Location: Cotton Fiber Bioscience Research2016 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.
Identified of deoxyribonucleic acid (DNA) markers associated with cotton fiber quality and yield using a multiparent advanced generation intercross (MAGIC) population: ARS scientists in New Orleans, Louisiana used a MAGIC population that consists of 550 recombinant inbred lines (RILs) derived from random mating of 11 cotton cultivars to identify DNA markers associated with fiber quality and yield traits. In 2014, 2015 and 2016, these 550 RILs along with their 11 parents were planted in three locations (Stoneville and Starkville, Mississippi, and Florence, South Carolina) to collect crop yield and fiber data. Fibers from 2014-2015 crop years were measured using a high volume instrument. The 550 RILs and their 11 parents were sequenced in order to identify single nucleotide polymorphism (SNP) markers covering the whole genome. About 1 million polymorphic SNP markers were identified. Future work will be to identify DNA markers associated with yield and fiber quality traits, validate and utilize them in breeding. Molecular mechanisms of cotton fiber elongation: Fiber length is an important agronomic trait at cotton that directly affects the quality of yarn and fabric. Understanding the molecular mechanisms regulating fiber elongation will enable breeders to breed a cultivar with good yield and fiber quality. ARS scientists at Cotton Fiber in New Orleans, Louisiana, used two short fiber mutants as a model system to study fiber elongation. Using mapping-by-sequencing, virus-induced gene silencing, and molecular modeling, they identified the causative gene of the dominant dwarf Ligon-lintless 1 (Li1) mutation. A single substitution in the actin gene, GhACT_LI1, disrupts cell polarity and membrane anchoring of F-actin resulting in dwarf, lintless cotton plants. As for Ligon-lintless 2 (Li2) mutation, they identified two putative gene candidates including aquaporin and Zinc finger transcription factor (ZnTF). Transformation of these two candidate genes into cotton cultivar Coker-312 is in progress. The first plant over-expressing ZnTF is setting seeds in a greenhouse. Identification of pentatricopeptide repeat (PPR) gene whose frame-shift deletion is linked to immature fiber mutant phenotype: To identify which gene is responsible for determining the degree of cotton fiber cell wall thickness that directly affects cotton yield and dye uptake, ARS scientists in New Orleans, Louisiana, compared genome sequences of the immature fiber mutant producing thin fibers with its near isogenic wild type cotton producing thick and mature fibers. They identified that a mutation of pentatricopeptide repeat (PPR) impeded mitochondrial biological processes in the immature fiber mutant from generating mature fibers. The 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, and therefore cotton textiles. Confirmation of this gene’s functionality in a transgenic cotton is in progress. Functional analyses of two near-isogenic upland cotton lines that differ in fiber strength: To determine molecular mechanisms regulating cotton fiber strength that is required for modern high speed spinning machinery, ARS scientists in New Orleans, Louisiana, compared phenotypic and transcriptomic profiles using two cotton lines showing different fiber strength. They discovered that crystalline cellulose assembly, phytohormones, and kinase signaling pathways were major candidates of regulating strength of individual fibers. The strength of bundle fibers was enhanced by fiber-to-fiber interaction in addition to the individual fiber strength. The results will help better understanding the regulatory mechanisms and relationships between individual and bundle strength of cotton fibers. 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, ARS scientists in Starkville, Mississippi, discovered that some cultivars such as Paymaster HS26 were susceptible. In order to identify the causative gene for this susceptibility, ARS scientists in New Orleans, Louisiana and Starkville, Mississippi, developed a large F2 population derived from a cross of STV474 and HS26. Using mapping by sequencing technique, they located the causative gene on chromosome 20, and identified DNA markers flanking this gene. A single base mutation altered the functionality of this gene. Transformation of this gene into Arabidopsis to confirm its function is in progress.
1. DNA markers associated to the superior fiber strength quantitative trait loci (QTL) from the cotton cultivar MD52ne. MD52ne has 15-30% higher fiber strength than many cotton cultivars. To transfer this superior fiber strength into other cotton cultivars has been difficult and long process if without assistance of DNA markers. Since 2011, ARS scientists in New Orleans, Louisiana, have been developing DNA markers associated to this superior fiber strength. They first identified three major QTL, and developed microsatellite markers. Then they confirmed the QTL using different populations and growing in a different environment. In addition, they developed more robust single nucleotide polymorphism (SNP) markers associated to these QTL. These markers are being used by an ARS cotton breeder in Stoneville, Mississippi, to transfer the fiber strength trait from MD52ne to other cotton germplasm lines.
Islam, M.S., Fang, D.D., Thyssen, G.N., Delhom, C.D., Liu, Y., Kim, H.J. 2016. Comparative fiber property and transcriptome analyses reveal key genes potentially related to high fiber strength in cotton (Gossypium hirsutum L.) line MD52ne. Biomed Central (BMC) Plant Biology. 16:36.
Islam, M.S., Zeng, L., Thyssen, G.N., Delhom, C.D., Kim, H.J., Li, P., Fang, D.D. 2016. Mapping by sequencing in cotton (Gossypium hirsutum) line MD52ne identified candidate genes for fiber strength and its related quality attributes. Theoretical and Applied Genetics. 129:1071-1086.
Naoumkina, M.A., Thyssen, G.N., Fang, D.D., Hinchliffe, D.J., Florane, C.B., Jenkins, J.N. 2016. Small RNA sequencing and degradome analysis of developing fibers of short fiber mutants Ligon-lintles-1 (Li1) and -2 (Li2) revealed a role for miRNAs and their targets in cotton fiber elongation. BMC Genomics. 17:360. https://doi.org/10.1186/s12864-016-2715-1.
Thyssen, G.N., Fang, D.D., Zeng, L., Song, X., Delhom, C.D., Condon, T.L., Li, P., Kim, H.J. 2016. The immature fiber mutant phenotype of cotton (Gossypium hirsutum) is linked to a 22-bp frame-shift deletion in a mitochondria targeted pentatricopeptide repeat gene. G3, Genes/Genomes/Genetics. 6:1627-1633.
Kim, H.J., Liu, Y., Dowd, M.K., Frelichowski, J.E., Delhom, C.D., Rodgers III, J.E., Thibodeaux, D.P. 2016. Comparative phenotypic analysis of Gossypium raimondii with Upland cotton. Journal of Cotton Science. 20:132-144.