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Recent progress and future prospect of cotton genome editing

Jin, Shuangxia
Sun, Lin
Qin, Lei
Rui, Hangping
Li, Jianying
Zhang, Xianlong
Recently, we successfully knock out several cotton genes by CRISPR-Cas 9 system with an average 65-85% efficiency. We also perform the whole genome sequencing to investigate the off-target in the CRISPR-Cas9 edited cotton plants. The results showed that of 2000+ potential off-targets sites (allowing ≤ 5 mismatches within the 20-bp sgRNA and 3-bp PAM sequences), the WGS data revealed that only a few (less than 10) are bona fide off-target mutations which suggested that CRISPR/Cas9 system is highly specific for the editing of genes of polyploid plant species.Then, we further developed a high-throughput genome editing system in cotton. A sgRNAs library (containing 1100 sgRNAs targeted to 600 independent genes ) was constructed and cloned into the CRISPR-Cas 9 vector. By this way, we can edit several hundred target genes in one transformation. This system need a very high efficient cotton genetic transformation system to generate thousands of regenerated plantlets by somatic embryogenesis. The data we obtained recently suggested that this system works pretty well in cotton. In addition, we start to work a new genome editing system in cotton by using a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method. Cpf1, a single-strand RNA-guided endonuclease of the class 2 CRISPR-Cas system that cleaves targeted DNA with features distinct from those of Cas9. For example, preferring a T-rich protospacer-adjencent motif (PAM) and cutting in staggered ends. This system has several advantages over the CRISPR-Cas 9 system including small Cas protein size, generating sticky end after cleaving the DNA, cutting the RNA target and lower off-target risk. Our data showed that CRISPR-Cpf1 system works very well with high efficiency and accuracy. At the same time, we developed a base editing system for cotton by fusion of nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) with activation-induced cytidine deaminase (AID). Guided by single guide (sg)RNAs, dCas9-AID will directly transfer cytidines or guanines (C or G) to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9- AIDx converted targeted cytidines (C) especifically to thymines (T), creating specific point mutations. By targeting GhCLA1 with dCas9-AID, we efficiently created known and new mutations showing various phenotype . Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution in cotton.

Systematic analysis of cotton non-specific lipid transfer proteins revealed a clade of genes that play an important role in fiber development

Systematic analysis of cotton non-specific lipid transfer proteins revealed a clade of genes that play an important role in fiber development Cheng-sheng Meng1,2, Yuan-yuan Yan1,2, Zheng-wen Liu1, Li-ting Chen1, Yan Zhang1, Xiu-xin Li1, Li-qiang Wu1, Gui-yin Zhang1, Xing-fen Wang1* and Zhi-ying Ma1* 1College of Agronomy, North China Key Laboratory for Germplasm Resources of Education Ministry, Co-Innovation Center For Cotton Industry of Hebei Province, Hebei Agricultural University, Baoding, Hebei 071001, China 2 These authors contributed equally to this work *Correspondence: mzhy@hebau.edu.cn; cotton@hebau.edu.cn It makes great sense to investigate the regulatory mechanism of fiber development of cotton as it supports an important economic industry-textile. Plant non-specific lipid transfer proteins (nsLTPs) are capable to bind in vitro to various phospholipids including phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and galactolipids with broad specificity. Multiple physiological functions of nsLTPs have been suggested, including membrane and liposome biogenesis, somatic embryogenesis, pollen development, stress resistance, defence and signal transduction. Although some nsLTPs were isolated from cotton fibers and supposed to regulate fiber development, little progress has been achieved on how nsLTPs regulate fiber development. In the present study, nsLTP genes were strictly identified from cotton genome with 138 members in G. hirsutum, 65 members in G. arboreum and 70 members in G. raimondii. These cotton nsLTP genes could be clustered into ten subgroups according to the number of flanking amino acid residues within the conserved ECM domain. Interestingly, type Ⅺ extremely expanded in G. hirsutum genome to be the largest subgroup, which is different from Arabidopsis, cacao,grape, rape and rice whose genome contain most members belonging to type Ⅰ or type Ⅱ. Sequence analysis revealed that GhLtpⅪs were evolutionally close to GhLtpⅡ12-15, and duplication pairs were indentified between GhLtpⅪs and GhLtpⅡs, indicating that type Ⅺ genes of G. hirsutum are likely to diverge from type Ⅱ genes. A large amount of tandem duplication events and non-reciprocal DNA exchanges were found within GhLtpⅪs, which might contribute to the tremendous expansion of type Ⅺ genes. Transcriptional analysis showed that GhLtps were highly transcribed in ovules and fibers, especially GhLtpⅪs whose transcription was significantly higher during fiber elongation in upland cotton cultivars with long fibers, suggesting an essential role of GhLtpⅪs in fiber elongation. Additionally, analysis of the published transcriptome data suggested that most of GhLtpⅪs, ignoring 12 genes that were scarcely detected, were significantly differentially transcribed in ovules at 10 and 20DPA compared with their orthologs in genome A or D, indicating a correlation of cotton type Ⅺ genes with fiber evolution. As fibers are trichomes of the outer epidermis of a single ovule, GhLtpⅪ17, 24, 27 and 28 were cloned and ectopically expressed in Arabidopsis. And significantly elongated trichomes of GhLtpⅪs overexpressed Arabidopsis suggested functional roles of GhLtpⅪs in promoting cell elongation. Our results implied a clade of nsLTP genes regulating fiber elongation and provide new insights into the phenotypic evolution of Gossypium species.

Temporal expression of MYB25-like homoeologs controls lint and fuzz development in cotton

Zhu, Qian-Hao
Stiller, Warwick
Llewellyn, Danny
Wilson, Iain
Cotton fibres arise from the epidermal cells of the seed coat and may be either long (lint) or very short (fuzz). Both lint and fuzz are single-celled and indistinguishable in appearance during the early stages of their growth, suggesting that their growth may involve the same physiological and biochemical processes that are regulated by the same set of genes. The dominant fuzzless mutation N1 of tetraploid Gossypium hirsutum has been demonstrated to be a defective allele of the At-subgenome homoeolog of MYB25-like, a master gene regulating fibre initiation as silencing MYB25-like resulted in lintless and fuzzless seeds. We recently identified five genetic loci, including a major contributing locus containing MYB25-like_Dt, associated with the recessive fuzzless seed trait n2 in G. barbadense based on genotyping (SNP chip and mapping-by-sequencing) of fuzzy and fuzzless near isogenic lines (NILs) derived from an interspecies cross (G. barbadense x G. hirsutum). We compared the expression changes of MYB25-like_At and MYB25-like_Dt during fibre development in cotton accessions with different fibre phenotypes. At 3 dpa (days-post-anthesis) when fuzz fibres are initiating, expression of MYB25-like_Dt was significantly lower in fuzzless NILs than in fuzzy seeded NILs, while higher MYB25-like_Dt expression was associated with more seed fuzz across different cotton genotypes. Phenotypic and genotypic analysis of MYB25-like homoeoalleles in cottons showing different fibre phenotypes and their crossing progeny indicated that both MYB25-like_At and MYB25-like_Dt are associated with lint development, and that fuzz development is mainly determined by the expression level of MYB25-like_Dt at ~3 dpa, suggesting that cotton lint and fuzz development are regulated by the temporal expression of MYB25-like homoeologs. In this presentation, we will propose a working model for the role of MYB25-like homoeologs in lint and fuzz development, and discuss strategies for confirmation of the contributions of MYB25-like homoeologs to lint and fuzz development.

The Role of Noncoding RNA Pattern and Function in Fiber Cell

Guan, Xueying
Zhao, Ting
For ICGI 2018. Cotton fiber is the most important sustainable fiber source for textile industry. It is a single cell organ derived from the epidermis of the cotton ovule or seed. To understand the molecular basis of plant cell differentiation pattern, the cotton fiber cell is a good model system. Non-coding RNA is emerging as one of the most important regulators for the gene expression in response to multiple biological transitions and environmental stimuli. We systematically investigate the non-coding RNA behavior in the fiber cell differentiation progress. The major data indicate both small RNA and long non-coding RNA (lncRNA) play critical roles in fiber cell development. First of all, the fiber cell generates a unique group of small RNA in the fiber initiation stage. For example, the miR828 and miR858 trigger the target gene GhMYB2 to generate tasiRNAs in fiber cell fate determination. Another MIXTA MYB transcriptional factor coding gene, GhMML3 can generate an antisense transcript on the 3’ end of the gene loci. Together with the sense and antisense transcripts, a double strand RNA come into being to derive small RNAs. These secondary generated small RNAs interfere the cell fate determination in the mml3 mutant in stimulating the fiberless seed phenotype. On the other hand, the long non-coding RNAs are also found to take parts in the fiber cell differentiation by small RNA generation. We therefore conclude noncoding RNAs directly impact the fiber cell development in multiple aspects of molecular regulation.

Upgraded genome assembly reveals the key importance of TEs, in-dels, SNPs and point mutations during the evolution of spinnable cotton fibres

Yuxian, Zhu
Gai, Huang
It is generally believed that the ancestors of Gossypium arboreum (A2 genome) or G. herbaceum (A1 genome) together with that of G. raimondii (D5 genome) provided the genetic basis for the modern, cultivated allotetraploid cotton species G. hirsutum (AtDt genome). Here we upgraded the A2 genome assembly by integrating SMRT sequencing and Hi-C technologies. We further resequenced 230 G. arboreum and 13 G. herbaceum accessions (average 6x depth) in China to generate a map of genome variation including ~18 million SNPs and ~2 million indels. Independent analysis suggested that Chinese G. arboreum originated in South China and was subsequently introduced to the Yangtze River and Yellow River regions. GWAS for 11 agronomically-important traits in G. arboreum identified a total of 98 significant peak associations. We found that a burst of transposable elements (TEs) contributed significantly to the 2-fold increase in the size of the G. arboreum genome, when compared to that of the D genome. Comparative genomics analysis suggested that an Ile/Val substitution in conserved catalytic motif DDVAE of 4 gossypol biosynthesis CDN1 genes renders the ability to produce gossypols only in cotton plants. Sequence alignments of the promoters of ACO gene, which is key to ethylene biosynthesis in cotton, revealed that specific deletions in both G. arboreum and in A-subgenome of G. hirsutum caused the loss of several cis-elements, including MYB binding sites, and may be responsible for the inactivation of ACO gene transcription in G. arboreum ovules. This reduced ACO gene expression correlated to the short-fibered phenotype in G. arboreum, whereas very high levels of ACO transcriptions, with a resultant ethylene burst, in G. raimondii ovules seems to force an early fiber senescence phenotype. A Cysteine/Arginine substitution of GaKASIII seems to have conferred significant alteration of the fatty acid composition (C16:0 and C16:1) in cotton seed. Also, gain of Fusarium wilt disease resistance in YZ and YR accessions is associated with GaGSTF9, whose expression is highly inducible upon fungal inoculation. The GaGSTF9-silenced cotton line shows sensitivity to fungal inoculation, whereas overexpression of cotton GaGSTF9 in A. thaliana displays resistance to Fusarium wilt disease compared to wild-type.

Biodiversity and the evolution of the cotton genome

Wendel, Jonathan F.
The cotton genus (Gossypium) is remarkable for its extraordinary natural diversity as well as its importance to humankind. More than 50 species are recognized, including several recently described, which collectively are distributed in arid to semi-arid regions of the tropics and subtropics. Diversity in Gossypium has been promoted by two seemingly unlikely processes: trans-oceanic, long-distance dispersal, and wide hybridization among lineages that presently are widely separated geographically. Included are four species that were independently domesticated for their seed fiber, two diploids from Africa-Asia and two allopolyploids from the Americas. As Gossypium spread worldwide, it experienced remarkable morphological, cytogenetic and genomic diversification, spawning eight monophyletic groups of diploid (n = 13) species (“genome groups” A through G, and K) and 8 allopolyploids (n = 26; AD-genome), the latter resulting from an improbable trans-oceanic dispersal of an A-genome taxon to the New World 1-2 million years ago and subsequent hybridization with an indigenous D-genome diploid. The extraordinary diversity in the genus represents a largely untapped genomic reservoir for agronomic exploration. From a genomic perspective, cotton has a marvelously complex evolutionary history. We now understand that modern allopolyploid species are descended from diploid ancestors which themselves were once polyploid, with this cycle of polyploidization followed by diploidization being repeated many times. This history of “genomic superimpositions” will be described, as will some of the many implications that derive from this understanding.

Cotton DNA methylation and its analysis under the salt- & draught-stress

Wuwei, Ye
DNA methylation, an important component of epigenetics induced usually by adversity, plays a vital role in the response to various stresses including drought and salt. A methylation-sensitive amplification polymorphism method based on capillary electrophoresis was used to explore the epigenetic mechanisms of salt tolerance and heterosis in Upland cotton (Gossypium hirsutum L.), and the results indicated that hypermethylation and demethylation could be an important mechanism to resist the stresses. And the demethylation could be the mechanism to explain heterosis in cotton hybrid. The results of whole genome methylation sequencing showed high DNA methylation density usually occurs in promoter regions and transposons areas. Methylated cytosines in different sequence contexts (CG, CHG and CHH) have different functions and methylation levels. And the results also showed methylated cytosines in asymmetric CHH sequence context are dynamic, being mostly related to stresses. Combined with transcriptome data, we found long non-coding RNAs (lncRNAs) may involve in the regulation of DNA methylation in response to drought stress. All these results could provide theoretical reference value for the mechanism research of tolerance in cotton. Keywords: cotton, salt, draught, methylation

Genome-wide association study identifies the loci and genes related to days to flowering, fiber length and strength in Gossypium hirsutum

Zhiying Ma1, Xingfen Wang1, Yan Zhang1, Guiyin Zhang1, Liqiang Wu1, Zhikun Li1, Xiongming Du2, Shoupu He2, Junling Sun2
Upland cotton (Gossypium hirsutum) is the most widely cultivated species, with over 90% of the global cotton production. Owing to long-term natural selection and artificial breeding under diverse climatic and cultivated conditions, plentiful germplasm resources have been produced for sustainable genetic improvement. Thus, to detect genetic factors contributing to the important traits in a genome-wide scale is indispensable based on these germplasm resources. In the present study, on the basis of approximately 3.66 million SNPs, we conducted genome-wide association study with 419 accessions in three important traits including days to flowering (FD), fiber length (FL) and strength (FS). The results showed that there were 1199, 1661 and 735 SNPs (P < 10-6) significantly associated with FD, FL and FS. We identified 528 FD, 456 FL and 301 FS genes in the 50-kb region surrounding the associated SNPs. For FD, we observed that 94.6% of the associated SNPs (1,199) were located on Dt03 with a strong peak. Among the identified FL-associated SNPs, 646 (38.9%) and 755 (45.5%) were located in At10 and Dt11 with strong association signals, respectively. For FS, 391 (53.1%) SNPs were located in At07. Therefore, we focused on these chromosomes to detect the new genes. We adopted the method that combines peak SNPs and SNP type in GWAS with transcriptome data, functional annotation of the orthologues in Arabidopsis to rapidly identify candidate genes associated with the traits. Through phenotypic significance analysis of haplotypes, qRT-PCR using two types of varieties with different haplotypes, transgenic method and VIGS technology, we found that GhCIP1 and GhUCE are the new genes contributing to FD and fiber initiation in cotton. GhFL1 in At10 and GhFL2 in Dt11 are the new genes controlling fiber elongation. Gh_A07G1769 is a causal gene underpinning the fiber strength of cotton. These results provide targets for molecular selection and genetic manipulation in cotton improvement.

Genome Wide QTL Mapping for Resistance to Verticillium Wilt, Fiber Quality and Yield Traits in Cotton Chromosome Segment Substitution Lines

Md, HarunorRashid
The development of Chromosome Segment Substitution Lines (CSSLs) from Gossypium barbadense in G. hirsutum background provided ideal materials for further genome research and crop improvement through MAS. We had developed BC5F3:5 population with the donor parent Hai1 and the recurrent parent CCRI36. In this study,300 CSSLs and their two parents CCRI36 and Hai1 were planted in a randomized complete block design with 2 replications in two Ecological locations(Anyang and Xinjiang) in 2015 and 2016, respectively. Phenotypic evaluation included Verticillium wilt disease resistance(disease index), fiber yield(plant height,boll weight, lint percentage and seed index) and fiber quaility(fiber length, fiber strength, micronaire,fiber uniformity and fiber elongation). Verticillium wilt disease resistance were collected at the time of July and August.A total of 597 pairs SSR markers screened from 2292 pairs of markers in the whole genome high density map from a BC2F1 population of G. hirsutum×G. barbadense were used to identify the polymorphisms among the BC5F3:5 lines.A total of 56 QTLs for Verticillium wilt disease resistance were detected, 30 of them are stable.,38 QTLs (68%) had negative additives effects, which indicate that the G. barbadense alleles increased Verticillium wilt resistance and decrease DI by about 2.64 to 13.23. The highest number of QTLs (15) for Verticillium wilt disease resistance was detected on Chromosome 05. By meta-analysis, 30 QTL hotspot regions for VW resistance were identified and 13 of them were new, unreported hotspot regions.191 QTLs have been detected for fiber yield and fiber quality, 98 for the fiber quality traits and 93 for the yield related traits, 54 of them are stable(12 for fiber length, 8 for fiber strength, 5 for micronaire,2 for fiber elongation, and 6 for plant height,10 for boll weight,6 for lint percentage and 5 for seed index). Three chromosomes Chr05, Chr10 and Chr20 contained more QTLs.30 clusters with disease index and fiber related traits were identified on 16 chromosomes.Most of the fiber traits were clustered with the disease index stable QTLs. We found 6 clusters namely, C01-cluster-1, C05-cluster-4, C07-cluster-1, C19-cluster-2, C22-cluster-1 and C22-cluster-2, which had positive correlation between VW resistance and fiber quality traits. Two clusters, C10-cluster-1 and C25-cluster-1 had also positive correlation between VW resistance and yield related traits( boll weight and lint percentage). One cluster, C20-cluster-1 is important for VW resistance, fiber quality and fiber yield.So, these clusters and related QTLs are very important for breeding improvement of fiber quality and yield, VW disease resistance. Key words: CSSL, Verticillium wilt disease index, fiber quality, yield, QTL, SSR markers. Correspondence: shiyuzhen@caas.cn,yuanyouluyuan@caas.cn

Genotypic Variations in Salinity Tolerance among BT-Cotton

Farooq, Muhammad Awais,
Shakeel, Amir
Atif, Rana Muhammad
Farrukh, Muhammad Saleem
Fifty cotton cultivars were evaluated for NaCl tolerance in the Department of Plant Breeding and Genetics in the University of Agriculture, Faisalabad. Plants were irrigated with a nutrient solution with an electrical conductivity (EC) of 10dSm-1 and 15dSm-1 with the addition of NaCl from 10-day seedlings to 40-day seedlings. The data for chlorophyll content, root length, shoot length, fresh root weight, fresh shoot weight, shoot dry weight, root dry weight, Na+, K+ and K+/Na+ were taken from the seedlings at the time of harvesting. Analysis of variance indicated that there were significant differences among the genotypes at control and both salt stress levels. Salinity negatively impacted the growth of all cotton cultivars, however, the magnitude of reduction varied among all cultivars. High heritability and the high genetic advance was noticed for the root length, fresh root weight, dry root weight, dry shoot weight and potassium to sodium ratio which indicated the presence of additive gene actions in the expression of these characters. NIAB-824, Mubarak, CIM-612, FH-114, and Kehkshan were conceived as salt tolerant genotypes. The existence of genetic variability in the cotton germplasm exhibited that it can be exploited for the genetic improvement in future breeding programs.