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

Location: Cotton Fiber Bioscience Research

2019 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 first 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. Under Objective 1, ARS researchers validated the previously-identified DNA markers associated with fiber length and strength in new populations with different genetic backgrounds. ARS researchers also made progress in acquiring agronomic trait and DNA sequence data on the newly created populations derived from crosses between 3 elite cotton cultivars and 18 chromosome substitution lines (an upland cotton line with one of its chromosomes replaced by one from pima cotton). Under Objective 2, ARS researchers identified a gene whose mutation could result in shortening of fiber length in cotton. ARS researchers are in the process of identifying the true function of this gene by adding the mutant gene back into a regular cotton line to see if the resulting cotton plant would have short fiber. Under Objective 3, ARS researchers used a CottonScope© (a new instrument) to measure the fiber maturity and fineness of a population that was further random-mated 5 times and self-pollinated 6 generations after the original crosses between 11 cotton cultivars. ARS researchers also identified tentative fiber maturity quantitative trait loci (QTL, a region of chromosome which is associated with a particular phenotypic trait) based on genome wide association study. Overall, our research progress is in line with Project Plan. Detail progress for each objective is shown below. Objective 1, Quantitative trait loci (QTL, a region of chromosome which is associated with a particular phenotypic trait) for fiber strength and length were validated in populations with different genetic backgrounds. A QTL can be mapped by identifying DNA markers associated with the trait of interest, therefore, marker-assisted selection can significantly reduce breeding cost as marker analysis is much cheaper than phenotypic evaluation in a field. In addition, MAS can shorten breeding cycle because a breeder may be able to select a progeny containing the trait of interest by skipping one or two generations based on the marker information. A key to the successful application of MAS in crop breeding is to identify a trait QTL that has relatively large effect and is stable in different genetic backgrounds. Previously, ARS researchers in New Orleans, Louisiana identified a significant fiber strength QTL on chromosome (a section of genome) A07 and a length QTL on chromosome D11 using a population derived from crosses between 11 parental cotton lines. The same group of researchers made new populations by crossing cotton lines that are different from the 11 parents. They grew the populations in Stoneville, MS, analyzed DNA markers on each progeny plant, and measured fiber quality attributes. The A07 fiber strength QTL was present in three different diverse populations, while the D11 length QTL was confirmed in the 4th population. The DNA markers associated with these QTLs are being used to breed high quality cotton lines by cotton breeders in ARS, universities and private industry. In addition, for Objective 1, the genomes (a genome is a combination of all chromosomes, i.e., DNA compositions, also called genetic factors within a cell) of the 180 recombinant inbred lines (RILs) derived from crosses between 21 cotton lines were sequenced. The 21 cotton parental lines include 3 elite upland cotton cultivars and 18 lines in which one of its 26 chromosomes is substituted by a corresponding chromosome from pima cotton (a cotton species with higher quality but lower yield than ordinary upland cotton). The RILs and their 21 parents were grown with one replicate in Stoneville and Starkville, Mississippi in 2018. Fiber quality and yield from each RIL x location were obtained. The ARS researchers in New Orleans, Louisiana are identifying DNA sequence variants between the RILs using the DNA sequencing data, and mapping QTLs by associating the DNA sequence variants with agronomic traits (yield and fiber quality). In regards to Objective 2, ARS researchers in New Orleans, Louisiana used a short fiber mutant to understand cotton fiber elongation process. Discovery of the altered genes responsible for short fiber phenotype and understanding how these genes control fiber elongation will provide researchers an opportunity to manipulate cotton fiber length through breeding or biotechnology. Previously, the same group of researchers identified a mutation in an actin gene as the cause of the short fiber phenotype in the Ligon-lintless 1 mutant. Actin proteins are essential components for cytoskeleton formation that makes up the structural framework inside a cell. To test the true function of the actin gene in cotton fiber elongation, ARS researchers inserted this gene along with two different fiber specific promoters (a piece of DNA able to promote the expression of the target gene) into a normal cotton line Coker 312. The DNA insertion (also called transformation) work has been started, which will take about 9-12 months to obtain transgenic plants. The transgenic plants will be used to evaluate the specific role of actin gene in cotton fiber growth. Additionally, for Objective 2, functional analysis of a candidate gene for the Ligon-lintless 2 (Li2) short fiber mutation was being conducted through transformation (transformation is a process to insert a piece of DNA from another plant or organism into the genome of a plant of interest). The Li2 mutant plants have normal vegetative phenotype, but produce seeds with short fiber. Previously, ARS researchers in New Orleans, Louisiana identified a candidate gene on chromosome D13 causing the Li2 short fiber phenotype. The same group of ARS researchers designed transformation experiments in order to observe the effects of over-expression or suppression of the putative Li2 gene on cotton fiber length. The expression level of putative Li2 gene was significantly reduced in Li2 mutant plants. Therefore, short fiber phenotype is being expected in transgenic plants when the expression of this gene is suppressed. Over-expression of the Li2 gene would expect to increase fiber length in theory. Transgenic plants containing the putative Li2 gene with fiber specific promoters are expected to be transplanted into soil in 9-12 months. For Objective 3, a QTL for fiber maturity was identified based on the phenotypic assessment of a random-mated population using a novel Cottonscope© instrument. Cotton breeders have long been looking for a new way to accurately measure fiber-thickness since the conventional methods like high volume instrument (HVI) failed to obtain consistent fiber maturity (thickness) data suitable for genetic and biological analyses. Cottonscope© is a newly-developed instrument for measuring fiber maturity and fineness. ARS researchers in New Orleans, Louisiana measured 550 recombinant inbred lines that were derived from crossing between 11 cotton cultivars and random-mating thereafter using a Cottonscope© as well as HVI and advanced fiber information system (AFIS). Genome wide association study failed to identify any QTLs for fiber-thickness related properties using phenotypes obtained by AFIS measurement, however, a QTL on chromosome A13 was identified when using the phenotypic data obtained by Cottonscope©, an indication of the new instrument’s merit. Validation of this fiber-thickness QTL using an F2 population is in progress. In addition, for Objective 3, functional characterization of immature fiber gene is being conducted via a transgenic approach. Fiber maturity (fiber cell wall thickness) is a key quality attribute of fiber and yarn fabric. ARS researchers in New Orleans, Louisiana previously identified a mutation in a pentatricopeptide repeat (PPR) gene on chromosome A03 as the cause of the immature fiber trait. To further characterize the PPR gene, these researchers made DNA constructs containing both PPR gene and a constitutive 35S promoter (a piece of DNA that helps the expression of a target gene). These constructs are being inserted into cotton line Coker-312 tissues. It is expected that transgenic plants containing the desired constructs will be generated in the next 9-12 months. They will be used to evaluate true functionality of the PPR gene on affecting fiber maturity.

Accomplishments
1. The gene responsible for the ethyl methanesulfonate (EMS)-induced short fiber phenotype 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 a fiber mutant (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. A mutant with very short fiber (<10 mm) was created by treating cotton seeds using EMS (a chemical that can cause mutation in DNA). The mutant was named Ligon-lintless y (liy). ARS researchers in New Orleans, Louisiana and Stoneville, Mississippi developed populations derived from crosses between the liy mutant and normal cotton lines, analyzed fiber phenotypes and DNA markers of more than 4000 progeny plants. They identified a nucleotide substitution in a tetratricopeptide repeat (TPR)-like superfamily protein gene on chromosome A12 as the cause of the liy short fiber mutation. The single nucleotide substitution in this TPR gene in liy mutant disrupts cell extension resulting in extremely short fibers. Identification of the liy 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
Thyssen, G.N., Jenkins, J.N., McCarty, J.C., Zeng, L., Campbell, B.T., Delhom, C.D., Islam, M.S., Li, P., Jones, D.C., Condon, B.D., Fang, D.D. 2018. Whole genome sequencing of a MAGIC population identified genomic loci and candidate genes for major fiber quality traits in upland cotton (Gossypium hirsutum L.). Journal of Theoretical and Applied Genetics. 132:989-999. https://doi.org/10.1007/s00122-018-3254-8.
Naoumkina, M.A., Thyssen, G.N., Fang, D.D., Jenkins, J.N., McCarty, J.C., Florane, C.B. 2019. Genetic and transcriptomic dissection of the fiber length trait from a cotton (Gossypium hirsutum L.) MAGIC population. BMC Genomics. 20:112. https://doi.org/10.1186/s12864-019-5427-5.
Hu, Y., Chen, J., Fang, L., Zhang, Z., Ma, W., Niu, Y., Ju, R., Zhao, T., Fang, D.D., Zhang, T., Lian, J., Baruch, K., Liu, X., Ruan, Y. 2019. Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton. Nature Genetics. 51:739-748. https://doi.org/10.1038/s41588-019-0371-5.
Gitz, D.C., Liu Gitz, L., Xin, Z., Baker, J.T., Payton, P.R., Lascano, R.J. 2017. Description of a novel allelic “thick leafed” mutant of sorghum. American Journal of Plant Sciences. 8:2956-2965.
Fang, D.D., Naoumkina, M.A., Kim, H.J. 2018. Unraveling cotton fiber development using fiber mutants in the post-genomic era. Crop Science. 58(6):2214-2228. https://doi.org/10.2135/cropsci2018.03.0184.
Kim, H.J., Delhom, C.D., Rodgers III, J.E., Jones, D.C. 2018. Effect of fiber maturity on bundle and single fiber strength of Upland cotton. Crop Science. 59(1):115-124. https://doi.org/10.2135/cropsci2018.05.0324.
Fang, D.D. 2018. General description of cotton. Cotton Fiber: Physics, Chemistry and Biology. pg. 1-11. https://doi.org/10.1007/978-3-030-00871-0_1.
French, A.D., Kim, H.J. 2018. Cotton fiber structure. Cotton Fiber: Physics, Chemistry and Biology. pp. 13-40. https://doi.org/10.1007/978-3-030-00871-0_2.
Kim, H.J. 2018. Cotton fiber biosynthesis. Cotton Fiber: Physics, Chemistry and Biology. pp. 133-150. https://doi.org/10.1007/978-3-030-00871-0_7.
Fang, D.D. 2018. Cotton fiber genes and stable quantitative trait loci. Cotton Fiber: Physics, Chemistry and Biology. pp. 151-178. https://doi.org/10.1007/978-3-030-00871-0_8.
Naoumkina, M.A. 2018. Advances in understanding of cotton fiber cell differentiation and elongation. Book Chapter. pp. 179-191. https://doi.org/10.1007/978-3-030-00871-0_8.
Kim, H.J. 2018. Cotton hairy root culture as an alternative tool for cotton functional genomics. Springer Verlag. pp. 213-221. https://doi.org/10.1007/978-1-4939-8952-2_18.
Kim, H.J., Liu, Y., Fang, D.D., Delhom, C.D. 2019. Feasibility assessment of phenotyping cotton fiber maturity using infrared spectroscopy and algorithms for genotyping analyses. Journal of Cotton Research. 2:8. https://doi.org/10.1186/s42397-019-0027-0.