Crop Science Research Laboratory
Crop Science Research Laboratory
Geng L.,Hebei Academy of Agriculture and Forestry science |
Deng D.D.,U.S. Department of Agriculture |
Wubben M.J.,U.S. Department of Agriculture |
Jenkins J.N.,U.S. Department of Agriculture |
And 2 more authors.
Frontiers in Plant Science | Year: 2017
In crop research programs that implement transgene-based strategies for trait improvement it is necessary to distinguish between transgene homozygous and hemizygous individuals in segregating populations.Direct methods for determining transgene zygosity are technically challenging, expensive, and require specialized equipment.In this report, we describe a standard PCR-based protocol coupled with capillary electrophoresis that can identify transgene homozygous and hemizygous individuals in a segregating population without knowledge of transgene insertion site.PCR primers were designed to amplify conserved T-DNA segments of the 35S promoter, OCS terminator, and NPTII kanamycin resistance gene in the pHellsgate-8 RNAi construct for the Gossypium hirsutum phytochrome A1 gene.Using an optimized multiplexed reaction mixture and an amplification program of only 10 cycles we could discriminate between transgene homozygous and hemizygous cotton control DNA samples based on PCR product peak characteristics gathered by capillary electrophoresis.The protocol was refined by evaluating segregating transgenic progeny from nine BC1 S1 populations derived from crosses between the transgenic cotton parent ‘E-1-7-6’ and other cotton cultivars.OCS PCR product peak height and peak area, normalized by amplification of the native cotton gene GhUBC1, revealed clear bimodal distributions of OCS product characteristics for each BC1 S1 population indicating the presence of homozygous and hemizygous clusters which was further confirmed via K-means clustering.BC1 S1 plants identified as homozygous or hemizygous were self-fertilized to produce BC1 S2 progeny.For the homozygous class, 19/20 BC1 S2 families confirmed the homozygous BC1 S1 prediction while 21/21 BC1 S2 families confirmed the hemizygous prediction of the original parent.This relatively simple protocol provides a reliable, rapid, and high-throughput way of evaluating segregating transgenic populations using methods and equipment common to crop molecular breeding labs. © 2017 Geng, Deng, Wubben, Jenkins, McCarty and Abdurakhmonov.
Shapulatov U.M.,REPUBLIC RESOURCES |
Buriev Z.T.,REPUBLIC RESOURCES |
Ulloa M.,Plant Stress and Germplasm Development Research |
Saha S.,Crop Science Research Laboratory |
And 7 more authors.
Plant Molecular Biology Reporter | Year: 2015
Genes for host-plant resistant do exist in cotton (Gossypium spp.) but improvement of cotton cultivars with resistance is difficult due to intensive breeding. Identifying molecular-genetic mechanisms associated with disease resistance can offer a new way to combat a serious threat such as Fusarium oxysporum f. sp. vasinfectum (FOV). Here, we captured and annotated “top-layer” of abundantly and specifically expressed cotton root small RNA (sRNA) including microRNA (miR) sequences during FOV pathogenesis using size-directed and adenylated linker-based sRNA cloning strategy. A total of 4116 candidate sRNA sequences with 16 to 30 nucleotide (nt) length were identified from four complementary DNA (cDNA) libraries of noninfected and FOV race 3-infected roots of susceptible (“11970”) versus resistant (“Mebane B-1”) cotton genotypes (G. hirsutum L.). The highest numbers of sRNA signatures were those with 19–24 nt long in all libraries, and interestingly, the number of sRNAs substantially increased during FOV infection in a resistant genotype, while it sharply decreased in a susceptible genotype. In BLAST analysis, more than 73 % of sRNAs matched Gossypium (G. arboretum L., G. hirsutum, and G. barbadense L.) ESTs. A small percentage of sRNAs matched A. thaliana (1.68 %), T. cacao (1.26 %), fungal (2 %), and other organism (21.33 %) ESTs. mirBase comparisons showed that 4 % of sRNAs were homologous to previously reported plant miRs, among which we predicted novel cotton Ghr-miR-160 that was not registered in the cotton miR database. These major representative sRNA signatures targeted proteins associated with the key biological processes and molecular functions, explaining the molecular mechanisms of the host defense response during the FOV pathogenesis in cotton. © 2015 Springer Science+Business Media New York
Bondalapati K.D.,South Dakota State University |
Jenkins J.N.,Crop Science Research Laboratory |
McCarty J.C.,Crop Science Research Laboratory |
Wu J.,South Dakota State University
Euphytica | Year: 2015
Field variation is one of the important factors that can have a significant impact on genetic data analysis. Ineffective control of field variation may result in an inflated residual variance and/or biased estimation of genetic variations and/or effects. In this study, we addressed this problem by merging genetic models with the information from a rectangular cotton field layout (referred to row and column directions). Data from a genetic mapping study in Upland cotton (Gossypium hirsutum L.) was used to validate the proposed methodology. This study included model evaluation based on simulations and actual data analysis on four agronomic traits (seed yield, lint yield, lint percentage, and boll weight) in cotton. Results based on simulations suggested that when there were no row and column effects, the conventional and the extended genetic models yielded similar results. However, when either field row and/or column effects were significant, the conventional genetic model yielded biased estimates for residual variance component with larger mean square error whereas the extended genetic models yielded more unbiased estimates. Actual data analysis revealed that lint yield and seed yield were significantly influenced by the systematic variation present in the field. With the extended model, the residual variance associated with these traits was reduced approximately 65 % compared to the conventional block model. Accordingly, the averaged heritability estimate increased by about 18 % for these traits. Thus, the results suggested that genetic data analysis can be improved when field variation is considered. © 2015 Springer Science+Business Media Dordrecht
Saha S.,Crop Science Research Laboratory |
Wu J.,Mississippi State University |
Wu J.,South Dakota State University |
Jenkins J.N.,Crop Science Research Laboratory |
And 3 more authors.
Theoretical and Applied Genetics | Year: 2011
Genetic diversity is the foundation of any cropimprovement program, but the most cultivated Uplandcotton [Gossypium hirsutum L., 2n = 52, genomic formula2(AD)1] has a very narrow gene pool resulting fromits evolutionary origin and domestication history. Cultivarsof this cotton species (G. hirsutum L.) are prized for theircombination of exceptional yield, other agronomic traits,and good fiber properties, whereas the other cultivated52-chromosome species, G. barbadense L. [2n = 52,genomic formula 2(AD)2], is widely regarded as havingthe opposite attributes. It has exceptionally good fiberqualities, but generally lower yield and less desirableagronomic traits. Breeders have long aspired to combinethe best attributes of G. hirsutum and G. barbadense, buthave had limited success. F1 hybrids are readily created andlargely fertile, so the limited success may be due to crypticbiological and technical challenges associated with theconventional methods of interspecific introgression. Wehave developed a complementary alternative approach forintrogression based on chromosome substitution line, followedby increasingly sophisticated genetic analyses ofchromosome-derived families to describe the inheritanceand breeding values of the chromosome substitution lines.Here, we analyze fiber quality traits of progeny familiesfrom a partial diallel crossing scheme among selectedchromosome substitution lines (CS-B lines). The resultsprovide a more detailed and precise QTL dissection of fibertraits, and an opportunity to examine allelic interactioneffects between two substituted chromosomes versus onesubstituted chromosome. This approach creates newgermplasm based on pair wise combinations of quasi-isogenicchromosome substitutions. The relative geneticsimplicity of two-chromosome interactions departs significantlyfrom complex or RIL-based populations, in whichhuge numbers of loci are segregating in all 26 chromosomepairs. Data were analyzed according to the ADAA geneticmodel, which revealed significant additive, dominance, andadditive-by-additive epistasis effects on all of the fiberquality traits associated with the substituted chromosomeor chromosome arm of CS-B lines. Fiber of line 3-79, thedonor parent for the substituted chromosomes, had thehighest Upper Half Mean length (UHM), uniformity ratio,strength, elongation, and lowest micronaire among allparents and hybrids. CS-B16 and CS-B25 had significantadditive effects for all fiber traits. Assuming a uniformgenetic background of the CS-B lines, the comparativeanalysis of the double-heterozygous hybrid combinations(CS-B 9 CS-B) versus their respective single heterozygouscombinations (CS-B 9 TM-1) demonstrated that interspecific epistatic effects between the genes in thechromosomes played a major role in most of the fiberquality traits. Results showed that fiber of several hybridsincluding CS-B16 9 CS-B22Lo, CS-B16 9 CS-B25 andCS-B16 9 TM-1 had significantly greater dominanceeffects for elongation and hybrid CS-B16 9 CS-B17 hadhigher fiber strength than their parental lines. Multipleantagonistic genetic effects were also present for fiberquality traits associated with most of the substituted chromosomesand chromosome arms. Results from this studyhighlight the vital importance of epistasis in fiber qualitytraits and detected novel effects of some cryptic beneficialalleles affecting fiber quality on the 3-79 chromosomes,whose effects were not detected in the 3-79 parental lines. © Springer-Verlag (outside the USA) 2011.
PubMed | Crop Science Research Laboratory
Type: Journal Article | Journal: TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik | Year: 2011
Genetic diversity is the foundation of any crop improvement program, but the most cultivated Upland cotton [Gossypium hirsutum L., 2n = 52, genomic formula 2(AD)(1)] has a very narrow gene pool resulting from its evolutionary origin and domestication history. Cultivars of this cotton species (G. hirsutum L.) are prized for their combination of exceptional yield, other agronomic traits, and good fiber properties, whereas the other cultivated 52-chromosome species, G. barbadense L. [2n = 52, genomic formula 2(AD)(2)], is widely regarded as having the opposite attributes. It has exceptionally good fiber qualities, but generally lower yield and less desirable agronomic traits. Breeders have long aspired to combine the best attributes of G. hirsutum and G. barbadense, but have had limited success. F(1) hybrids are readily created and largely fertile, so the limited success may be due to cryptic biological and technical challenges associated with the conventional methods of interspecific introgression. We have developed a complementary alternative approach for introgression based on chromosome substitution line, followed by increasingly sophisticated genetic analyses of chromosome-derived families to describe the inheritance and breeding values of the chromosome substitution lines. Here, we analyze fiber quality traits of progeny families from a partial diallel crossing scheme among selected chromosome substitution lines (CS-B lines). The results provide a more detailed and precise QTL dissection of fiber traits, and an opportunity to examine allelic interaction effects between two substituted chromosomes versus one substituted chromosome. This approach creates new germplasm based on pair wise combinations of quasi-isogenic chromosome substitutions. The relative genetic simplicity of two-chromosome interactions departs significantly from complex or RIL-based populations, in which huge numbers of loci are segregating in all 26 chromosome pairs. Data were analyzed according to the ADAA genetic model, which revealed significant additive, dominance, and additive-by-additive epistasis effects on all of the fiber quality traits associated with the substituted chromosome or chromosome arm of CS-B lines. Fiber of line 3-79, the donor parent for the substituted chromosomes, had the highest Upper Half Mean length (UHM), uniformity ratio, strength, elongation, and lowest micronaire among all parents and hybrids. CS-B16 and CS-B25 had significant additive effects for all fiber traits. Assuming a uniform genetic background of the CS-B lines, the comparative analysis of the double-heterozygous hybrid combinations (CS-B CS-B) versus their respective single heterozygous combinations (CS-B TM-1) demonstrated that interspecific epistatic effects between the genes in the chromosomes played a major role in most of the fiber quality traits. Results showed that fiber of several hybrids including CS-B16 CS-B22Lo, CS-B16 CS-B25 and CS-B16 TM-1 had significantly greater dominance effects for elongation and hybrid CS-B16 CS-B17 had higher fiber strength than their parental lines. Multiple antagonistic genetic effects were also present for fiber quality traits associated with most of the substituted chromosomes and chromosome arms. Results from this study highlight the vital importance of epistasis in fiber quality traits and detected novel effects of some cryptic beneficial alleles affecting fiber quality on the 3-79 chromosomes, whose effects were not detected in the 3-79 parental lines.