A genome-wide association study for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in diploid cotton (Gossypium arboreum) and resistance transfer to tetraploid Gossypium hirsutum.

Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f. sp. vasinfectum (FOV), is a devastating disease affecting cotton (Gossypium spp.) worldwide. Understanding the genetic basis of resistance in diploid cotton and successfully transferring the resistance to tetraploid Upland cotton (G. hirsutum) are crucial for developing resistant cotton cultivars. Although numerous studies have been conducted to investigate the genetic basis of Fusarium wilt in tetraploid cotton, little research has been conducted on diploid species. In this study, an association mapping panel consisting of 246 accessions of G. arboreum, was used to identify chromosomal regions for FOV race 4 (FOV4) resistance based on foliar disease severity ratings in four greenhouse tests. Through a genome-wide association study (GWAS) based on 7,009 single nucleotide polymorphic (SNP) markers, 24 FOV4 resistance QTLs, including three major QTLs on chromosomes A04, A06, and A11, were detected. A validation panel consisting of 97 diploid cotton accessions was employed, confirming the presence of several QTLs. Evaluation of an introgressed BC2F7 population derived from G. hirsutum/G. aridum/G. arboreum showed significant differences in disease incidence and mortality rate, as compared to susceptible and resistant controls, suggesting that the resistance in G. arboreum and/or G. aridum was transferred into Upland cotton for the first time. The identification of novel major resistance QTLs, along with the transfer of resistance from the diploid species, expands our understanding of the genomic regions involved in conferring resistance to FOV4 and contributes to the development of resilient Upland cotton cultivars.

Mapping of dynamic QTLs for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in a backcross inbred line population of Upland cotton.

KEY MESSAGE: A backcross inbred line population of cotton was evaluated for Fusarium wilt race 4 resistance at different days after inoculation (DAI). Both constitutively expressed and developmentally regulated QTLs were detected. The soil-borne fungus Fusarium oxysporum f. sp. vasinfectum (FOV) race 4 (FOV4) causes Fusarium wilt including seedling mortality in cotton. A backcross inbred line (BIL) population of 181 lines, derived from a bi-parental cross of moderately resistant non-recurrent Hai 7124 (Gossypium barbadense) and recurrent parent CCRI 36 (G. hirsutum), was evaluated under temperature-controlled conditions for FOV4 resistance with artificial inoculations. Based on three replicated tests evaluated at 7, 14, 21, and 28 days after inoculation (DAI), only 2-5 BILs showed lower disease severity ratings (DSR) than the parents while 22-50 BILs were more susceptible, indicating transgressive segregation toward susceptibility. Although DSR were overall congruent between DAI, there were many BILs displaying different responses to FOV4 across DAI. Genetic mapping using 7709 SNP markers identified 42 unique QTLs for four evaluation parameters- disease incidence (DI), DSR, mortality rate (MR), and area under disease progress curve (AUDPC), including 26 for two or more parameters. All five QTLs for AUDPC were co-localized with QTLs for DI, DSR, and/or MR at one or two DAI, indicating the unnecessary use of AUDPC in QTL mapping for FOV4 resistance. Those common QTLs explained the significant positive associations between parameters observed. Ten common QTLs with negative or positive additive effects were detected between DAI. DAI-specific and consistent QTLs were detected between DAI in cotton for the first time, suggesting the existence of both constitutively expressed and developmentally regulated QTLs for FOV4 resistance and the importance of evaluating genetic populations for FOV4 resistance at different growth stages.

A GWAS identified a major QTL for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in a MAGIC population of Upland cotton and a meta-analysis of QTLs for Fusarium wilt resistance.

KEY MESSAGE: A major QTL conferring resistance to Fusarium wilt race 4 in a narrow region of chromosome D02 was identified in a MAGIC population of 550 RILs of Upland cotton. Numerous studies have been conducted to investigate the genetic basis of Fusarium wilt (FW, caused by Fusarium oxysporum f. sp. vasinfectum, FOV) resistance using bi-parental and association mapping populations in cotton. In this study, a multi-parent advanced generation inter-cross (MAGIC) population of 550 recombinant inbred lines (RILs), together with their 11 Upland cotton (Gossypium hirsutum) parents, was used to identify QTLs for FOV race 4 (FOV4) resistance. Among the parents, Acala Ultima, M-240 RNR, and Stoneville 474 were the most resistant, while Deltapine Acala 90, Coker 315, and Stoneville 825 were the most susceptible. Twenty-two MAGIC lines were consistently resistant to FOV4. Through a genome-wide association study (GWAS) based on 473,516 polymorphic SNPs, a major FOV4 resistance QTL within a narrow region on chromosomes D02 was detected, allowing identification of 14 candidate genes. Additionally, a meta-analysis of 133 published FW resistance QTLs showed a D subgenome and individual chromosome bias and no correlation between homeologous chromosome pairs. This study represents the first GWAS study using a largest genetic population and the most comprehensive meta-analysis for FW resistance in cotton. The results illustrated that 550 lines were not enough for high resolution mapping to pinpoint a candidate gene, and experimental errors in phenotyping cotton for FW resistance further compromised the accuracy and precision in QTL localization and identification of candidate genes. This study identified FOV4-resistant parents and MAGIC lines, and the first major QTL for FOV4 resistance in Upland cotton, providing useful information for developing FOV4-resistant cultivars and further genomic studies towards identification of causal genes for FOV4 resistance in cotton.

Comparative Analysis of Infection Process in Pima Cotton Differing in Resistance to Fusarium Wilt Caused by Fusarium oxysporum f. sp. vasinfectum Race 4.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) causes an early season cotton disease including seedling deaths. This study compared two Pima cottons (Gossypium barbadense) in the infection process of FOV4 using a confocal and a scanning electron microscope. Seedlings were grown in a hydroponic system and inoculated with a virulent local FOV4 isolate. As compared with the susceptible Pima S-7, the resistant Pima PHY 841 RF had significantly fewer conidia attached and germinated on the root surface. FOV4 penetration into the root epidermis of PHY 841 RF was delayed until 24 h postinoculation (hpi) as compared with 8 hpi in Pima S-7. In Pima S-7, hyphae progressed to the xylem through the cortex between 5 and 7 days postinoculation. However, hyphae grew much slower in the cortex with no apparent hyphae observed in the xylem of PHY 841 RF. At plant maturity, no FOV4 was detected through fungal isolation and PCR in the stem of PHY 841 RF and its resistance donor parents PHY 800 and Pima S-6, as compared with Pima S-7 and DP 744 with positive results. The results demonstrate that PHY 841 RF is resistant to FOV4, due to delayed infection, reduced fungal growth and reproduction, and prevention of the fungus from invading the xylem.

GWAS reveals consistent QTL for drought and salt tolerance in a MAGIC population of 550 lines derived from intermating of 11 Upland cotton (Gossypium hirsutum) parents.

Cotton is grown in arid and semi-arid regions where abiotic stresses such as drought and salt are prevalent. There is a lack of studies that simultaneously address the genetic and genomic basis of tolerance to drought and salt stress. In this study, a multi-parent advanced generation inter-cross (MAGIC) population of 550 recombinant inbred lines (RILs) together with their 11 Upland cotton parents with a total of 473,516 polymorphic SNP markers was used to identify quantitative trait loci (QTL) for drought tolerance (DT) and salt tolerance (ST) at the seedling stage based on two replicated greenhouse tests. Transgressive segregation occurred in the MAGIC-RILs, indicating that tolerant and sensitive alleles recombined for tolerance to the abiotic stress during the intermating process for the population development. A total of 20 QTL were detected for DT including 13 and 7 QTL based on plant height (PH) and dry shoot weight (DSW), respectively; and 23 QTL were detected for ST including 12 and 11 QTL for PH and DSW, respectively. There were several chromosomes with QTL clusters for abiotic stress tolerance including four QTL on chromosome A13 and three QTL on A01 for DT, and four QTL on D08 and three QTL on A11 for ST. Nine QTL (21% of the 43 QTL) detected were in common between DT and ST, indicating a common genetic basis for DT and ST. The narrow chromosomal regions for most of the QTL detected in this study allowed identification of 53 candidate genes associated with responses to salt and drought stress and abiotic stimulus. The QTL identified for both DT and ST have significantly augmented the repertoire of QTL for abiotic stress tolerance that can be used for marker-assisted selection to develop cultivars with resilience to drought and/or salt and further genomic studies towards the identification of drought and salt tolerance genes in cotton.

Evaluation and genome-wide association study of resistance to bacterial blight race 18 in U.S. Upland cotton germplasm.

Bacterial blight (BB), caused by Xanthomonas citri pv. malvacearum (Xcm), is a destructive disease to cotton production in many countries. In the U.S., Xcm race 18 is the most virulent and widespread race and can cause serious yield losses. Planting BB-resistant cotton cultivars is the most effective method of controlling this disease. In this study, 335 U.S. Upland cotton accessions were evaluated for resistance to race 18 using artificial inoculations by scratching cotyledons on an individual plant basis in a greenhouse. The analysis of variance detected significant genotypic variation in disease incidence, and 50 accessions were resistant including 38 lines with no symptoms on either cotyledons or true leaves. Many of the resistant lines were developed in the MAR (multi-adversity resistance) breeding program at Texas A&M University, whereas others were developed before race 18 was first reported in the U.S. in 1973, suggesting a broad base of resistance to race 18. A genome-wide association study (GWAS) based on 26,301 single nucleotide polymorphic (SNP) markers detected 11 quantitative trait loci (QTL) anchored by 79 SNPs, including three QTL on each of the three chromosomes A01, A05 and D02, and one QTL on each of D08 and D10. This study has identified a set of obsolete Upland germplasm with resistance to race 18 and specific chromosomal regions delineated by SNPs for resistance. The results will assist in breeding cotton for BB resistance and facilitate further genomic studies in fine mapping resistance genes to enhance the understanding of the genetic basis of BB resistance in cotton.

Detection and Characterization of Fusarium Wilt (Fusarium oxysporum f. sp. vasinfectum) Race 4 Causing Fusarium Wilt of Cotton Seedlings in New Mexico.

Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. vasinfectum (Atk.) W.C. Snyder & H.N. Hans (FOV), is one of the most destructive diseases of cotton (Gossypium spp.) worldwide. FOV race 4 (FOV4) is a highly virulent nominal race of this pathogen and a significant threat to cotton production in the western and southwestern USA and, potentially, the entire Cotton Belt. A field survey to identify FOV4 was performed in three southern counties of New Mexico in 619 cotton fields from 2018 to 2020. From 132 samples of cotton plants that exhibited wilt symptoms, Fusarium spp. were the most frequently isolated group of fungal species, with an isolation frequency of 57.4%. Eighty-four Fusarium spp. isolates were subsequently characterized by a DNA sequence analysis of three genes, EF-1α, PHO, and BT, encoding for translation elongation factor, phosphate permease, and ß-tubulin, respectively. Forty-two isolates from 10 cotton fields were identified as FOV4 and confirmed with a positive 500-bp fragment diagnostic for FOV4. Twenty-six (62%) of the 42 FOV4 isolates were T type and the remainder (38%) were null type with and without a Tfo1 insertion in PHO, respectively. Each FOV4-infested field contained the same FOV4 genotype. Ten representative FOV4 isolates (one each from the 10 FOV4-infested fields) were evaluated for their pathogenicity on resistant Pima PHY 841 RF and susceptible Upland PHY 725 RF at 7, 14, 21, and 28 days after inoculation under temperature-controlled conditions at 21 to 22°C. Based on the disease severity rating, mortality rate, and area under the disease progress curve value, all 10 isolates were pathogenic to both cotton cultivars and differed in virulence; four isolates of the T genotype as a whole were more virulent than the six isolates of the N genotype. PHY 841 RF had significantly higher levels of resistance than PHY 725 RF to all FOV4 isolates. The results provide the first comprehensive account of the occurrence, distribution, and virulence of FOV4 in cotton production in New Mexico and will be useful for developing an effective strategy to manage FW in the state of New Mexico and the entire western and southwestern Cotton Belt.

A genome-wide association study uncovers consistent quantitative trait loci for resistance to Verticillium wilt and Fusarium wilt race 4 in the US Upland cotton.

KEY MESSAGE: A high-resolution GWAS detected consistent QTL for resistance to Verticillium wilt and Fusarium wilt race 4 in 376 U.S. Upland cotton accessions based on six independent replicated greenhouse tests. Verticillium wilt (VW, caused by Verticillium dahliae Kleb.) and Fusarium wilt (FOV, caused by Fusarium oxysporum f.sp. vasinfectum Atk. Sny & Hans) are the most important soil-borne fungal diseases in cotton. To augment and refine resistance quantitative trait loci (QTL), we conducted a genome-wide association study (GWAS) using high-density genotyping with the CottonSNP63K array. Resistance of 376 US Upland cotton accessions to a defoliating VW and virulent FOV4 was evaluated in four and two independent replicated greenhouse tests, respectively. A total of 15 and 13 QTL for VW and FOV4 resistances were anchored by 30 (on five chromosomes) and 56 (on six chromosomes) significant single nucleotide polymorphic (SNPs) markers, respectively. QTL on c8, c10, c16, and c21 were consistent in two or more tests for VW resistance, while two QTL on c8 and c14 were consistent for FOV4 resistance in two tests. Two QTL clusters on c16 and c19 were observed for both VW and FOV4 resistance, suggesting that these genomic regions may harbor genes in response to both diseases. Using BLAST search against the sequenced TM-1 genome, 30 and 35 candidate genes were identified on four QTL for VW resistance and on three QTL for FOV4 resistance, respectively. These genomic regions were rich in NBS-LRR genes presented in clusters. The results create opportunities for further studies to determine the correlations of field resistance with these QTL, molecular examinations of VW and FOV4 resistances, marker-assisted selection (MAS) and eventual cloning of QTL for disease resistance in cotton.

A meta-analysis of quantitative trait loci for abiotic and biotic stress resistance in tetraploid cotton.

The number and location of mapped quantitative trait loci (QTL) depend on genetic populations and testing environments. The identification of consistent QTL across genetic backgrounds and environments is a pre-requisite to marker-assisted selection. This study analyzed a total of 661 abiotic and biotic stress resistance QTL based on our previous work and other publications using the meta-analysis software Biomercator. It identified chromosomal regions containing QTL clusters for different resistance traits and hotspots for a particular resistance trait in cotton from 98 QTL for drought tolerance under greenhouse (DT) and 150 QTL in field conditions (FDT), 80 QTL for salt tolerance in the greenhouse conditions (ST), 201 QTL for resistance to Verticillium wilt (VW, Verticillium dahliae), 47 QTL for resistance to Fusarium wilt (FW, Fusarium oxysporum f. sp. vasinfectum), and 85 QTL for resistance to root-knot nematodes (RKN, Meloiodogyne incognita) and reniform nematodes (RN, Rotylenchulus reniformis). The traits used in QTL mapping for abiotic stress tolerance included morphological traits-plant height and fresh and dry shoot and root weights, physiological traits-chlorophyll content, osmotic potential, carbon isotope ratio, stomatal conductance, photosynthetic rate, transpiration, canopy temperature, and leaf area index, agronomic traits-seedcotton yield, lint yield, boll weight, and lint percent, and fiber quality traits-fiber length, uniformity, strength, elongation, and micronaire. The results showed that resistance QTL are not uniformly distributed across the cotton genome; some chromosomes carried disproportionally more QTL, QTL clusters, or hotspots. Twenty-three QTL clusters were found on 15 chromosomes (c3, c4, c5, c6, c7, c11, c14, c15, c16, c19, c20, c23, c24, c25, and c26). Moreover, 28 QTL hotshots were associated with different resistance traits including one hotspot on c4 for Verticillium wilt resistance, two QTL hotspots on c24 for chlorophyll content measured under both drought and salt stress conditions, and three other hotspots on c19 for the resistance to Verticillium wilt and Fusarium wilt, and micronaire under drought stress conditions. This meta-analysis of stress tolerance QTL provides an important foundation for cotton breeding and further studies on the genetic mechanisms of abiotic and biotic stress resistance in cotton.

Quantitative Trait Locus Mapping for Fusarium Wilt Race 4 Resistance in a Recombinant Inbred Line Population of Pima Cotton (Gossypium Barbadense).

Fusarium oxysporum f. sp. vasinfectum (FOV) race 4 (FOV4) causes seedling death immediately after emergence, in addition to leaf chlorosis and necrosis, vascular discoloration, plant wilting, defoliation, and plant death at late stages. Breeding for FOV4 resistance is the most cost effective management method. In this study, 163 recombinant inbred lines (RILs) of FOV4-resistant Pima S-6 × susceptible 89590, together with the two parents (Gossypium barbadense), were artificially inoculated with FOV4 and assayed for resistance based on foliar disease severity ratings (DSR) at 30 days post inoculation (dpi) in two replicated tests in the greenhouse or controlled conditions. Significant genotypic variations were detected for FOV4 resistance in a combined analysis of variance. Although a significant genotype × test interaction was detected for DSR, the 10 most resistant RILs had significantly and consistently lower DSR than the susceptible parent in both tests. The heritability estimate for DSR was 0.65, indicating that two-thirds of the phenotypic variation for FOV4 resistance in this Pima RIL population was due to genetic factors. Based on 404 polymorphic SSR markers, five and four quantitative trait loci (QTL) on six chromosomes (c14, c17, c19, c21, c24, and c25) were detected in Tests 1 and 2, respectively, and each explained 15 to 29% of the phenotypic variation. Three QTL on c17, c24, and c25 were in common between the two tests, accounting for 60% and 75% of the QTL detected in Tests 1 and 2, respectively. The three QTL were also reported in previous studies and will be useful for marker-assisted selection for FOV4 resistance in Pima cotton.

Studies of Evaluation Methods for Resistance to Fusarium Wilt Race 4 (Fusarium oxysporum f. sp. vasinfectum) in Cotton: Effects of Cultivar, Planting Date, and Inoculum Density on Disease Progression.

Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is an early season disease causing root rot, seedling wilt, and death. To develop an appropriate field evaluation method for resistance to FOV4 in cotton breeding, the objectives of this study were to investigate the effects of cultivar, planting date, and inoculum density on disease progression in 2020-2021. Results showed that the usual local mid-April planting had the lowest disease severity (DSR) or mortality rate (MR) in 2020 and 2021. DSR or MR increased at the late April and early May plantings in both years and reached the highest at the early May planting in 2020, while MR in 2021 was followed by a decrease in the late May planting and reached the highest in the mid-June planting. Local daily low temperatures between mid-April and mid-June were favorable for FOV4 infections, whereas daily high temperatures at 35°C or higher suppressed wilt severity. When seedlings at the 2-true leaf stage were inoculated with 104, 105, 106, and 107 conidia ml-1 per plant in 2020, DSR was low but a linear relationship between inoculum density and DSR was observed. When a FOV4-infested soil supplemented with artificial inoculation was used, disease progression in three moderately susceptible or moderately resistant cultivars followed a linear model, while it followed a quadratic model in the highly susceptible Pima S-7 cultivar only. Among the other three cultivars, FM 2334GLT had the lowest DSR or MR except for one planting date in both years, followed by PHY 725 RF and Pima PHY 881 RF in ascending order, which were consistent with the difference in regression coefficients of the linear models. This study demonstrates that disease progression curves due to FOV4 can be used to compare responses to FOV4 infections among cotton genotypes in cotton breeding and genetic studies, regardless of planting date and inoculation method.