Location: Cotton Fiber Bioscience Research2017 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.
Immature cotton fiber phenotype is linked to a gene targeting the cell organelle mitochondria: Fiber maturity directly affects yield and dye uptake, and is determined by fiber cell wall thickness. To identify which genes are responsible for determining the cotton fiber cell wall thickness, ARS scientists in New Orleans, Louisiana, compared deoxyribonucleic acid (DNA) sequences of the immature fiber mutant producing thin fibers with its near isogenic (nearly identical) wild type cotton producing thick fibers. They identified a mutation of pentatricopeptide repeat (PPR) gene in the immature fiber mutant. The PPR gene may affect cell function by targeting mitochondria (a cell organelle). A mutated PPR gene impedes mitochondrial biological processes such as energy distribution in the immature fiber mutant from producing mature fibers. This discovery provides insight on how fiber thickness is regulated during fiber development and allows cotton breeders to better understand and improve the fineness and maturity of cotton fibers. Confirmation of this gene’s functionality in a transgenic cotton is in progress. Objective 2. Roles of xyloglucan in affecting cotton fiber length: Xyloglucan is a polysaccharide (complex carbohydrate) found embedded in the cell walls of all land plants. In growing cells, xyloglucan is thought to connect cellulose complex and regulate their separation during cell growth. To understand the role of xyloglucan in cotton fiber elongation, ARS scientists in New Orleans, Louisiana, used the short fiber mutant called ligon-lintless-2 (Li2) and its long fiber wild type (a normal cotton) for analysis of xyloglucan content and expression levels of xyloglucan-related genes in developing fibers. Accumulation of xyloglucan was significantly higher in Li2 developing fibers than in wild type. Ribonucleic acid (RNA) sequencing analysis revealed that Li2 fiber cells had much higher expression level of xyloglucan-related genes than the wild type fiber cells. These results suggest that early activation and higher expression of xyloglucan-related genes during the fiber elongation phase lead to elevated accumulation of xyloglucan that restricts elongation of fiber cells in short fiber mutant Li2. Objective 3. Validation of infrared method to quantitatively characterize cotton fiber cell wall development: Secondary cell wall (SCW) development is an important factor that affects fiber thickness and strength. However, SCW has not been well characterized due to technical difficulties in determining its development quantitatively, especially in young developing fibers. To address this issue, ARS scientists in New Orleans, Louisiana, compared several previously used conventional methods including chemical, X-ray diffraction, and spectroscopic method with a newly-developed infrared method to estimate crystallinity index (refers to the relative amount of crystalline material in cellulose) of cotton fiber. Compared to the conventional methods that require lengthy and laborious processes, the infrared method enables to characterize SCW cellulose development in a quantitative, rapid, and non-invasive way. The new method will facilitate researchers to quantitatively characterize cotton fiber SCW development, and to identify SCW-related genes. Objective 1. The Ligon-lintless 1 short fiber mutant gene is identified: Cotton fiber length is an important agronomic trait that directly affects the quality of yarn and fabric. Identification of genes regulating fiber elongation will enable researchers to better understand the mechanisms of fiber development, and possibly to devise a strategy to improve fiber length. In cotton, genes directly regulating fiber length are seldom identified. ARS scientists in New Orleans, Louisiana, identified an actin gene (GhACT_LI1) as the cause of the extremely short fiber mutant known as Ligon-lintless 1 (Li1). A single nucleotide substitution in this actin gene in Li1 mutant disrupts cell polarity and membrane anchoring resulting in lintless fibers. Confirmation of this gene’s functionality through cotton transformation is in progress. Objective 2.
1. A major cotton fiber quality trait cluster on chromosome 7 is identified. Identification of genes or genomic locations affecting an agronomic trait is prerequisite to use DNA markers to improve a specific trait in plant breeding. Improving fiber quality is a top breeding goal in almost all cotton breeding programs. However, utilization of DNA markers to improve fiber quality in practical breeding is still rare. Using a unique random-mated population that was derived from crosses involving 11 cotton cultivars, ARS scientists in New Orleans, Louisiana, identified a major fiber quality trait cluster (i.e., a group of genes) on chromosome 7 of cotton genome. This fiber trait cluster has large effect on fiber strength, uniformity and short fiber content, and moderate effect on length. They developed DNA markers to mark this cluster. Selection based on these DNA markers can simultaneously improve fiber strength, length, and uniformity in Upland cotton. The fiber cluster and associated DNA markers identified in this research are being used in cotton breeding programs to improve fiber quality while maintaining yield via marker assisted selection strategy.
2. Identification of a gene susceptible to Envoke® herbicide in cotton. Envoke® herbicide is widely used in cotton production to control broad-leaf weeds. Although most cotton cultivars are tolerant to this herbicide, some cultivars such as Paymaster HS26 are susceptible. Use of this herbicide in a field where a susceptible cultivar is grown may cause a total loss of production. By sequencing the whole genomes of resistant and susceptible cotton cultivars, ARS scientists in New Orleans, Louisiana, identified the causative gene for Envoke® herbicide susceptibility in cotton, and located this gene on chromosome 20 of the cotton genome. It was determined that a single nucleotide mutation in a P450 gene altered the functionality of this gene. Silencing this gene in cotton using a technology known as virus induced gene silencing confirmed that this gene indeed allows cotton plants to be susceptible to this herbicide. With further research, scientists may develop a new safer herbicide controlling broad-leaf weeds in cotton production.
Thyssen, G.N., Fang, D.D., Turley, R.B., Florane, C.B., Li, P., Mattison, C.P., Naoumkina, M.A. 2017. A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants. Plant Journal. 90(1):111-121. https://doi.org/10.1111/tpj.13477.
Islam, M.S., Thyssen, G.N., Jenkins, J.N., Zeng, L., Delhom, C.D., McCarty, J.C., Deng, D.D., Hinchliffe, D.J., Jones, D.C., Fang, D.D. 2016. A MAGIC population-based genome-wide association study reveals functional association of GhRBB1_A07 gene with superior fiber quality in cotton. BMC Genomics. 17:903. https://doi.org/10.1186/s12864-016-3249-2.
Fang, L., Gong, H., Lui, C., Zhou, B., Huang, T., Wang, Y., Chen, S., Fang, D.D., Du, X., Chen, H., Chen, J., Wang, S., Wang, Q., Wan, Q. 2017. Genomic insights into divergence and dual domestication of cultivated allotetraploid cottons. Genome Biology. 18:33. doi:10.1186/s13059-017-1167-5.
Kim, H.J., Lee, C.M., Dazen, K., Delhom, C.D., Liu, Y., Rodgers III, J.E., French, A.D., Kim, S.H. 2017. Comparative physical and chemical analyses of cotton fibers from two near isogenic upland lines differing in fiber wall thickness. Cellulose. 24:2385-2401.
Naoumkina, M.A., Hinchliffe, D.J., Fang, D.D., Florane, C.B., Thyssen, G.N. 2017. Role of xyloglucan in cotton (Gossypium hirsutum L.) fiber elongation of the short fiber mutant Ligon-lintless-2 (Li2). Gene. 626:227-233.
Naoumkina, M.A., Bechere, E., Fang, D.D., Thyssen, G.N., Florane, C.B. 2017. Genome-wide analysis of gene expression of EMS-induced short fiber mutant Ligon lintless-y (liy) in cotton (Gossypium hirsutum L.). Genomics. 109:320-329.