Location: Cotton Fiber Bioscience Research2014 Annual Report
The overall goal of the project is to develop novel molecular tools and approaches to enhance the development of new cotton genotypes with improved fiber properties. Specific objectives are: 1) Identify molecular markers associated with fiber quality and yield quantitative trait loci (QTL) through genome-wide association analysis, and to implement the markers in breeding to improve cotton fiber. 2) Identify genes controlling fiber elongation and maturation, confirm their functionality through transformation in cotton, and develop improved cotton germplasm with novel quality trait genes. 3) Determine gene networks, phytohormones, and molecular mechanisms directly involved in cellulose and xyloglucan biosynthesis in cotton fibers, and elucidate how these genes function to develop effective ways to use them in breeding. Sub-objective: 3a) Determine gene networks, phytohomones and molecular mechanisms involved in cellulose biosynthesis in developing cotton fibers. Sub-objective: 3b) Identify xyloglucan biosynthetic enzymes related to cotton fiber elongation.
Fiber quality and yield are controlled by multiple genes that physically reside on chromosomes. Selection of DNA markers physically associated with superior alleles of these genes will enable breeders to more efficiently and effectively breed a cotton genotype with improved fiber quality and yield. In this project, a recombinant inbred population resulting from random-mating of 11 Upland cotton cultivars will be used to develop molecular markers associated with the quantitative trait loci (QTLs). Simple sequence repeat and single nucleotide polymorphism markers will be developed using approaches such as genotyping-by-sequencing. Agronomic traits will be acquired from multiple year x location test. Associations between markers and traits will be established through a rigorous analysis using statistical softwares. Marker-trait associations will be validated, and transferred to breeders for implementation. Genes, by way of their products such as transcripts or proteins, affect fiber development and physical properties. Therefore, manipulation of these genes or their products will alter fiber development. Three naturally-occurred fiber mutants (Ligon-lintless 1, Ligon-lintless 2 and immature fiber) will be used to study the fiber elongation and maturation. Genetic mapping techniques will be employed to identify the chromosomal locations of these genes. Functional genomics analysis such as nucleic acid sequencing will be used to identify genes or gene-networks affected by the mutations. Potential genes that affect fiber development will be transformed into cotton to validate their functionality. Cellulose biosynthesis is transcriptionally regulated in developing cotton fibers, and phytohormone levels regulate cellulose biosynthesis and secondary cell wall development in cotton fibers. Gene networks and molecular mechanisms involved in cellulose biosynthesis in developing cotton fibers will be determined, and phytohormones promoting second cell wall cellulose biosynthesis in cotton fibers will be identified. Xyloglucan biosynthetic enzymes by regulating xyloglucan affect cotton fiber quality. Members of cellulose synthase like family from Gossypium (G.) hirsutum will be identified and analyzed through functional analysis using heterologous expression and virus induced gene silencing.
In FY 2014 ARS scientists were responsible for the development of Deoxyribonucleic acid (DNA) markers associated with cotton fiber quality and yield, using recombinant inbred lines (RILs). The 550 RILs derived from random mating of 11 cotton cultivars demonstrated wide range of variations in fiber quality and yield. The RILs were planted in Mississippi in 2013 to increase seeds. In 2014, these RILs were planted in three locations (Stoneville and Starkville, Mississippi, and Florence, South Carolina) to collect yield and fiber data. In addition, the 11 parents and 84 RILs were used to develop single nucleotide polymorphism (SNP) markers (a kind of DNA marker) via sequencing technologies. A total of 4663 polymorphic SNP markers were developed, and 2435 of them were included in the first Cotton SNP Chip that has been manufactured by Illumina, Inc. Future work will be to identify DNA markers associated with yield and fiber quality traits, and utilize them in breeding. Progress was made on the validation of the fiber strength quantitative trait locus (QTL) from the cotton cultivar MD52ne. MD52ne has 15-30% higher fiber strength than its parent MD90ne. In 2013, a major fiber strength QTL was identified as residing on chromosome 3. DNA markers associated with this QTL were developed. To validate this QTL, crosses were made between MD52ne and three cultivars including MD25, SG747 and STV474 in the summer of 2013. F2 seeds were planted in 2014 in Stoneville, Mississippi, to obtain fiber data. The existence and effect of the fiber strength QTL will be evaluated using the resulting F2 populations. If confirmed, DNA markers will be used to assist transferring this high strength trait into other cultivars. ARS scientists continuing research on short fiber mutants ligon-lintless 1 and 2 (Li1 and Li2) were used as a model system to study fiber elongation. Next generation sequencing techniques were used to identify the genetic mechanisms that underlie mutant phenotypes. A new approach to design subgenome specific single nucleotide polymorphisms (SNPs) in upland cotton was developed. Large F2 mapping populations (2000 and 1200 individuals for Li1 and Li2, respectively) were analyzed to determine mutation loci. Based on results of mapping of Li2 F2 population the mutation locus was narrow down to 26 genes. Analysis of expression level of genes in the Li2 locus suggested 2 putative candidates, including aquaporin and C2H2 zinc finger family protein (C2H2 zf) transcription factor. Functions of these genes will be tested in cotton using virus induced gene silencing approach. The fine mapping of Li1 mutation locus is in process. Differentiation of fiber initials from cotton ovules is a crucial biological process. Fiber yield depends on the numbers of fiber initials of each ovule. To understand molecular mechanisms of differentiating fiber initials from epidermal tissues of cotton ovules, transcriptome profiles were determined from cotton ovules differing in fiber initial numbers. The differentially expressed genes (DEGs) involved in fiber initiation from the in vitro cultured cotton ovules were verified from the field grown cottons. The results showed that genes involved in 1) auxin signal, 2) ethylene biosynthesis, and 3) abscisic acid catabolism are responsible for the differentiation of fiber initials from cotton ovules. ARS scientists continue research on Genome-wide characterization of cellulose synthase catalytic subunits (CesAs) in two different diploid cotton species, Gossypium raimondii (Gr) and Gossypium arborium (Ga). CesA genes play a pivotal role in cellulose biosynthesis of cotton fibers. In the cotton reference genome of G. raimondii (D5 genome) containing little fibers, ARS scientiests identified 17 GrCesAs, classifying 6 GrCesAs for secondary cell wall (SCW) cellulose biosynthesis and 11 GrCesAs for primary cell wall (PCW) cellulose biosynthesis were identified. All GrCesAs were developmentally and tissue-specifically regulated. In G. arborium (A2 genome), another cotton species containing lint fibers, 17 GaCesAs were also identified. During fiber development, PCW GaCesAs were abundantly expressed while fiber were elongating, whereas SCW GaCesAs were specifically expressed while fiber cell walls were thickening.
1. Identification of quantitative trait loci (QTL) controlling cotton fiber quality. Negative correlation between yield and fiber quality is an obstacle for cotton improvement. Identification of stable cotton fiber QTL is essential in order to improve cotton cultivars with superior quality using marker-assisted selection (MAS) strategy. ARS scientists at New Orleans, Louisiana, identified 131 fiber QTLs and 37 QTL clusters using a random-mated recombinant inbred population. Two major QTL clusters were observed on chromosomes 7 and 16. The fiber QTLs and QTL clusters identified in this research can be readily implemented in a cotton breeding program to improve fiber quality while maintain yield via MAS strategy.
2. Roles of mitochondrial genes in cotton fiber development. Although mitochondrial DNA plays important roles in plant growth and development, little is known on how it affects cotton fiber development. ARS scientists at New Orleans, Louisiana, used Ligon-lintless 2 (Li2) short fiber mutant and its wild type cotton to study the roles of mitochondrial DNA in fiber development. It was found that mitochondrial genes related to energy production such as aderosine triphosphate (ATP) synthase and cytochrome genes are actively involving in the fiber length development. Fiber quality, especially fiber length, could be improved through manipulating the respiratory system of cotton cells, more specifically increasing mitochondrial sublimon replication.
3. Short fiber mutation results in reduced subgenome expression bias in elongating cotton fibers. Short fiber mutation such as Ligon-lintless 2 (Li2) results in extremely short cotton fiber. However, it is unclear how the mutation affects the network related to fiber development. ARS scientists at New Orleans, Louisiana, evaluated the effects of the Li2 mutation on transcriptomes of both subgenomes of cotton as compared to its near isogenic wild type. The mutation affected both subgenomes; however, Li2 had a significantly greater effect on the Dt than on the At subgenome. This was the first report to explore the effects of a single mutation on homeologous gene expression in allotetraploid cotton. These results provide deeper insights into the evolution of allotetraploid cotton gene expression and cotton fiber development.
4. Common mechanisms pertinent to fiber elongation in cotton were revealed. It is important to identify key mechanisms related to fiber development in order to improve the fiber quality through molecular and genomic approaches. ARS scientists at New Orleans, Louisiana, compared gene expression profiles of two short fiber mutants and their near isogenic wild type (WT) cotton grown under both field and greenhouse environments. Analyses of the gene expression profiles showed that most differentially expressed genes (DEGs) were affected by growth conditions. Under field conditions, short fiber mutants commanded higher expression of genes related to energy production, manifested by the increasing of mitochondrial electron transport activity or responding to reactive oxygen species when compared to the WT. Eighty-eight DEGs were identified to have altered expression patterns common to both short fiber mutants regardless of growth conditions. These 88 genes were likely involved in fiber elongation without being affected by growth conditions, and would be good candidates for further investigation.
Kim, H.J., Rodgers III, J.E., Delhom, C.D., Cui, X. 2014. Comparisons of methods measuring fiber maturity and fineness of Upland cotton fibers containing different degree of fiber cell wall development. Textile Research Journal. 84:1622-1633.
Naoumkina, M.A., Thyssen, G.N., Fang, D.D., Hinchliffe, D.J., Florane, C.B., Yeater, K.M., Page, J.J., Udall, J.A. 2014. The Li2 mutation results in reduced subgenome expression bias in elongating fibers of allotetraploid cotton (Gossypium hirsutum L.). PLoS One. 9(3):e90830.
Fang, H., Zhou, H., Sanogo, S., Lipka, A.E., Fang, D.D., Percy, R.G., Hughs, S.E., Jones, D.C., Gore, M.A., Zhang, J. 2014. Quantitative trait locus analysis of Verticillium wilt resistance in an introgressed recombinant inbred population of Upland cotton. Molecular Breeding. 33:709-720.
Islam, M.S., Zeng, L., Delhom, C.D., Song, X., Kim, H.J., Li, P., Fang, D.D. 2014. Identification of cotton fiber quality quantitative trait loci using intraspecific crosses derived from two near-isogenic lines differing in fiber bundle strength. Molecular Breeding. 34:373-384.
Gilbert, M.K., Kim, H.J., Tang, Y., Naoumkina, M.A., Fang, D.D. 2014. Comparative transcriptome analysis of short fiber mutants ligon-lintless 1 and 2 reveals common mechanisms pertinent to fiber elongation in cotton (Gossypium hirsutum L.). PLoS One. 9:e95554.
Thyssen, G.N., Song, X., Naoumkina, M.A., Kim, H.J., Fang, D.D. 2014. Independent replication of mitochondrial genes supports the transcriptional program in developing fiber cells of cotton (Gossypium hirsutum L.). Gene. 544:41-48.
Gilbert, M.K., Bland, J.M., Shockey, J.M., Cao, H., Hinchliffe, D.J., Fang, D.D., Naoumkina, M.A. 2013. A transcript profiling approach reveals an abscisic acid-specific glycosyltransferase (UGT73C14) induced in developing fiber of Ligon lintless-2 mutant of cotton (Gossypium hirsutum L.). PLoS One. 8(9):e75268.
Kim, H.J., Tang, Y., Moon, H.S., Delhom, C.D., Fang, D.D. 2013. Functional analyses of cotton (Gossypium hirsutum L.) immature fiber (im) mutant infer that fiber cell wall development is associated with stress responses. Biomed Central (BMC) Genomics. 14,889.
Thyssen, G.N., McCarty, J.C., Li, P., Jenkins, J.N., Fang, D.D. 2014. Genetic mapping of non-target-site resistance to a sulfonylurea herbicide (Envoke®) in Upland cotton (Gossypium hirsutum L.). Molecular Breeding. 33:341-348.
Fang, D.D., Jenkins, J.N., Deng, D.D., McCarty Jr, J.C., Li, P., Wu, J. 2014. Quantitative trait loci analysis of fiber quality traits using a random-mated recombinant inbred population in Upland cotton (Gossypium hirsutum L.). Biomed Central (BMC) Genomics. 15:397.
Gore, M.A., Fang, D.D., Poland, J.A., Zhang, J., Percy, R.G., Cantrell, R.G., Thyssen, G.N. 2014. Linkage map construction and QTL analysis of agronomic and fiber quality traits in cotton. The Plant Genome. 7(1):1-10.