Project : USDA ARS

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Research Project: Molecular Characterization and Phenotypic Assessments of Cotton Fiber Quality Traits

Location: Cotton Fiber Bioscience Research

2020 Annual Report

Objectives
1. Use genome-wide association analysis to identify genes and molecular markers that are positively associated with cotton fiber quality and yield traits, and work with breeders to evaluate their effectiveness for simultaneous improvement of fiber quality and yield in diverse genetic backgrounds. 1.A. Use genome-wide association analysis to identify genes and molecular markers that are associated with cotton fiber quality and yield trait QTL. 1.B. Validate the stability and transferability of fiber QTL in diverse genetic backgrounds and work with breeders to evaluate their effectiveness for simultaneous improvement of fiber quality and yield. 2. Use short fiber mutants to evaluate cotton fiber elongation to discover and characterize biochemical pathways and genes controlling fiber elongation. 3. Use cotton mutants to determine impacts of genes and environment on cotton fiber maturity and fineness.

Approach
Fiber quality and yield are controlled by multiple genes that physically reside on chromosomes. Re-sequencing the genomes of a population of recombinant inbred lines (RILs) that differ in fiber quality and yield will identify genes or genomic regions controlling these traits. Fiber quality is controlled by genes that physically reside on chromosomes. Selection of DNA markers physically adjacent to the superior alleles of these genes will enable breeders to more efficiently and effectively breed a cotton genotype with improved fiber quality. 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. Biological processes affecting fiber maturity and fineness are regulated by genes, and are significantly affected by environmental factors. Manipulation of these genes will alter fiber maturity and fineness and may reduce the influence of environmental factors.

Progress Report
This is the second annual progress report of the Project 6054-21000-018-00D that started on May 29, 2018. Progress was made on all three objectives and their sub-objectives, which fall under National Program 301, Component 1: Crop Genetic Improvement; Problem Statement 1A: Trait discovery, analysis and superior breeding methods; and Component 3: Crop Biological and Molecular Processes; Problem Statement 3A: Fundamental knowledge of plant biological and molecular processes. Objective 1, ARS researchers at New Orleans, Louisiana, evaluated the stability and transferability of two loci responsible for cotton fiber strength and length, respectively. A locus is the position of a gene on a chromosome. ARS researchers at New Orleans, Louisiana, also further revealed the possible identities of the genes. Objective 2, ARS researchers at New Orleans, Louisiana, inserted a gene that is responsible for short fiber phenotype into a regular cotton line using fiber specific promoters. A promoter is a piece of DNA that promotes and helps the function of a gene. ARS researchers at New Orleans, Louisiana, sequenced fiber ribonucleic acid (RNA, one form of nucleic acid in living organism) to identify genes controlling cotton fiber elongation. Under Objective 3, we identified a locus for fiber maturity using the fiber data obtained from a CottonScope. CottonScope is a new fiber measuring instrument that can provide more accurate readings on fiber thickness. Overall, our research progress is in line with the Project Plan. Detail progress for each objective is shown below. Objective 1, Genes responsible for cotton fiber strength are being identified. Previously, ARS researchers in New Orleans, Louisiana, identified two loci responsible for fiber strength and length, respectively. They also developed DNA tools to help breeders incorporate these two loci in their breeding programs. To further identify the genes responsible for the improved fiber strength, ARS researchers in New Orleans, Louisiana, analyzed the entire genome DNA sequences (i.e., the order and reading of DNA letters on a chromosome) of 550 cotton lines. They identified three genes that are possibly responsible for the stronger fiber. To confirm the gene function, the researchers started a process called transformation by inserting the superior version of a gene into a normal cotton variety. Transformation is a procedure whereby a piece of DNA from one organism is inserted into the genome of another organism of interest, and the resulting plant is called transgenic. Transgenic cotton plants will be generated in 12-18 months. In addition, for Objective 1, ARS researchers in New Orleans, Louisiana, assessed the stability and breeding utility of a locus responsible for fiber length. This fiber length locus was previously identified by the same researchers using a population derived from crosses involving 11 cotton varieties. The researchers made two new populations by crossing cotton varieties that differ from the previously-used 11 cotton varieties. They grew cotton plants in Stoneville, Mississippi, analyzed DNA markers and measured fiber length of each progeny plant. They confirmed that the fiber length locus was stable in the two new populations, and suitable for use in a breeding program to improve fiber length. Incorporation of this fiber length locus into new breeding materials is being carried out by an ARS cotton breeder in Stoneville, Mississippi. Part of this progress was made under the agreement # 66165 with Cotton Incorporated. Objective 2, ARS researchers in New Orleans, Louisiana, analyzed the function of a gene responsible for the short fiber mutation through transformation. Previously, ARS researchers in New Orleans, Louisiana, identified a gene possibly responsible for the short fiber phenotype in a naturally-occurring mutant called Li2. In order to confirm the effect of this gene on fiber length reduction, the researchers either suppressed or enhanced the expression of the target gene in their transformation experiments. The researchers suppressed the target gene only during fiber development by using a fiber specific promoter. Promoter is a piece of DNA promoting and helping the function of a gene. Transformation procedure was started and is in process. Transgenic plants over-expressing the target gene were obtained, however, fiber length in transgenic plants was not significantly different from a normal cotton line. To further prove that the gene is correct and responsible for the short fiber phenotype, ARS researchers crossed a transgenic plant with a mutant plant. The first generation hybrid seeds were planted in a greenhouse, and plants are growing. Additionally, for Objective 2, ARS researchers in New Orleans, Louisiana, used RNA sequences of a population consisting of 550 progeny lines to identify genes controlling fiber length. RNA (ribonucleic acid) is a form of nucleic acid carrying instructions from DNA for controlling the production of proteins. The researchers grew 275 progeny lines along with their 11 parents in a field in New Orleans. They collected young fibers from cotton fruits at three different days after flower opening, and isolated RNA from young fibers. These RNAs are being sequenced (an analytic process to determine the order of each nucleotide letter along the RNA string), and the expression of active genes can be determined based on the RNA sequence reads. Association of gene expressions with gene locations on chromosomes and fiber length measurement of each progeny line will enable researchers to identify genes controlling fiber elongation. Analysis of RNA sequence data is in progress. Part of this progress was made under the agreement # 66156 with Cotton Incorporated. Objective 3, A locus responsible for fiber maturity was identified based on the phenotypic measurement of a cotton population using CottonScope. CottonScope is a newly-developed instrument that can more accurately measure fiber maturity (an indication of fiber cell wall thickness) and fineness than the commonly-used instrument such as high volume instrument (HVI) or advanced fiber information system (AFIS). ARS researchers in New Orleans, Louisiana, measured fiber properties of 550 cotton lines using a CottonScope, HVI and AFIS. Genetic analysis failed to detect any locus responsible for fiber maturity using either HVI or AFIS measurements. However, using the measurements obtained by CottonScope, ARS researchers at New Orleans, Louisiana, identified a locus that is highly likely to house genes controlling fiber maturity. Validation of this fiber maturity locus is being conducted using two new populations derived from crosses between cotton lines showing significant fiber maturity difference. In addition, for Objective 3, ARS researchers in New Orleans, Louisiana, studied the effect of environmental factors on phenotypic variations between the immature fiber mutant and a normal cotton line. The immature fiber mutant produces weak and thin fibers. When grown under ideal environmental conditions in a growth chamber, there was little phenotypic variation in the fibers and leaves of the mutant and normal cotton plants. However, the variation in fiber thickness and biomass of non-fiber tissues became obvious when they were grown under high temperatures. The results revealed that thermo-tolerance is associated with the phenotypic and functional variation of immature fiber mutant as compared to the normal cotton plants. Part of this progress was made under the agreement # 66180 with Cotton Incorporated.

Accomplishments
1. A DNA region responsible for cotton fiber strength is transferrable, stable, and strongly expressed in new cotton varieties.. ARS researchers in New Orleans, Louisiana, successfully showed that a DNA region they had identified as responsible for the stronger fiber was transferrable, stable, and strongly expressed. When they crossed 14 cotton lines and generated eight new populations, seven of the eight contained the DNA region of interest and the improvement in fiber strength was clearly observed in all of the seven. This confirms their previous results when eleven different initial varieties were used with the same results. ARS researchers at New Orleans, Louisiana, also developed DNA markers for how to find this DNA region in the cotton genome. The finding is important because some breeders use a molecular genetic technique called marker-assisted selection to look for this DNA region using the DNA markers when cotton plants are at seedling stage. The knowledge that early detection and analysis of this DNA region will most likely result in a stable trait that will generate strong fibers is invaluable to cotton breeders as they can make early assessment of crosses. The DNA markers to tag the specific DNA region responsible for cotton fiber strength are now being used by cotton breeders in ARS, university, and private industry.

U.S. DEPARTMENT OF AGRICULTURE

Cotton Fiber Bioscience Research: New Orleans, LA