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Evolutionary, transcriptomic and functional analyses revealed a GST cluster played an important role in Verticillium wilt resistance in Gossypium hirsutum

Li, Zhikun
Chen, Bin
Li, Xiuxin
Wang, Jinpeng
Yan, Yuanyuan
Yang, Jun
Ke, Huifeng
Zhang, Yan
Wang, Xingfen
Zhang, Guiyin
Wu, Liqiang
Wang, Xiyin
Ma, Zhiying
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. 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.

Exploitation of favorable gene resource by a synthetic hexaploid derived from an interspecific hybrid between Gossypium hirsutum and G. anomalum

Xinlian, Shen
Caijiao, Zhai
Xia, Zhang
Zhenzhen, Xu
Peng, Xu
Shan, Meng
Qi, Guo
Xianggui, Zhang
Fang, Huang
Wild species of Gossypium present an impressive range of variation in many characters, all of which can potentially be exploited in cotton improvement programs. G. anomalum possesses several desirable characters such as extremely fine fibers, immunity to black arm and bacterial blight disease and tolerance to water deficit, as this species is endemic to relatively dry areas. To effectively introgression desirable traits into cultivated cotton from G. anomalum, we made crosses between G. hirsutum (A1A1D1D1) and G. anomalum (B1B1) and obtained triploid hybrids with the genome composition A1D1Bl. Then we treated triploid hybrids with 0.15% colchicine and obtained a putative fertile hexaploid (A1A1D1D1B1B1) in order to resolve interspecific hybrid sterility problems. We demonstrated the hybridity and doubled status of a (G. anomalum × G. hirsutum)2 hexaploid using morphological, cytological and molecular marker methods. To effectively monitor G. anomalum genome components in the G. hirsutum background, we developed 5974 non-redundant G. anomalum derived SSR primer pairs using RNA-Seq technology, which were combined with a publicly available physical map. Based on this combined map and segregation data from the BC2F1 population, we identified a set of 230 informative G. anomalum-specific SSR markers distributed on the chromosomes, which cover 95.72 % of the cotton genome. After analyzing BC2F1 segregation data, 50 recombination types from 357 recombination events were identified, which cover 81.48 % of the corresponding G. anomalum genome. A total of 203 recombination events occurred on chromosome 11, accounting for 56.86 % of the recombination events on all chromosomes. By successive backcrosses and selfing combined with marker assisted selection, about 60 chromosome segment substitution lines (CSSLs) were developed. Genomic regions that affect fiber quality, plant height and boll size were identified. This study represents an important step towards introgressing desirable traits into cultivated cotton from the wild cotton species G. anomalum.

Identifying QTL for Fiber Yield and Fiber Quality related Traits in Upland RIL population with a High-density Genetic Map on the whole genome

Palanga,Koffi Kibalou
A recombinant inbred line (RIL) population derived from an intraspecific cross between two elite upland cotton cultivars, 0–153 with excellent fiber quanlity and sGK9708 as commercial variety. A genetic map which covered the whole genome of upland cotton was constructed by three types of markers (SLAF-SNP markers, chip-SNP makers and SSR markers). The consensus genetic map harbored 8295 markers included 5521 SLAF SNP markers, 2398 chip –SNP markers and 733 SSR markers). The total distance of genetic map were 5197.17cM and the avenge distance between adjust markers was 0.88 cM. Combined the 22 phenotype data of the RIL population and the genetic map, the QTLs for fiber quality traits (fiber strength, fiber length and micronaire) and yield traits (boll weight, lint percentage and seed index) were identified. The results showed that there were total of 177 QTLs for fiber length, 156 for fiber strength, 201 for micronaire, 253 for boll weight, 179 for lint percentage and 187 for seed index. Among them, 44 for fiber length, 39 for fiber strength, 42 for micronaire, 58 for boll weight, 35 for lint percentage and 38 for seed index were the stable ones which could be detected in at least three environments. And there were 32 QTLs cluster related to fiber length, 29 related to fiber strength, 18 related to micronaire, 30 related to boll weight, 18 related to lint percentage and 29 related to seed index. And there were 18 negtive related QTLs cluster for fiber length, 14 for fiber strength,11 for micronaire, 14 for boll weight, 18 for lint percentage and 12 for seed index.There were also eight QTL clusters between fiber strength and lint percentage and nine QTL clusters between fiber length and lint percentage, all the QTL clusters showed the different additive effect direction for the related traits, which was in accordance with the significant negative genetic corelation between the fiber quality and lint percentage. The QTL cluster of qCluster-chr7-1 could increase fiber length, fiber strength and seed index the qCluster-chr25-1 could increase the fiber length and fiber strength and reduce the lint percentage and boll weight. The further research including function of genes located on the QTL clusters and the interaction between these genes,which would lay foundations for improving the cotton fiber quality and yield simultaneously. Key words: upland cotton; fiber quality, High-density Genetic Map, QTL,RIL population Youlu Yuan as Corresponding authors

Laccase gene, GhLAC15, enhanced Verticillium wilt resistance via increasing defense-induced lignification and arabinose and xylose accumulation in the cell wall of Gossypium hirsutum

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.

Proactive Breeding for Cotton Diseases Through International Collaborations

Scheffler, Jodi
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.

Progress in Cotton Functional Genomics and structural Genomics

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.

Sequencing and comparative analyses of polyploid cotton genomes

Chen, Z. Jeffrey
Schmutz, Jeremy
Grimwood, Jane
Saski, Chris
Scheffler, Brian
Stelly, David
Peterson, Daniel
Jones, Don
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.

Transcriptome Analysis of Upland Cotton Roots at Two Critical stages of Meloidogyne incognita infection and development.

Kumar, Pawan
Da Silva, Mychele
Singh, Rippy
Davis, Richard F.
Nichols, Robert L.
Chee, Peng W.
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.

A novel virus induced gene silencing method and its application in functional analysis of cotton BAX inhibitor 1 in cotton

Zhang, Jingxia
Wang, Furong
Zhang, Chuanyun
Zhang, Junhao
Liu, Guodong
Chen, Yu
Hao, Fushun
Zhang, Jun
Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not in very young seedlings and roots in cotton. Here, we developed a simple seed soak inoculation (SSI) agroinoculation VIGS (SSI-VIGS) method by soaking naked cotton seeds into Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. Obvious photo-bleaching symptom in newly emerging leaves inoculated with pTRV:GhCLA1 vectors by SSI-VIGS method was observed after 12-14 day post inoculation, while the virus control plants remained green at the same period. Molecular analysis indicated that the photo-bleaching symptom resulted from the reduction of endogenous GhCLA1 transcript abundances, suggesting that SSI-VIGS was able to elicit gene silencing in young seedlings. Naked seeds, Agrobacterium cultures with an OD 600 of 1.5 and 90 min for inoculation time were optimal for SSI-VIGS efficiency of GhCLA1. Besides, SSI-VIGS was able to knock down the expression of gene expressed in roots at the early stage of development, by employing cotton GhPGF (pigment gland formation) as a target. In addition, we readily reduced the expression of cotton Bax inhibitor 1 (GhBI-1) using SSI-VIGS system, The seedlings inoculated with pTRV:GhBI-1 vectors were more sensitive to salt stress compared with viral controls, indicating that GhBI-1 played a positive role in salt tolerance in cotton. Collectively, SSI is a novel and simple agroinoculation method for VIGS, and very useful in elucidating functions of genes, especially genes expressed in roots and young seedlings, in cotton and other seed plants.