhanced Verticillium wilt resistance via increasing defense-induced lignification and arabinose and xylose accumulation in the cell wall of Gossypium hirsutum YanZhang#, Li-zhuWu#, Xing-fenWang, Zhi-kun Li, Jun Yang, Guo-ningWang, Yuan-yuanYan, Jin-huaWu, Li-qiangWu, Gui-yinZhang, Zhi-yingMa* College of Agronomy, North China Key Laboratory for Crop Germplasm Resources of Education Ministry, Co-Innovation Center For Cotton Industry of Hebei Province, Hebei Agricultural University, Baoding 071001, China. #These authors contributed equally to this work. *Correspondence: mzhy@hebau.edu.cn Tel: +86-312-7528401 Fax: +86-312-7521279 Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. Enhancing the immune system of plants is regarded as useful for controlling this disease. Lignification of cell wall appositions is a conserved basal defense mechanism in plant innate immune response. Significant roles of laccase family in cotton fiber development had been reported. However, the function of laccase involved in defense-induced lignification has not been described to date. During screening the SSH library of resistant cultivar Jimian20 inoculated by V. dahliae, a laccase gene displayed strongly induced by the pathogen, which was phylogenetically related to AtLAC15 gene, containing conserved domains that laccase commonly possessed, thus named it as GhLAC15. Overexpression GhLAC15 gene in Arabidopsis enhanced cell wall lignification, resulting in increasing the total lignin and G monolignol as well as the radio of G/S, which significantly improved the Verticillium wilt resistance of transgenic plants. In addition, the predominant carbohydrate constituents of cell wall, arabinose (Ara), xylose (Xyl) and glucose (Glc), displayed obvious difference between transgenic plants and wild type. The levels of Ara and Xyl distinctly increased, whereas the Glc level decreased in transgenic Arabidopsis. Based on the high levels of Ara and Xyl representing a high content of arabinose-substituted xylan (arabinoxylan) in enhancing plant resistance to pathogen, the high levels of Ara and Xyl provided the biochemical evidence in defense against V. dahliae in our transgenic Arabidopsis. Furthermore, suppressing the transcriptional level of GhLAC15, via virus-induced gene silencing, resulted in the increase of diseased plant rate and disease index in cotton. More fungal colonies were found in the roots of silent plants, suggesting that the extent of fungal colonization in silent plants was much more severe than in control plants. The content of monolignol and the G/S radio also decreased in the silent cotton plants, which led to resistant cotton cv. Jimian20 susceptible to the disease. These results demonstrated that GhLAC15 gene enhanced Verticillium wilt resistance via increasing defense-induced lignification and arabinose and xylose accumulation in the cell wall of G. hirsutum. This broadens our knowledge in defense-induced lignifications and cell wall modification as a defense mechanism against V. dahliae.

Abstract：Cotton fiber is the most important natural textile raw material in the world. It also represents an outstanding single-celled model for understanding plant cell elongation and cell wall and cellulose biosynthesis. Compared to extensive studies on fiber initiation and elongation processes, much less is known about the molecular basis underlying secondary cell wall cellulose deposition, a process profoundly impacting mature fiber properties. In this study, we identified a gene, GhBG1A, encoding glycosyl hydrolase family 3 beta-glucosidase, which coordinates cotton fiber elongation and cellulose deposition through regulating sugar metabolism. GhBG1A was preferentially expressed in cotton fibers and significantly associated with fiber length and strength. Overexpression of GhBG1A at fiber elongation stage significantly reduced fiber length, but remarkably increased cellulose biosynthesis leading to thicker fiber cell wall. Molecular and biochemical studies indicate that sucrose content and expression levels of cellulose biosynthesis related genes was evidently increased in transgenic cotton fibers. Moreover, using two cultivated tetraploid cotton species with distinctly different fiber qualities, higher transcript levels and enzyme activities of GhBG1A homologs were detected at fiber secondary cell wall (SCW) deposition stage in G. barbadense cv. H7124, which possesses elite fibers, than that in G. hirsutum acc. TM-1. Taken together, GhBG1A plays an important role in fiber transition from elongation to SCW deposition, and fine-tune overexpression of GhBG1A homolog at fiber SCW stage could improve fiber qualities in cotton fiber quality breeding program.

High-throughput genotyping platforms play important roles in plant genomic studies. Cotton (Gossypium pp.) is the world’s important natural textile fiber and oil crop. Upland cotton accounts for more than 90% of the world’s cotton production, however, modern upland cotton cultivars have narrow genetic diversity. The amounts of genomic sequencing and re-sequencing data released make it possible to develop a high-quality single nucleotide polymorphism (SNP) array for intraspecific genotyping detection in cotton. Here we report a high-throughput CottonSNP80K array and its utilization in genotyping detection and targeted genes mining. 82,259 SNP markers were selected from the re-sequencing data of 100 cotton cultivars and used to produce the array on the Illumina Infinium platform. 77,774 SNP loci (94.55%) were successfully synthesized on the array. Of them, 77,252 (99.33%) had call rates of >95% in 352 cotton accessions and 59,502 (76.51%) were polymorphic loci. Application tests using 22 cotton accessions with parent/F1 combinations or with similar genetic backgrounds showed that CottonSNP80K array had high genotyping accuracy, good repeatability, and wide applicability. Phylogenetic analysis of 312 cotton cultivars and landraces with wide geographical distribution showed that they could be classified into ten groups, irrelevant of their origins. We found that the different landraces were clustered in different subgroups, indicating that these landraces were major contributors to the development of different breeding populations of modern G. hirsutum cultivars in China. We integrated a total of 54,588 SNPs (MAFs >0.05) associated with 10 salt stress traits into 288 G. hirsutum accessions for genome-wide association studies (GWAS), and eight significant SNPs associated with three salt stress traits were detected. Further, two genes located in the SNP regions were detected to distinctly related to salt tolerance, with significantly upregulated expression after salt treatment and significantly enhanced cotton susceptibility to salt stress after their silencing. Taken together, CottonSNP80K play important roles in germplasm genotyping, variety verification, functional genomics studies, and molecular breeding in cotton.

Cotton (Gossypium hirsutum) is an important cash crop that can produce the fiber and oilseed. It often undergoes various biotic or abiotic stresses in growth periods. Verticillium wilt, caused by Verticillium dahliae, is a severe disease in cotton. The molecular mechanism of cotton resistance to Verticillium wilt needs to be further investigated. In the present study, we firstly revealed a tau Glutathione S-transferases (GSTs, E.C.2.5.1.18) cluster (including Gh_A09G1508, Gh_A09G1509 and Gh_A09G1510) participating in the Verticillium wilt resistance in cotton based on evolutionary, transcriptomic and functional analyses. This gene cluster located in chromosome 09 of A –subgenome. Evolutionary analysis showed that the cluster originated from the unbalancing gene losses in genetic innovation during the formation of allotetraploid (G. hirsutum). Transcriptome analysis revealed that the cluster took part in Verticillium wilt resistance. Based on the GhGST gene Gh_A09G1509 which was highest differentially expressed in the resistant cultivar under V. dahliae stress, we overexpressed the gene of the cluster in tobacco, and it can greatly enhance Verticillium wilt resistance. When the genes of the cluster suppressed via virus-induced gene silencing (VIGS), the plants of resistant cultivar Nongda601 showed significantly susceptible to the disease. These results demonstrated that the GST cluster played an important role in Verticillium wilt resistance in G. hirsutum. Further investigation of the molecular mechanism showed that the cluster regulated the delicate equilibrium between production and scavenging of H2O2 during V. dahliae stress.

A short fiber mutant Ligon-lintless-y was created through seed treatment with ethyl methanesulfonate (EMS). Genetic analysis indicated that the mutation is controlled by a single recessive locus designated liy. Besides causing short seed fibers, the mutation also affects other traits including plant height. From an F2 mapping population, we selected 100 short fiber progeny (liy/liy), and bulked their DNAs. We sequenced this DNA bulk along with the two parents of the mapping population. The liy locus was mapped on chromosome A07. Flanking SNP/InDel markers were developed. We also evaluated global transcriptional changes during cotton fiber development at 3, 8 and 16 days post anthesis triggered by liy mutation. 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.