Targeted development of diagnostic SNP markers for resistance to Fusarium wilt race 4 in Upland cotton (Gossypium hirsutum).

Fusarium wilt caused by the soil-borne fungus Fusarium oxysporum f. sp. vasinfectum (FOV) race 4 (FOV4) has become one of the most important emerging diseases in US cotton production. Numerous QTLs have been reported for resistance to FOV; however, no major FOV4-resistance QTL or gene has been identified and used in breeding Upland cotton (Gossypium hirsutum) for FOV4 resistance. In this study, a panel of 223 Chinese Upland cotton accessions was evaluated for FOV4 resistance based on seedling mortality rate (MR) and stem and root vascular discoloration (SVD and RVD). SNP markers were developed based on targeted genome sequencing using AgriPlex Genomics. The chromosome region at 2.130-2.292 Mb on D03 was significantly correlated with both SVD and RVD but not with MR. Based on the two most significant SNP markers, accessions homozygous for AA or TT SNP genotype averaged significantly lower SVD (0.88 vs. 2.54) and RVD (1.46 vs. 3.02) than those homozygous for CC or GG SNP genotype. The results suggested that a gene or genes within the region conferred resistance to vascular discoloration caused by FOV4. The Chinese Upland accessions had 37.22% homozygous AA or TT SNP genotype and 11.66% heterozygous AC or TG SNP genotype, while 32 US elite public breeding lines all had the CC or GG SNP genotype. Among 463 obsolete US Upland accessions, only 0.86% possessed the AA or TT SNP genotype. This study, for the first time, has developed diagnostic SNPs for marker-assisted selection and identified FOV4-resistant Upland germplasms with the SNPs.

A Rapid and Reliable Method for Evaluating Cotton Resistance to Fusarium Wilt Race 4 Based on Taproot Rot at the Seed Germination Stage.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a soilborne fungal pathogen threatening U.S. cotton production. The objective of this study was to develop a reliable and efficient method to evaluate cotton for FOV4 resistance based on taproot rot during seed germination through five growth chamber tests and two greenhouse tests. Seeds from eight cotton cultivars (Set 1) were germinated in a paper towel for 6 days, and taproots were inoculated with a FOV4 conidial suspension using three inoculation methods (i.e., taproot dipping, taproot wounding, and paper towel drenching), in addition to seed soaking before germination. The taproot rot-based disease incidence (DI) and disease severity rating (DSR), seed germination percentage (SGP), and plant fresh weight (PFW) were measured 7 days after inoculation. Taproot dipping was the most efficient and reliable evaluation method. The SGP and PFW were not significantly correlated with the DI and DSR, making them inappropriate to use in resistance evaluation. Pima DP 359 RF and PHY 881 RF were the most resistant with the lowest root rot. The taproot dipping method was repeated in another test and confirmed in two tests using another set of eight cultivars (Set 2). The taproot rot-based DSR at germination was significantly correlated with the DSR at the seedling stage in the greenhouse in both sets and with previous results in seedling mortality in the greenhouse and field in Set 2. The results suggest that the response to FOV4 infections at the seed germination stage is overall congruent with that at the seedling stage.

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.

Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection.

Investigation of cotton response to nematode infection will allow us to better understand the cotton immune defense mechanism and design a better biotechnological approach for efficiently managing pest nematodes in cotton. In this study, we firstly treated cotton by root knot nematode (RKN, Meloidogyne incognita) infections, then we employed the high throughput deep sequencing technology to sequence and genome-widely identify all miRNAs in cotton; finally, we analyzed the functions of these miRNAs in cotton response to RKN infections. A total of 266 miRNAs, including 193 known and 73 novel miRNAs, were identified by deep sequencing technology, which belong to 67 conserved and 66 novel miRNA families, respectively. A majority of identified miRNA families only contain one miRNA; however, miR482 family contains 14 members and some others contain 2-13 members. Certain miRNAs were specifically expressed in RKN-infected cotton roots and others were completely inhibited by RKN infection. A total of 50 miRNAs were differentially expressed after RKN infection, in which 28 miRNAs were up-regulated and 22 were inhibited by RKN treatment. Based on degradome sequencing, 87 gene targets were identified to be targeted by 57 miRNAs. These miRNA-targeted genes are involved in the interaction of cotton plants and nematode infection. Based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, 466 genes from all 636 miRNA targets were mapped to 6340 GO terms, 181 genes from 228 targets of differentially expressed miRNAs were mapped to 1588 GO terms. The GO terms were then categorized into the three main GO classes: biological processes, cellular components, and molecular functions. The targets of differentially expressed miRNAs were enriched in 43 GO terms, including 22 biological processes, 10 cellular components, and 11 molecular functions (p < 0.05). Many identified processes were associated with organism responses to the environmental stresses, including regulation of nematode larval development, response to nematode, and response to flooding. Our results will enhance the study and application of developing new cotton cultivars for nematode resistance.

Production of tocotrienols in seeds of cotton (Gossypium hirsutum L.) enhances oxidative stability and offers nutraceutical potential.

Upland cotton (Gossypium hirsutum L.) is an economically important multi-purpose crop cultivated globally for fibre, seed oil and protein. Cottonseed oil also is naturally rich in vitamin E components (collectively known as tocochromanols), with α- and γ-tocopherols comprising nearly all of the vitamin E components. By contrast, cottonseeds have little or no tocotrienols, tocochromanols with a wide range of health benefits. Here, we generated transgenic cotton lines expressing the barley (Hordeum vulgare) homogentisate geranylgeranyl transferase coding sequence under the control of the Brassica napus seed-specific promoter, napin. Transgenic cottonseeds had ~twofold to threefold increases in the accumulation of total vitamin E (tocopherols + tocotrienols), with more than 60% γ-tocotrienol. Matrix assisted laser desorption ionization-mass spectrometry imaging showed that γ-tocotrienol was localized throughout the transgenic embryos. In contrast, the native tocopherols were distributed unequally in both transgenic and non-transgenic embryos. α- Tocopherol was restricted mostly to cotyledon tissues and γ-tocopherol was more enriched in the embryonic axis tissues. Production of tocotrienols in cotton embryos had no negative impact on plant performance or yield of other important seed constituents including fibre, oil and protein. Advanced generations of two transgenic events were field grown, and extracts of transgenic seeds showed increased antioxidant activity relative to extracts from non-transgenic seeds. Furthermore, refined cottonseed oil from the two transgenic events showed 30% improvement in oxidative stability relative to the non-transgenic cottonseed oil. Taken together, these materials may provide new opportunities for cottonseed co-products with enhanced vitamin E profile for improved shelf life and nutrition.

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.

Homology-based identification of candidate genes for male sterility editing in upland cotton (Gossypium hirsutum L.).

Upland cotton (Gossypium hirsutum L.) accounts for more than 90% of the world's cotton production, providing natural material for the textile and oilseed industries worldwide. One strategy for improving upland cotton yields is through increased adoption of hybrids; however, emasculation of cotton flowers is incredibly time-consuming and genetic sources of cotton male sterility are limited. Here we review the known biochemical modes of plant nuclear male sterility (NMS), often known as plant genetic male sterility (GMS), and characterized them into four groups: transcriptional regulation, splicing, fatty acid transport and processing, and sugar transport and processing. We have explored protein sequence homology from 30 GMS genes of three monocots (maize, rice, and wheat) and three dicots (Arabidopsis, soybean, and tomato). We have analyzed evolutionary relationships between monocot and dicot GMS genes to describe the relative similarity and relatedness of these genes identified. Five were lowly conserved to their source species, four unique to monocots, five unique to dicots, 14 highly conserved among all species, and two in the other category. Using this source, we have identified 23 potential candidate genes within the upland cotton genome for the development of new male sterile germplasm to be used in hybrid cotton breeding. Combining homology-based studies with genome editing may allow for the discovery and validation of GMS genes that previously had no diversity observed in cotton and may allow for development of a desirable male sterile mutant to be used in hybrid cotton production.

Conservation and Divergence of Phosphoenolpyruvate Carboxylase Gene Family in Cotton.

Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme in plants, which regulates carbon flow through the TCA cycle and controls protein and oil biosynthesis. Although it is important, there is little research on PEPC in cotton, the most important fiber crop in the world. In this study, a total of 125 PEPCs were identified in 15 Gossypium genomes. All PEPC genes in cotton are divided into six groups and each group generally contains one PEPC member in each diploid cotton and two in each tetraploid cotton. This suggests that PEPC genes already existed in cotton before their divergence. There are additional PEPC sub-groups in other plant species, suggesting the different evolution and natural selection during different plant evolution. PEPC genes were independently evolved in each cotton sub-genome. During cotton domestication and evolution, certain PEPC genes were lost and new ones were born to face the new environmental changes and human being needs. The comprehensive analysis of collinearity events and selection pressure shows that genome-wide duplication and fragment duplication are the main methods for the expansion of the PEPC family, and they continue to undergo purification selection during the evolutionary process. PEPC genes were widely expressed with temporal and spatial patterns. The expression patterns of PEPC genes were similar in G. hirsutum and G. barbadense with a slight difference. PEPC2A and 2D were highly expressed in cotton reproductive tissues, including ovule and fiber at all tested developmental stages in both cultivated cottons. However, PEPC1A and 1D were dominantly expressed in vegetative tissues. Abiotic stress also induced the aberrant expression of PEPC genes, in which PEPC1 was induced by both chilling and salinity stresses while PEPC5 was induced by chilling and drought stresses. Each pair (A and D) of PEPC genes showed the similar response to cotton development and different abiotic stress, suggesting the similar function of these PEPCs no matter their origination from A or D sub-genome. However, some divergence was also observed among their origination, such as PEPC5D was induced but PEPC5A was inhibited in G. barbadense during drought treatment, suggesting that a different organized PEPC gene may evolve different functions during cotton evolution. During cotton polyploidization, the homologues genes may refunction and play different roles in different situations.

Gossypolhemiquinone, a dimeric sesquiterpenoid identified in cotton (Gossypium).

The report that the cotton leaf perforator, Bucculatrix thurberiella, is one of the few insect herbivores to attack Gossypium thurberi prompted an investigation of the terpenoids present in the leaves of this wild species of cotton. Members of Gossypium produce subepidermal pigment glands in their leaves that contain the dimeric sesquiterpenoid gossypol as well as other biosynthetically related terpenoids. In addition to gossypol, a previously unknown dimeric sesquiterpenoid, gossypolhemiquinone (GHQ), was identified in trace amounts in G. thurberi, a member of the D genome. Other members of the D genome of Gossypium were subsequently found to contain this compound, but GHQ was not detected in commercial cotton cultivars. When fed to Helicoverpa zea in an artificial diet, GHQ delayed days-to-pupation, reduced pupal weights, and survival to adulthood to a lesser or equal extent than gossypol in comparison to the control diet. However, GHQ had a synergistic effect on survival and days-to-pupation when combined with gossypol at the highest dosage tested (0.18%; 15.5:84.5 GHQ:gossypol). Because gossypol exhibits anti-cancer activity, GHQ was also evaluated for its anti-cancer activity against the National Cancer Institute's 60-Human Tumor Cell Line Screen. Significant inhibitory activity against most of these cell lines was not observed, but the results may offer some promise against leukemia cancer cell lines.