Molecular mapping of a new source of Fusarium wilt resistance in tetraploid cotton (Gossypium hirsutum L.)

Fusarium wilt (FW) disease is an economically important disease of cotton worldwide and a major cause of crop losses in Australia and many other cotton-producing countries. Symptoms include wilting, vascular browning and death. Australian races of the causal agent Fusarium oxysporum f. sp. vasinfectum (Fov) are genetically distinct from those in other countries and are thought to have evolved from indigenous races. New sources of resistance for breeding are rare, as cotton cultivars with significant FW resistance against Fov isolates from other cotton-producing regions are usually susceptible to Australian Fov races. MCU-5, an Upland Indian cotton cultivar, has been identified as having improved resistance to Australian Fov and is being used to breed new commercial cultivars with higher resistance to FW. To investigate the genetic basis of the FW resistance in MCU-5, QTL analysis was performed on 244 F3 and 244 F4 families derived from an intraspecific cross between MCU-5 and Siokra 1-4, a cultivar highly sensitive to Australian Fov races. Resistance, as measured by leaf symptoms, vascular browning and survival, showed low to moderate heritability between generations. MCU-5 resistance to FW was found to be complex with three quantitative trait loci (QTL) identified in the F3, and eight in the F4, that explained between 9 and 41% of the phenotypic variation. The QTL were located on four linkage groups including chromosomes A6 (Chr 6), D4 (Chr 22) and D6 (Chr 25), with two QTL located in similar regions to previously identified FW resistance from the Sea Island cultivar Pima 3-79. The QTL identified in this study represent the first targets for marker-assisted selection of FW resistance in Australia.     

Genetic diversity and population structure in the US Upland cotton (Gossypium hirsutum L.)

Key message

Genetic diversity and population structure in the US Upland cotton was established and core sets of allelic richness were identified for developing association mapping populations in cotton.

Abstract

Elite plant breeding programs could likely benefit from the unexploited standing genetic variation of obsolete cultivars without the yield drag typically associated with wild accessions. A set of 381 accessions comprising 378 Upland (Gossypium hirsutum L.) and 3 G. barbadense L. accessions of the United States cotton belt were genotyped using 120 genome-wide SSR markers to establish the genetic diversity and population structure in tetraploid cotton. These accessions represent more than 100 years of Upland cotton breeding in the United States. Genetic diversity analysis identified a total of 546 alleles across 141 marker loci. Twenty-two percent of the alleles in Upland accessions were unique, specific to a single accession. Population structure analysis revealed extensive admixture and identified five subgroups corresponding to Southeastern, Midsouth, Southwest, and Western zones of cotton growing areas in the United States, with the three accessions of G. barbadense forming a separate cluster. Phylogenetic analysis supported the subgroups identified by STRUCTURE. Average genetic distance between G. hirsutum accessions was 0.195 indicating low levels of genetic diversity in Upland cotton germplasm pool. The results from both population structure and phylogenetic analysis were in agreement with pedigree information, although there were a few exceptions. Further, core sets of different sizes representing different levels of allelic richness in Upland cotton were identified. Establishment of genetic diversity, population structure, and identification of core sets from this study could be useful for genetic and genomic analysis and systematic utilization of the standing genetic variation in Upland cotton.     

Genome‐wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.)

Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield‐limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome‐wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific‐locus amplified fragment sequencing (SLAF‐seq) approach. Trait–SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 10^–5 (P = 1/85 630, –log₁₀P = 4.93); the peaks of SNPs associated with VW resistance on A10 were continuous and common in three environments (RDIG2015, RDIF2015 and RDIF2016). Haplotype block structure analysis predicted 22 candidate genes for VW resistance based on A10_99672586 with a minimum P‐value (–log₁₀P = 6.21). One of these genes (CG02) was near the significant SNP A10_99672586 (0.26 Mb), located in a 372‐kb haplotype block, and its Arabidopsis AT3G25510 homologues contain TIR‐NBS‐LRR domains that may be involved in disease resistance response. Real‐time quantitative PCR and virus‐induced gene silencing (VIGS) analysis showed that CG02 was specific to up‐regulation in the resistant (R) genotype Zhongzhimian2 (ZZM2) and that silenced plants were more susceptible to V. dahliae. These results indicate that CG02 is likely the candidate gene for resistance against V. dahliae in cotton. The identified locus or gene may serve as a promising target for genetic engineering and selection for improving resistance to VW in cotton.     

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.     

Mapping and genomic targeting of the major leaf shape gene (L) in Upland cotton (Gossypium hirsutum L.)

Key message

A major leaf shape locus (L) was mapped with molecular markers and genomically targeted to a small region in the D-genome of cotton. By using expression analysis and candidate gene mapping, two LMI1 -like genes are identified as possible candidates for leaf shape trait in cotton.

Abstract

Leaf shape in cotton is an important trait that influences yield, flowering rates, disease resistance, lint trash, and the efficacy of foliar chemical application. The leaves of okra leaf cotton display a significantly enhanced lobing pattern, as well as ectopic outgrowths along the lobe margins when compared with normal leaf cotton. These phenotypes are the hallmark characteristics of mutations in various known modifiers of leaf shape that culminate in the mis/over-expression of Class I KNOX genes. To better understand the molecular and genetic processes underlying leaf shape in cotton, a normal leaf accession (PI607650) was crossed to an okra leaf breeding line (NC05AZ21). An F₂ population of 236 individuals confirmed the incompletely dominant single gene nature of the okra leaf shape trait in Gossypium hirsutum L. Molecular mapping with simple sequence repeat markers localized the leaf shape gene to 5.4 cM interval in the distal region of the short arm of chromosome 15. Orthologous mapping of the closely linked markers with the sequenced diploid D-genome (Gossypium raimondii) tentatively resolved the leaf shape locus to a small genomic region. RT-PCR-based expression analysis and candidate gene mapping indicated that the okra leaf shape gene (L ^o ) in cotton might be an upstream regulator of Class I KNOX genes. The linked molecular markers and delineated genomic region in the sequenced diploid D-genome will assist in the future high-resolution mapping and map-based cloning of the leaf shape gene in cotton.     

Rapid in-vitro plant regeneration of cotton (Gossypium hirsutum L.)

A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 μm) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitro-grown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 μm) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated shoots at 0.3 μm BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other high-fiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date have initiated flowers and set viable R₁ seeds. Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

Transformation of cotton (Gossypium hirsutum L.) via particle bombardment

Embryogenic suspension cultures of cotton (Gossypium hirsutum L.) were subjected to particle bombardment, where high density particles carrying plasmid DNA were accelerated towards the embryogenic plant cells. The plasmid DNA coating the particles encoded hygromycin resistance. One to two weeks following bombardment, embryogenic cotton cells were placed in proliferation medium containing 100 g/ml hygromycin. Clumps of tissue which grew in the presence of hygromycin were subcultured at low density into fresh hygromycin-containing proliferation medium. Following sequential transfer of embryogenic tissue to development and then germination media, plants were recovered from transgenic embryogenic tissue. Southern hybridization confirmed the presence of the hygromycin resistance gene in embryogenic suspension culture tissue and regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Aph IV aminoglycoside phosphotransferase type IV Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC and USDA-ARS. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC or USDA, and also does not imply approval to the exclusion of other products that may also be suitable. Journal Article No. 354-89     

A study of heterosis in upland cotton (Gossypium hirsutum L.)

Summary Heterosis (over mid parent) and useful heterosis (over commercial variety H14) estimates were obtained from a line x tester analysis of crosses involving thirteen diverse female parents with two locally adapted varieties H14 (local standard) and J34. Marked heterosis was observed for seed cotton yield, boll number and halo length. The values of positive heterosis and useful heterosis for seed cotton yield ranged from 28.1 to 87.0% and 20.1 to 45.5%, respectively. The overall study of heterosis revealed that female parents PRS-72 (USSR), 5904F (USSR) and MCU-5 (Madras Cambodian Uganda Selection, Coimbatore) were among the top three females, showing considerable heterosis in crosses with H14 and J34 for seed cotton yield and fibre properties. The practical difficulties in exploiting the phenomenon of heterosis and possible experimental approaches in upland cotton are discussed.     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine     

An RFLP linkage map of Upland cotton, Gossypium hirsutum L.

 Ninety-six F₂.F₃ bulked sampled plots of Upland cotton, Gossypium hirsutum L., from the cross of HS46×MARCABUCAG8US-1-88, were analyzed with 129 probe/enzyme combinations resulting in 138 RFLP loci. Of the 84 loci that segregated as co-dominant, 76 of these fit a normal 1 :  2 : 1 ratio (non-significant chi square at P=0.05). Of the 54 loci that segregated as dominant genotypes, 50 of these fit a normal 3: 1 ratio (non-significant chi square at P=0.05). These 138 loci were analyzed with the MAPMAKER∖ EXP program to determine linkage relationships among them. There were 120 loci arranged into 31 linkage groups. These covered 865 cM, or an estimated 18.6% of the cotton genome. The linkage groups ranged from two to ten loci each and ranged in size from 0.5 to 107 cM. Eighteen loci were not linked. Received: 31 March 1998 / Accepted: 29 April 1998     

Effects of fusicoccin on intact cotton plants (Gossypium hirsutum L.)

Fusicoccin (FC) was applied as a spray to shoots of intact field- and glasshouse-grown cotton plants. Distortions of shoot morphology resulted. Stems and petioles of FC-treated plants were irregular in diameter and twisted, whereas leaf laminae were curled and crinkled. Shoot elongation was inhibited by FC; the effect was dependent upon the concentration and timing of the applications.Abbreviation FC fusicoccin     

Wide-cross whole-genome radiation hybrid mapping of cotton (Gossypium hirsutum L.)

We report the development and characterization of a "wide-cross whole-genome radiation hybrid" (WWRH) panel from cotton (Gossypium hirsutum L.). Chromosomes were segmented by gamma-irradiation of G. hirsutum (n = 26) pollen, and segmented chromosomes were rescued after in vivo fertilization of G. barbadense egg cells (n = 26). A 5-krad gamma-ray WWRH mapping panel (N = 93) was constructed and genotyped at 102 SSR loci. SSR marker retention frequencies were higher than those for animal systems and marker retention patterns were informative. Using the program RHMAP, 52 of 102 SSR markers were mapped into 16 syntenic groups. Linkage group 9 (LG 9) SSR markers BNL0625 and BNL2805 had been colocalized by linkage analysis, but their order was resolved by differential retention among WWRH plants. Two linkage groups, LG 13 and LG 9, were combined into one syntenic group, and the chromosome 1 linkage group marker BNL4053 was reassigned to chromosome 9. Analyses of cytogenetic stocks supported synteny of LG 9 and LG 13 and localized them to the short arm of chromosome 17. They also supported reassignment of marker BNL4053 to the long arm of chromosome 9. A WWRH map of the syntenic group composed of linkage groups 9 and 13 was constructed by maximum-likelihood analysis under the general retention model. The results demonstrate not only the feasibility of WWRH panel construction and mapping, but also complementarity to traditional linkage mapping and cytogenetic methods.     

Chilling injury in cotton (Gossypium hirsutum L.) Prevention by abscisic acid

Cotton (Gossypium hirsutum L.) intact seedlings and isolatedcotyledonary discs were exposed to chilling (4?C) under humidconditions which prevented dehydration. The damage resultingfrom chilling was estimated by means of electrolyte leakageand survival in whole seedlings and by the electrolyte leakageand necrotic areas in isolated cotyledonary discs. Also, theeffect of chilling on membrane phospholipids and cellular reducedglutathione was determined. Within the first two and three daysof chilling, there was a marked reduction in the reduced glutathioneand membrane phospholipid levels without electrolyte loss andnecrosis. This reduction was completely prevented by pretreatmentwith abscisic acid. Prolonging the chilling period resultedin decreased survival in whole seedlings and in progressiveincrease in electrolyte leakage and necrosis in isolated cotyledonarydiscs. Pretreatment with abscisic acid prior to chilling almostcompletely prevented this chilling injury when exposure to 4?Cwas less than 5 days. Even with longer chilling periods, theabscisic acid pretreatment greatly reduced the damage. ³Incumbent of the Seagram Chair in Plant Science. (Received July 21, 1979; )     

Inheritance and QTL mapping of Fusarium wilt race 4 resistance in cotton

Diseases such as Fusarium wilt [Fusarium oxysporum f.sp. vasinfectum (FOV) Atk. Sny & Hans] represent expanding threats to cotton production. Integrating disease resistance into high-yielding, high-fiber quality cotton (Gossypium spp.) cultivars is one of the most important objectives in cotton breeding programs worldwide. In this study, we conducted a comprehensive analysis of gene action in cotton governing FOV race 4 resistance by combining conventional inheritance and quantitative trait loci (QTL) mapping with molecular markers. A set of diverse cotton populations was generated from crosses encompassing multiple genetic backgrounds. FOV race 4 resistance was investigated using seven parents and their derived populations: three intraspecific (G. hirsutum × G. hirsutum L. and G. barbadense × G. barbadense L.) F₁ and F₂; five interspecific (G. hirsutum × G. barbadense) F₁ and F₂; and one RIL. Parents and populations were evaluated for disease severity index (DSI) of leaves, and vascular stem and root staining (VRS) in four greenhouse and two field experiments. Initially, a single resistance gene (Fov4) model was observed in F₂ populations based on inheritance of phenotypes. This single Fov4 gene had a major dominant gene action and conferred resistance to FOV race 4 in Pima-S6. The Fov4 gene appears to be located near a genome region on chromosome 14 marked with a QTL Fov4-C14 ₁ , which made the biggest contribution to the FOV race 4 resistance of the generated F₂ progeny. Additional genetic and QTL analyses also identified a set of 11 SSR markers that indicated the involvement of more than one gene and gene interactions across six linkage groups/chromosomes (3, 6, 8, 14, 17, and 25) in the inheritance of FOV race 4 resistance. QTLs detected with minor effects in these populations explained 5–19 % of the DSI or VRS variation. Identified SSR markers for the resistance QTLs with major and minor effects will facilitate for the first time marker-assisted selection for the introgression of FOV race 4 resistance into elite cultivars during the breeding process.     

A new SNP haplotype associated with blue disease resistance gene in cotton (Gossypium hirsutum L.)

Resistance to cotton blue disease (CBD) was evaluated in 364 F_2.3 families of three populations derived from resistant variety ‘Delta Opal’. The CBD resistance in ‘Delta Opal’ was controlled by one single dominant gene designated Cbd. Two simple sequence repeat (SSR) markers were identified as linked to Cbd by bulked segregant analysis. Cbd resides at the telomere region of chromosome 10. SSR marker DC20027 was 0.75 cM away from Cbd. DC20027 marker fragments amplified from 3 diploid species and 13 cotton varieties whose CBD resistance was known were cloned and sequenced. One single nucleotide polymorphism (SNP) was identified at the 136th position by sequence alignment analysis. Screening SNP markers previously mapped on chromosome 10 identified an additional 3 SNP markers that were associated with Cbd. A strong association between a haplotype based on four SNP markers and Cbd was developed. This demonstrates one of the first examples in cotton where SNP markers were used to effectively tag a trait enabling marker-assisted selection for high levels of CBD resistance in breeding programs.     

Gene effects for fibre properties in upland cotton (Gossypium hirsutum L.)

Summary Six populations — P₁,P₂,F₁,F₂,B₁ and B₂ — each of five Upland Cotton (Gossypium hirsutum L.) crosses were used to evaluate gene effects in the inheritance of fibre properties by Gamble's six-parameter model for the analysis of generation means. Partial dominance of long fibres over short fibres and of mature fibres over immature fibres was found in the material studied. Overdominance in gene action governed fibre fineness while additive gene action governed the fibre strength. Besides additive and dominance effects, significant epistasis was present in all crosses. These results indicate a significant potential for improving fibre properties through reciprocal recurrent selection.     

Chronology of the differentiation of cotton (Gossypium hirsutum L.) fiber cells

Cotton fibers are single elongated cells that develop from epidermal cells of the ovule. The chronology of fiber differentiation was investigated using cultured ovules. Epidermal cells differentiate into fiber cells approx. 3 d before anthesis. When ovules were cultured on a defined medium, fiber growth could be initiated on ovules any time between 2 d preanthesis and the time of anthesis by adding indole-3-acetic acid and gibberellic acid to the medium. In the absence of phytohormones, fibers did not grow, and when ovules between 2 d preanthesis and anthesis were cultured without hormones past the day of anthesis and hormones then added, most ovules failed to produce fibers. The results define the timing of fiber differentiation from epidermal cells, and also define a window of time when differentiated cells are capable of further development. During this window, fiber cells are latent awaiting appropriate stimulation which in the intact plant is apparently associated with anthesis.Abbreviations GA₃ gibberellic acid - IAA indole-3-acetic acid     

Chilling injury in cotton (Gossypium hirsutum L.) : Effects of antimicrotubular drugs

When exposed to 4°C for more than three days, intact cotton(Gossypium hirsutum L.) seedlings and isolated cotyledonarydiscs suffered chilling injury as shown by the leakage of electrolytesfrom the tissue and the development of necrotic areas. Applicationof antimicrotubular drugs such as colchicine, demecolcine orpodophyllotoxin during chilling significantly accelerated andenhanced tissue damage. Lumicolchicine, the stereoisomer ofcolchicine, was ineffective. Non-chilled tissues showed hardlyany damage when treated with the same levels of antimicrotubulardrugs. Prior treatment with 10^–5 M abscisic acid (ABA)prevented the appearance of symptoms of damage caused by chillingand the antimicrotubular drugs during the first 2 to 3 daysand greatly reduced it at later stages. Our present resultssuggest that chilling damage may be due at least in part, tothe cold-induced disassembly of microtubules. Furthermore, themode of action of ABA might be related to factors which influencethe physiological stability of the microtubule network. ¹Preliminary report of this work was presented at the 10th InternationalConference on Plant Growth Substances, Madison, Wisconsin, 1979. ³Incumbent of the Seagram Chair in Plant Sciences. (Received April 15, 1980; )