Mapping of dwarfing QTL of Ari1327, a semi-dwarf mutant of upland cotton.

BACKGROUND: Upland Cotton (Gossypium hirsutum L.) has few cotton varieties suitable for mechanical harvesting. The plant height of the cultivar is one of the key features that need to modify. Hence, this study was planned to locate the QTL for plant height in a 60Co γ treated upland cotton semi-dwarf mutant Ari1327. RESULTS: Interestingly, bulk segregant analysis (BSA) and genotyping by sequencing (GBS) methods exhibited that candidate QTL was co-located in the region of 5.80-9.66 Mb at D01 chromosome in two F2 populations. Using three InDel markers to genotype a population of 1241 individuals confirmed that the offspring's phenotype is consistent with the genotype. Comparative analysis of RNA-seq between the mutant and wild variety exhibited that Gh_D01G0592 was identified as the source of dwarfness from 200 genes. In addition, it was also revealed that the appropriate use of partial separation markers in QTL mapping can escalate linkage information. CONCLUSIONS: Overwhelmingly, the results will provide the basis to reveal the function of candidate genes and the utilization of excellent dwarf genetic resources in the future.

Analysis of genetic diversity and population structure in upland cotton (Gossypium hirsutum L.) germplasm using simple sequence repeats.

Improvement of cotton fibre yield and quality is challenging due to the narrow genetic base of modern cotton cultivars, which emphasizes the great need to effectively explore the existing germplasm resources. With major objective to assess the genetic diversity and population structure at DNA level, 302 elite upland cotton germplasm accessions (253 Chinese and 49 different exotic origins), were genotyped using 198 simple sequence repeats (SSRs) markers. Each of the 198 markers differed greatly in its ability to detect variations in the panel of cotton germplasm. The SSRs amplified 897 alleles, of which 77.7% were polymorphic. The number of alleles varied from 2 to 12 (mean 4.53). Gene diversity ranged from 0.020 to 0.492 with a mean of 0.279. The polymorphic information content (PIC) values ranged from 0.371 to 0.019 (mean 0.225). Genetic distances in the whole cotton germplasm ranged from 0.451 to 0.052 (mean 0.270), demonstrating relatively wider genetic diversity range. Chinese-origin cotton germplasm showed the highest level of SSR polymorphisms (gene diversity=0.268, PIC=0.218), whereas American-origin revealed the highest mean genetic distance (0.274). Model-based Bayesian analysis clustered the whole cotton germplasm into three subpopulations, and the highest molecular variation ws revealed between subpopulations (4%, P<0.001). The SSRs revealed moderate level of genetic diversity at DNA level, identified three structured subpopulations, suggesting a potential use of these markers for genomewide association mapping studies and for identifying and conserving useful alleles in upland cotton germplasm.

EcoTILLING revealed SNPs in GhSus genes that are associated with fiber- and seed-related traits in upland cotton.

Cotton is the most important textile crop in the world due to its cellulose-enriched fibers. Sucrose synthase genes (Sus) play pivotal roles in cotton fiber and seed development. To mine and pyramid more favorable alleles for cotton molecular breeding, single nucleotide polymorphisms (SNPs) of GhSus family genes were investigated across 277 upland cotton accessions by EcoTILLING. As a result, a total of 24 SNPs in the amplified regions of eight GhSus genes were identified. These SNPs were significantly associated with at least one fiber- or seed-related trait measured in Nanjing, Anyang and Kuche in 2007-2009. Four main-effect quantitative trait nucleotides (QTNs) and five epistatic QTNs, with 0.76-3.56% of phenotypic variances explained by each QTN (PVE), were found to be associated with yield-related traits; six epistatic QTNs, with the 0.43-3.48% PVE, were found to be associated with fiber quality-related traits; and one main-effect QTN and one epistatic QTN, with the PVE of 1.96% and 2.53%, were found to be associated with seed oil content and protein content, respectively. Therefore, this study provides new information for molecular breeding in cotton.

[Homologous simple sequence repeats (SSRs) analysis in tetraploid (AD1) and diploid (A2, D5) genomes of Gossypium].

Simple sequence repeats (SSRs)are a class of repetitive DNA sequences, which are commonly used for genome analysis. Comparison of the homologous SSRs among different genomes is helpful to understand the evolutionary process in relative species. In this study, SSR scanning was performed to investigate their distribution and length variation among the genomes of G. raimondii (D5), G. arboretum (A2) and G. hirsutum (AD1). The results demonstrated that the distribution of SSRs in A genome was very similar with that in D genome, while the length variation of homologous SSRs between A and AD genome was more conserved than that between D and AD genome. Compared with SSRs in AD genome, the number of SSRs with longer motif length in A genome was about five times of those with shorter motif length, while it was about three times in D genome. This implied that the length variation rates of homologous SSRs between diploid cotton and tetraploid cotton were different during the parallel evolution due to the subgenome fusion, and the motif length of most SSRs in tetraoploid genome tended to become shorter than homologous SSRs in diploid genome during the process of evolution. This study comprehensively compared the SSRs in three cotton genomes and revealed the significant difference among them, providing a foundation for further evolutionary study of Gossypium genome.

Identification and characterization of cotton genes involved in fuzz-fiber development.

Cotton fuzz fibers, like Arabidopsis trichomes, are elongated unicells. It is postulated that a transcriptional complex of GLABRA1 (GL1), GLABRA3 (GL3), and TRANSPARENT TESTAGLABRA1 (TTG1) might be in existence in Arabidopsis as evidenced by their physical interaction in yeast, and the complex regulates expression of GLABRA2 (GL2) controlling trichome cell differentiation; it is also assumed that TRIPTYCHON (TRY) and CAPRICE (CPC) counteract the complex formation in neighboring cells. Here, the homologs GaMYB23 (a homolog of GL1), GaDEL65 (a homolog of GL3), GaTTG1, GaCPC and GaTRY were identified in Gossypium arboreum. We show that GaMYB23 can bind to and activate the promoters of GaCPC, GaGL2 and GaTRY, and that GaMYB23, GaTRY and GaTTG1 could interact with GaDEL65 in yeast and in planta. In situ analysis showed that GaMYB23, GaGL2, GaDEL65, and GaTRY were predominantly expressed in fuzz fiber, but GaTRY proteins were primarily found in undeveloped epidermal cells. A G. arboreum fuzzless mutant with consistently high level GaMYB23 transcript has lost the detectable GaMYB23-promoter of GaGL2 complex, corresponding to sharply reduced transcription of GaGL2. Our results support that cotton homologs to the genetic molecules regulating Arabidopsis trichome differentiation interacted in the epidermis of ovules and the redundant GaMYB23 serves as a negative regulator in fuzz-fiber patterning.

Comparative proteomic analysis reveals differentially expressed proteins correlated with fuzz fiber initiation in diploid cotton (Gossypium arboreum L.).

In this study, a comparative proteomic analysis was employed to identify fuzz fiber initiation-related proteins in wild-type diploid cotton (Gossypium arboreum L.) and its fuzzless mutant. Temporal changes in global proteomes were examined using 2-DE at five developmental time points for fuzz fiber initiation, and 71 differentially expressed protein species were identified by MS, 45 of which were preferentially accumulated in the wild-type. These proteins were assigned to several functional categories, mainly in cell response/signal transduction, redox homeostasis, protein metabolism and energy/carbohydrate metabolism. It was remarkable that more than ten key proteins with high-abundance were involved in gibberellic acid (GA) signaling and ROS scavenging, and increasing concentrations of active GAs and H2O2 were also detected approximately 5dpa in wild type ovules. Furthermore, in vivo GA and H2O2 treatments of ovules inside young bolls showed that these compounds can synergistically promote fuzz fiber initiation. Our findings not only described a dynamic protein network supporting fuzz initiation in diploid cotton fiber ovules, but also deepened our understanding of the molecular basis of cotton fiber initiation. BIOLOGICAL SIGNIFICANCE: Our study reported the identification of differentially expressed proteins in wild-type diploid cotton (G. arboreum L.) and its fuzzless mutant by comparative proteomic approach. In total, 71 protein species related to fuzz initiation were identified by MS. These proteins were assigned to several functional categories, mainly in energy/carbohydrate metabolism, protein metabolism, signal transduction, redox homeostasis etc. Importantly, a number of key proteins were found to be associated with GA signaling and ROS scavenging. In consistence with these findings, we detected the increase of GAs and H2O2 concentrations during fiber initiation, and our in vivo ovule experiments with GA and H2O2 injection and following microscopy observation of fuzz fiber initiation supported promoting effects of GA and H2O2 on cotton fiber initiation. These findings depicted a dynamic protein network supporting cotton fiber initiation in diploid cotton ovules. Our study is of major significance for understanding the molecular mechanisms controlling fuzz initiation and also provides a solid basis for further functional research of single nodes of this network in relation to cotton fiber initiation.

[Analysis of cis-regulatory element distribution in gene promoters of Gossypium raimondii and Arabidopsis thaliana].

Cotton genomic studies have boomed since the release of Gossypium raimondii draft genome. In this study, cis-regulatory element (CRE) in 1 kb length sequence upstream 5' UTR of annotated genes were selected and scanned in the Arabidopsis thaliana (At) and Gossypium raimondii (Gr) genomes, based on the database of PLACE (Plant cis-acting Regulatory DNA Elements). According to the definition of this study, 44 (12.3%) and 57 (15.5%) CREs presented "peak-like" distribution in the 1 kb selected sequences of both genomes, respectively. Thirty-four of them were peak-like distributed in both genomes, which could be further categorized into 4 types based on their core sequences. The coincidence of TATABOX peak position and their actual position ((-) -30 bp) indicated that the position of a common CRE was conservative in different genes, which suggested that the peak position of these CREs was their possible actual position of transcription factors. The position of a common CRE was also different between the two genomes due to stronger length variation of 5' UTR in Gr than At. Furthermore, most of the peak-like CREs were located in the region of -110 bp-0 bp, which suggested that concentrated distribution might be conductive to the interaction of transcription factors, and then regulate the gene expression in downstream.

[Genetic analysis of fuzzless in cotton germplasm].

The present study was conducted to evaluate genetic analysis of fuzzless seed trait in cotton. One hundred and two upland cotton (G. hirsutum) and eighty-five island cotton (G. barbadense) were used to cross with the same lines, TM-1 (G. hirsutum) and Xinhai 13 (G. barbadense), respectively. Two different F1 populations obtained were assessed to specify the dominant and recessive inheritance of fiber fuzziness in these lines. Three F1 populations (Kuguangzi × TM-1, Luwuxu × TM-1, and SA65 × TM-1) displaying recessive fiber fuzziness inheritance were selected to construct the F2 population for a further genetic study of fuzzless seed trait. The results of this study indicated that (1) the same materials showed different quantities of fuzzy fiber in different environments. Less fuzzy fiber was found in Xinjian and Hainan compared to Anyang. Thus, the quantity of fuzzy cotton seed depends on ecological environment. (2) In upland cotton, the inheritance of fiber fuzziness was dominant for 26 accessions (25.49%), incompletely dominant for 8 accessions (7.84%), and recessive for 22 accessions (21.57%). The inheritance of fiber fuzziness in island cotton was dominant for 5 accessions (5.88%), incompletely dominant for 16 accessions (18.82%), and recessive for 9 accessions (10.59%). Analysis of F2 population indicated that the fiber fuzziness of Kuguangzi was controlled by two recessive complementary effect alleles. The fiber fuzziness of Luwuxu was controlled by two recessive additive effect alleles, and a single recessive gene controlled the same trait for SA65. Fiber fuzziness evaluation in cotton germplasm provides the genetic and basic information for cotton fiber development study and breeding.

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from Gossypium bickii.

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated 2000 genomic and 800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.

Proteomic identification of differentially expressed proteins in the Ligon lintless mutant of upland cotton (Gossypium hirsutum L.).

Cotton fiber is an ideal model for studying plant cell elongation. To date, the underlying mechanisms controlling fiber elongation remain unclear due to their high complexity. In this study, a comparative proteomic analysis between a short-lint fiber mutant (Ligon lintless, Li(1)) and its wild-type was performed to identify fiber elongation-related proteins. By 2-DE combined with local EST database-assisted MS/MS analysis, 81 differentially expressed proteins assigned to different functional categories were identified from Li(1) fibers, of which 54 were down-regulated and 27 were up-regulated. Several novel aspects regarding cotton fiber elongation can be illustrated from our data. First, over half of the down-regulated proteins were newly identified at the protein level, which is mainly involved in protein folding and stabilization, nucleocytoplasmic transport, signal transduction, and vesicular-mediated transport. Second, a number of cytoskeleton-related proteins showed a remarkable decrease in protein abundance in the Li(1) fibers. Accordingly, the architecture of actin cytoskeleton was severely deformed and the microtubule organization was moderately altered, accompanied with dramatic disruption of vesicle trafficking. Third, the expression of several proteins involved in unfolded protein response (UPR) was activated in Li(1) fibers, indicating that the deficiency of fiber cell elongation was related to ER stress. Collectively, these findings significantly advanced our understanding of the mechanisms associated with cotton fiber elongation.

Co-expression and preferential interaction between two calcineurin B-like proteins and a CBL-interacting protein kinase from cotton.

The CBL/CIPK signaling system mediates a variety of responses to environmental stimuli in plants. In this work, we identified four CBL genes from Gossypium hirsutum, two of which (designated GhCBL2 and GhCBL3) showed preferential expression in the elongating fiber cells. Moreover, the expression patterns of these two CBL genes coincided with that of a putative CBL-interacting protein kinase gene (GhCIPK1) that we isolated in a previous study. Yeast two-hybrid assay indicated that among the four CBLs, GhCIPK1 interacted selectively with GhCBL2 and GhCBL3. The co-expression and interactions of these proteins suggest that they are components of the same signaling pathway. These findings strengthen our previous prediction that CBL/CIPK signaling plays a critical role in the regulation of cotton fiber elongation.

Genetic diversity of source germplasm of Upland cotton in China as determined by SSR marker analysis.

The genetic diversity of 43 sources of Upland cotton germplasm with different parental origins, breeding periods, and ecological growing areas in China were studied on the basis of simple sequence repeat (SSR) markers. A total of 130 gene alleles with 80% polymorphism were detected from 36 SSR primers. The number of alleles per primer ranged from two to eight with an average of 3.6. The polymorphism information content (PIC) range was 0.278-0.865, with an average of 0.62. The average genotype diversity index (H') was 1.102, the highest was 2.039 and the lowest was 0.451. The average coefficient of the genetic similarity of SSR markers among source germplasm was 0.610, ranging from 0.409 to 0.865. These indicated that the genetic diversity at the genomic level of the selected source germplasm was rich, and was representative of the diversity of the germplasms, in general. The diversity at the genome level of the base germplasm from the second and third breeding periods was decreased compared to that of the first period, indicating that the cotton genetic background in China became narrow gradually. The diversity of SSR markers among the base germplasm from early maturity cotton growing areas in the north was higher than those from the Huanghe and Yangtze growing areas. The molecular marker genetic similarity index of the domestic varieties was higher than that in the introduced varieties, which indicates that the genetic diversity in domestic cultivars was lower than that in the introduced varieties. This study gives an overview of the genetic diversity of the cotton germplasm base in China, and provides a guide for breeders to develop new cultivars efficiently.