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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Cotton Fiber Bioscience Research » Research » Research Project #424867

Research Project: Molecular Approaches for More Efficient Breeding to Improve Cotton Fiber Quality Traits

Location: Cotton Fiber Bioscience Research

2018 Annual Report


Objectives
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.


Approach
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.


Progress Report
This is the final report of the Project 6054-21000-017-00D, which has been replaced by the new project 6054-21000-017-00D entitled “Molecular Characterization and Phenotypic Assessments of Cotton Fiber Quality Traits”. The new project started on May 29, 2018. For additional information, see the new project report. Progress was made on all three objectives and their sub-objectives, all of which fall under National Program 301, Component 3: Crop Biological and Molecular Processes. Progress on this project focuses on Problem 3A: Fundamental knowledge of plant biological and molecular processes. Under Objective 1; we made significant progress in identifying molecular markers associated with fiber quality quantitative trait loci. We also transferred the markers to cotton breeders for use in breeding. Under Objective 2, we identified the causative genes of the ligon-lintless 1 short fiber mutation and immature fiber mutation, determined the candidate gene for the ligon-lintless 2 short fiber mutation, and conducted genetic analysis on the ligon-lintless-y short fiber mutation. Transformation of the identified ligon-lintless 1 and immature fiber genes in cotton is in progress. Under Objective 3, we determined which cellulose synthase genes play key roles in cellulose production of cotton fibers. In addition, we also identified the relationship between xyloglucans and fiber elongation based on the analysis of short fiber mutants. Overall, our research progress is in line with Project Plan. Objective 1, Genes that are associated with superior cotton fiber traits were identified. Identification of genetic variants that cause cotton fiber quality differences is of keen interest to breeders and molecular biologists. ARS researchers in New Orleans, Louisiana, sequenced the genomes (genetic factor, i.e., a combination of all chromosomes and their DNA compositions) of 550 cotton progeny plants that were derived from crosses between 11 cotton varieties. This resulted in more than 500,000 DNA markers that segregate (present in some, and absent in other) among the progeny plants. By analyzing the DNA markers and the fiber quality parameters of the 550 progeny plants, this same group of researchers identified regions of the genome affecting cotton fiber length, strength, elongation, maturity, fineness and short fiber index. Of the 500,000 segregating markers, about 8,000 result in non-synonymous (not equivalent in function) changes to amino acid sequences of the genes whose functions are known or partially known. Of those, about a dozen genes are significantly associated with one or more of the cotton fiber quality traits. The DNA markers identified in this research will be used in breeding to improve fiber quality. Characterizing the actions of the target genes will further identify genes and gene networks that influence cotton fiber development and quality. In regards to Objective 2, ARS researchers characterized cotton plants producing immature fibers. Fiber maturity is represented by the degree of fiber cell wall thickness. This trait is a key determinant of the cotton fiber value. Both genetic factors and environmental conditions affect fiber maturity. Previously, ARS researchers in New Orleans, Louisiana, reported that a gene called pentatricopeptide repeat (PPR) is linked to the immature fiber phenotype. This same group of researchers further characterized the role of PPR gene in affecting fiber maturity in various environmental conditions. When the immature fiber mutant (a plant showing the effect of mutation, here the immature fiber) and its near isogenic (nearly identical) wild type (WT) cotton were grown under normal temperature and light conditions, the immature fiber mutant plants managed to maintain equivalent net photosynthesis (a process by which green plants use sunlight to produce foods from carbon dioxide and water) to the WT cotton. Heat stress significantly suppressed the photosynthesis of the immature fiber mutant plants compared to the WT plants, whereas light stress showed insignificant effects. The results provide insight into how environmental stress conditions and genetic factors synergistically affect fiber maturity in cotton. In addition, for Objective 2, the genetic cause of Ligon-lintless 2 short fiber mutation was identified. Cotton fiber length is an important agronomic trait that directly affects the quality of yarn and fabric. A mutation called Ligon-lintless 2 (Li2) causes very short cotton fiber. Identification of the gene or genetic factor that causes the Li2 short fiber mutation may enable cotton researchers to better understand the mechanisms of fiber elongation, and possibly to devise a strategy to improve fiber length through biotechnology or breeding. ARS researchers in New Orleans, Louisiana, identified a structural rearrangement (specifically, a piece of DNA segment reversed its direction on the chromosome) in the 18th (also called D13) chromosome, one of the total 26 chromosomes of the cotton genome as the possible cause of the Li2 mutation. Functionality confirmation of this mutation through cotton transformation is in progress. Additionally, an ethyl methanesulfonate (EMS)-induced short cotton fiber mutant Ligon lintless-y was genetically maped. A short fiber mutant called Ligon-lintless-y (Liy) was created by treating cotton seeds with EMS (a chemical that causes mutations in DNA). Liy mutant plants have much short fibers than its wild type cotton. Genetic analysis indicated that the short fiber mutation is controlled by a single recessive (heritable characteristic expressed in offspring only when inherited from both parents, i.e., when not masked by a dominant characteristic inherited from one parent) locus (the position of the mutation on a chromosome) designated liy. Besides causing short fibers, this mutation also affects other traits including plant height. ARS researchers in New Orleans, Louisiana, sequenced a DNA bulk consisting of 100 short fiber progeny plants along with the DNAs of two parental cotton lines. The liy locus was mapped on the 12th (also called A12) chromosome. DNA markers flanking the liy locus were developed. Mapping this locus with more DNA markers and characterization of the genes around the liy locus are in progress. With regard to Objective 3, the cellulose synthase genes in two cotton species differing in fiber properties were characterized. Cellulose is an organic substance that is the major constituent of cotton fibers. Cellulose synthase (CESA) is an enzyme that plays a pivotal role in cellulose synthesis of cotton fibers. Although 17 CESA genes are found in cotton genomes, it is not well known which CESA genes are responsible for the cellulose synthesis in cotton fibers. ARS researchers in New Orleans, Louisiana, compared the expression levels of each CESA gene in various cotton tissues from two cotton species including an uncultivated cotton species (Gossypium raimondii) with inferior fiber and a cultivated cotton species (Gossypium arboreum) with good fiber. Among the 17 CESA genes, 14 were actively expressed in both species. All actively-expressed CESA genes were universally expressed at most tissues of the uncultivated cotton species, whereas tissue specific expression pattern of CESA genes was found in the cultivated cotton species. This discovery provides insight into how cellulose biosynthesis is regulated by different CESA genes during fiber development and allows cotton breeders to better understand and improve cotton fiber properties.


Accomplishments
1. A DNA chip that assists cotton researchers to develop desirable cotton lines with reduced cost. The traditional approach for crop improvement involves identifying a desired trait and making selection in a plant population. This process is laborious and time-consuming, and highly dependent on the experience of a breeder. With the advent of DNA technologies, and the ability of scientists to determine the DNA composition of a plant at the molecular level, the process of identifying the genetic variation in a gene or a set of genes that are responsible for a particular trait has been greatly simplified. Single nucleotide polymorphisms (SNPs, pronounced “snips”) are the most common type of genetic variation among organisms. Each SNP represents a difference in a single DNA building block called nucleotide. There are only four nucleotides (A, C, G, T, in abbreviation) in DNAs of any organisms. SNPs can act as biological markers, helping scientists to locate genes that are associated with a desired trait. Thus, breeders can make selections based on DNA markers. ARS researchers in New Orleans, Louisiana, first developed more than 4,000 SNP markers that reveal polymorphisms (differences) within the cultivated cotton. Subsequently, these markers were placed on a DNA chip/array (a collection of microscopic DNA spots embedded in a solid surface) containing 63,000 SNP markers. This chip enables cotton researchers to quickly analyze the presence of genes in a cotton line at a very nominal cost. This data is enabling cotton researchers worldwide to characterize cotton lines that possess the desired trait so that breeders can then develop a cotton line with this trait.

2. Superior fiber strength quantitative trait loci from the cotton line MD52ne were identified. The cotton line MD52ne has 15-30% higher fiber strength than many other cotton cultivars. Cotton breeders have been trying to transfer this superior fiber strength trait into other cotton cultivars through conventional breeding. However, this process has been difficult and long when not knowing where the underlying genes are located in the cotton genome (a combination of chromosomes and their DNA compositions). Identifying the genomic locations of the underlying genes and developing DNA markers adjacent to these locations would greatly assist cotton breeders to bring this superior fiber trait into other cotton lines. ARS researchers in New Orleans, Louisiana, crossed MD52ne and its near isogenic (nearly identical) line MD90ne, measured fiber quality of their offspring plants, and analyzed all offspring plants with DNA markers. This group of researchers identified three major genomic locations (called quantitative trait loci, or QTL in short) that contain genes controlling this superior fiber strength trait, and developed DNA markers to tag these QTLs. They confirmed the QTL effects using different populations as well as by growing them in a different environment. These markers are being used by an ARS cotton breeder in Stoneville, Mississippi, to transfer the superior fiber strength trait from MD52ne into other cotton lines.

3. Major quantitative trait loci for cotton fiber strength and length were identified. Use of DNA markers to assist selection in plant breeding can significantly reduce cost and improve breeding efficiency. In order to use DNA markers in breeding, genes or genomic locations affecting an agronomic trait need to be identified, and DNA markers associated with these locations are required to be developed. In cotton, traditional approach for fiber quality improvement relies on measurement of fiber quality traits of each plant in a population, and selecting the best plant for advancing to the next generations. This process is not only long, but also a desirable fiber trait may get lost during the advancing process. To address this issue, ARS researchers in New Orleans, Louisiana, used a unique random-mated population that was derived from crosses among 11 cotton cultivars. They identified a genomic region on the 7th chromosome (also called A07) that greatly affects fiber strength. This genomic location (called quantitative trait locus, or QTL in short) could increase fiber strength by 25%. In addition, this same group of ARS researchers also identified a major fiber length QTL on the 21st chromosome (also called D11). They developed single nucleotide polymorphism (SNP) (one form of DNA marker) markers for both QTLs. Selection based on these DNA markers can significantly improve fiber quality traits in upland cotton. The DNA markers identified in this research are being used in the U.S. cotton breeding programs to improve fiber quality while maintaining yield via a marker assisted selection strategy.

4. The causative gene of the Ligon-lintless 1 short fiber mutation was identified. Understanding the molecular mechanisms of fiber development is important in order to improve fiber quality through an approach of gene manipulation. Comparative analyses of fiber mutants (a cotton plant showing the effects of fiber mutation) and a normal cotton line has been an excellent model system to study fiber growth and development. One of the cotton fiber mutants is Ligon-lintless 1 (Li1) short fiber mutation. This mutant has extremely short fiber (<6mm). Although this mutant was discovered in 1929, the causative gene of this mutation had not been identified until 2017 by a group of ARS researchers in New Orleans, Louisiana. They developed populations derived from crosses between the Li1 mutant and normal cotton lines, analyzed fiber phenotypes and DNA markers of more than 3000 progeny plants. They identified an actin gene (GhACT_LI1) as the cause of the Li1 short fiber mutation. Actin is a protein that functions mainly as cytoskeleton component in plant cells. This gene is located on the 22nd (also called D04) chromosome. A single nucleotide substitution in this actin gene in Li1 mutant disrupts cell extension resulting in extremely short fibers. Identification of the Li1 mutation gene laid the foundation for cotton researchers to better understand the mechanisms of fiber development, and possibly to devise a strategy to improve fiber length.


Review Publications
Abdelraheem, A., Fang, D.D., Zhang, J. 2017. Quantitative trait locus mapping of drought and salt tolerance in as introgressed recombinant inbred line population of upland cotton under the greenhouse and field conditions. Journal of Euphytica. (2018)214:8. https://doi.org/10.1007/s10681-017-2095-x.
Kim, H.J., Liu, Y., French, A.D., Lee, C.M., Kim, S.H. 2018. Comparison and validation of Fourier transform infrared spectroscopic methods for monitoring secondary cell wall cellulose from cotton fibers. Cellulose. 25:49-64.
Fang, D.D., Kim, H.J. 2018. History of cotton fiber bioscience research at USDA-ARS Southern Regional Research Center. Journal of Cotton Science. 22:12-23.