Location: Cotton Fiber Bioscience Research2015 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.
Development of deoxyribonucleic acid (DNA) markers associated with cotton fiber quality and yield using recombinant inbred lines (RILs): ARS scientists in New Orleans are using 550 RILs derived from random mating of 11 cotton cultivars to develop DNA markers associated with fiber quality and yield. In 2014 and 2015, these 550 RILs were planted in three locations (Stoneville and Starkville, Mississippi, and Florence, South Carolina) to collect yield and fiber data. All fibers from 2014 crop year in three locations were measured using high volume instrument. The 11 parents and 550 RILs were used to develop single nucleotide polymorphism (SNP) markers via genome-by-sequencing technologies. More than 4000 polymorphic SNP markers were identified. Furthermore, the genomes of 130 RILs and the 11 parents were deep-sequenced in order to identify SNP markers covering the whole genome. Sequencing the genomes of the remaining 420 RILs is in progress. Future work will be to identify DNA markers associated with yield and fiber quality traits, and utilize them in breeding. Research will continue to validate the markers identified. Validation of the fiber strength quantitative trait locus (QTL) from the cotton cultivar MD52ne: MD52ne has 15-30% higher fiber strength than its parent MD90ne. In 2013, a major fiber strength QTL was identified on chromosome 3. DNA markers associated with this QTL were developed. To validate this QTL, F3 progeny of the cross MD52ne x MD90ne were planted in 2014 in Stoneville, Mississippi, to obtain fiber data. The fiber strength QTLs were confirmed in the F3 progenies. In addition, new single nucleotide polymorphism (SNP) markers were developed to enrich the genomic regions harboring QTLs in order to improve the mapping accuracy. High resolution genetic mapping of the QTLs using SNP markers is in progress. High resolution genetic mapping of immature (im) mutant gene affecting cotton fiber maturity: Maturity is an important attribute of cotton fiber quality. Understanding the genetic control of fiber maturity has been very difficult because environmental factors greatly affect the eventual fiber maturity. To overcome this challenge, ARS scientists in New Orleans, Louisiana, used a naturally-occurring immature fiber mutant (im) to study the molecular mechanism regulating maturity development. Previously, they mapped the im mutant gene on chromosome 3. In 2014, they developed a new F2 population of 2400 progeny to fine map the im gene. They also sequenced the genomes of two parental varieties, TM-1 and im mutant. Single nucleotide polymorphism (SNP) markers are being developed to provide a high resolution coverage of the im gene region. High resolution genetic mapping of two mutant genes affecting cotton fiber length: Length is an important attribute of cotton fiber quality. Understanding how fiber elongation is regulated at molecular and genomic levels will enable researchers to increase fiber length through genetic manipulation of important fiber-related genes. ARS scientists in New Orleans, Louisiana, used two short fiber mutants Ligon-lintless 1 (Li1) and -2 (Li2), as a model system to unlock the molecular mechanisms of fiber elongation. Using modern sequencing technologies and genetic mapping, they mapped the Li1 and Li2 genes within a genomic interval of 255 kilobase (kb) and 153 kb, respectively. Gene annotation indicates that a large family of proteins including sugar transporters and an aquaporin (responsible for transporting water and nutrients) are possible candidate of Li1 and Li2 gene, respectively. These results will facilitate a more in-depth research of cotton fiber length development. Comparative analyses of two near-isogenic upland cotton lines that differ in bundle and single fiber strength: To determine the genetic and physical factors affecting cotton fiber strength, bundle fiber strength between two near isogenic (almost identical) cotton lines was compared, and differentially expressed genes (DEG) between the lines were identified. The results showed that the stronger line has higher molecular weight of celluloses than the weaker line and the bundle strength resulted from fiber to fiber interaction in addition to the strength of individual fibers. Future research will be to study the roles of the DEGs in fiber strength development.
1. A new 63K single nucleotide polymorphism (SNP) marker chip for cotton. To effectively use DNA markers to assist breeding requires characterizing a cotton line by analyzing many (usually thousands) markers. It has been very difficult and expensive to fingerprint a cotton line using moderate number of, such as 500, markers. ARS scientists in New Orleans, Louisiana, first developed more than 4000 SNP markers that reveal polymorphisms (differences) within the cultivated upland cotton. Then, as a member of International Cotton SNP Chip Consortium, the ARS researchers assisted Texas A&M University and Illumina, Inc. to develop the first cotton SNP chip containing 63K SNP markers. This chip enables cotton researchers to conveniently obtain 63000 marker data for a cotton line at a price of about 0.1 cent per marker datum. The SNP chip is being used by cotton breeders to characterize cotton lines and to assist breeding.
2. Characterization of fiber initiation process in cotton. The number of cotton fibers initiated from ovules is an important factor affecting cotton yield and fiber quality. Therefore, the fiber initiation process is considered a potential target for improving yield by biotechnological manipulations. However, the molecular mechanisms regulating cotton fiber initiation are not well-characterized due to technical difficulties in monitoring the rapid process of fiber cell differentiation on cotton ovaries. Using in vitro culture techniques and transcriptome analyses of wild type and fiberless mutants, ARS scientists in New Orleans, Louisiana, identified candidate genes involved in fiber initiation. Their findings provide an insight on molecular mechanisms controlling when and how fiber initiation occurs on cotton ovules.
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Thyssen, G.N., Fang, D.D., Turley, R.B., Florane, C.B., Li, P., Naoumkina, M.A. 2015. Mapping-by-sequencing of Ligon-lintless-1 (Li1) reveals a cluster of neighboring genes with correlated expression in developing fibers of Upland cotton (Gossypium hirsutum L.). Journal of Theoretical and Applied Genetics. 128:1703-1712.
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