2012 Annual Report
1a.Objectives (from AD-416):
Objective 1: To characterize spatial, temporal, and/or genotypic variation in cotton fiber gene expression and the mechanisms leading to this variation to identify strategies for producing cotton fiber with enhanced or novel properties.
Objective 2: Determine the consequences of environmental factors on cotton fiber development and develop tools to mitigate these effects.
1b.Approach (from AD-416):
Determine global patterns of genetic differences and gene expression in genotypes that differ in fiber properties. Determine global patterns of gene expression in cotton ovule cultures that are induced to express secondary cell wall CesA genes prematurely. Validate transcription factor candidates by functional genomic approaches in model systems and cotton. Compare the gene expression profiles from controls and cotton plants undergoing heat and/or drought stresses. Determine the effects of heat units on fiber strength and expression of genes specifically involved in the transition period. Evaluate the effects of drought on the fiber quality in a group of varieties (germplasm lines).
Recombinant inbred lines (RILs) are made to increase the cotton germplasm gene base so that cotton breeders will have the maximum available genes and traits to work with in cotton germplasm improvement for any trait of interest. The 550 RILs derived from random mating of 11 cotton varieties demonstrated a wide range of variations in fiber quality and yield. ARS scientists at New Orleans, LA, analyzed 275 RILs with 798 DNA molecular markers. The RILs were planted in Mississippi in 2009-2012 to collect yield and fiber data. We are analyzing the associations between molecular markers and fiber quality traits. Future work will be to validate the marker-fiber trait associations using the remaining 275 RILs, and utilize the markers in breeding.
Cotton hybrids were developed by a series of crosses and chromosome doubling using upland cotton cultivar SG747 and three diploid (double set of chromosomes)cotton species. Although Gossypium amourianum is fiberless, it is a major contributor to fiber strength when introgressed into upland cotton resulting in an 80 to 146% increase in strength. Pollens from synthetic tetraploid (four sets of chromosomes) hybrid plants were crossed with cotton varieties SG747 plants in a greenhouse. In FY 12, ARS scientists at Stoneville, MS and New Orleans, LA, planted F2 progeny in Stoneville, MS, to evaluate the segregation of fiber strength.
In FY 11, ARS scientists at New Orleans, LA, identified one DNA marker NAU3991 that was completely co-segregated with the Li2 gene. In FY 12, we screened a bacterial artificial chromosome (BAC) library using the NAU3991 gene as a probe, and identified 9 clones. One largest clone was sequenced. The clone of 160 kilo base pairs (kb) was from the chromosome 13, while the 130 kb clone was from chromosome 18 that harbors the Li2 gene. Many single nucleotide polymorphisms (SNPs) which can be used as DNA markers were identified. Currently, we are analyzing 50 SNPs in a 400 progeny of F2 population to confirm the linkage between the markers and the Li2 gene. The overall goal is to clone the Li2 gene and understand the mechanism that will enable us to manipulate the fiber length.
Gene expressions between two cotton lines, TM-1 and its near isogenic (almost identical) immature mutant were compared using ribonucleic acid (RNA) sequencing techniques. Genes related to fiber fineness and maturity were differentially expressed between these two lines. Next step will be to verify the identified genes using a technique called real-time quantitative polymerase chain reaction (RT-qPCR).
Isolation and characterization of cellulose synthase catalytic subunit 2 genes from allotetraploid cotton. ARS scientists at New Orleans, LA, isolated genes and transcripts of two cellulose synthase catalytic subunits (GhCesA2-AT and GhCesA2-DT) that are essentially responsible for cellulose production in cotton fiber. In contrast to the previous reports that GhCesA2-DT without consensus sequences for splice was suggested to be a pseudo-gene, the report showed that GhCesA2-DT was a functional gene that could produce transcripts. The results also suggested that GhCesA2-AT and GhCesA2-DT might be involved in regulating cotton fiber properties.
Identification of molecular markers associated with the immature fiber (im) gene affecting cotton fiber maturity. ARS scientists at New Orleans, LA, compared fiber properties between two near isogenic (almost identical) lines (NILs), wild type TM-1 and immature mutant (im) showing a great difference of fiber maturity. A comprehensive fiber property analyses of TM-1 and im fibers showed that the lower maturity of im fibers was due to the lower degree of fiber cell wall thickness as compared to the TM-1 fibers. Using an F2 population comprising 366 progeny derived from a cross between TM-1 and im mutant, the scientists confirmed that the immature fiber phenotype present in a mutant plant was controlled by one single recessive gene im. Furthermore, the researchers identified molecular markers that were closely linked to the im gene within a region of 4.3 centiMorgans on chromosome 3. This research will enable scientists to understand the molecular mechanisms that can be used to improve fiber fineness and maturity.
Construction of an ultra-dense (UD) consensus cotton genetic map. In collaboration with scientists from the U.S. and France, ARS scientists at New Orleans, LA, constructed a UD consensus genetic map of tetraploid cotton using six high density component maps. This UD map consisted of more than 8,200 unique DNA marker loci. Mutual blast alignment (a method to compare DNA sequences) between the marker sequences and the Gossypium raimondii genome sequence indicates high homology between Gossypium raimondii and tetraploid cotton, which will permit localizing genes or repetitive elements on chromosomes once gene annotation of the Gossypium raimondii genome, and of the future diploid A-genome Gossypium arboreum becomes available. This UD map will serve as a valuable resource for quantitative trait mapping, map-based cloning of important genes, and better understanding genome structure and tetraploid cotton evolution. This UD map will also serve as an anchor map to suit the needs of individual scientists who may use different populations.
Fang, D.D., Stetina, S.R. 2011. Improving Cotton (Gossypium hirsutum L.) Plant Resistance to Reniform Nematodes by Pyramiding Ren1 and Ren2. Plant Breeding. 130:673-678.
Hinchliffe, D.J., Turley, R.B., Naoumkina, M.A., Kim, H.J., Tang, Y., Yeater, K.M., Li, P., Fang, D.D. 2011. A combined functional and structural genomics approach identified an EST-SSR marker with complete linkage to the Ligon lintless-2 genetic locus in cotton (Gossypium hirsutum L.). Biomed Central (BMC) Genomics. 12:445.
Kim, H.J., Triplett, B.A., Zhang, H., Lee, M., Hinchliffe, D.J., Li, P., Fang, D.D. 2012. Cloning and characterization of homeologous cellulose synthase catalytic subunit 2 genes from allotetraploid cotton (Gossypium hirsutum L.). Gene. 494(2):181-189.
Han,, X.Y., Xu, X.Y., Fang, D.D., Zhang, T.Z., Guo, W.Z. 2012. Cloning and expression analysis of novel Aux/IAA family genes in Gossypium hirsutum. Gene. 503:83-91.