Detection and verification of quantitative trait loci for resistance to Fusarium ear rot in maize

Fusarium ear rot caused by Fusarium verticillioides is a prevalent disease in maize which can severely reduce grain yields and quality. Identification of stable quantitative trait loci (QTL) for resistance to Fusarium ear rot is a basic prerequisite for understanding the genetic mechanism of resistance and for the use of marker-assisted selection. In this study, two hundred and ten F _2:3 families were developed from a cross between resistant inbred line BT-1 and susceptible inbred line Xi502, and were genotyped with 178 simple sequence repeat markers. The resistance of each line was evaluated in two environments by artificial inoculation using the nail-punch method. The resistance QTL were detected using the composite interval mapping method. Three QTL were detected on chromosomes 4, 5 and 10. Of them, the QTL on chromosome 4 (bin 4.05/06) had the largest resistance to Fusarium ear rot, and could explain 17.95?% of the phenotypic variation. For further verification of the QTL effect, we developed near-isogenic lines (NILs) carrying the QTL region on chromosome 4 using parental line Xi502 as the recurrent parent. In the NIL background, this QTL can increase the resistance by 33.7?C35.2?% if the resistance region is homozygous, and by 17.8?C26.5?% if the resistance region contains the heterozygous allele. The stable and significant resistance effect of the QTL on chromosome 4 lays the foundation for further marker-assisted selection and map-based cloning in maize.     

Transcriptional and Metabolic Changes Associated to the Infection by Fusarium verticillioides in Maize Inbreds with Contrasting Ear Rot Resistance

Fusarium verticillioides causes ear rot and grain mycotoxins in maize (Zea mays L.), which are harmful to human and animal health. Breeding and growing less susceptible plant genotypes is one alternative to reduce these detrimental effects. A better understanding of the resistance mechanisms would facilitate the implementation of strategic molecular agriculture to breeding of resistant germplasm. Our aim was to identify genes and metabolites that may be related to the Fusarium reaction in a resistant (L4637) and a susceptible (L4674) inbred. Gene expression data were obtained from microarray hybridizations in inoculated and non-inoculated kernels from both inbreds. Fungal inoculation did not produce considerable changes in gene expression and metabolites in L4637. Defense-related genes changed in L4674 kernels, responding specifically to the pathogen infection. These results indicate that L4637 resistance may be mainly due to constitutive defense mechanisms preventing fungal infection. These mechanisms seem to be poorly expressed in L4674; and despite the inoculation activate a defense response; this is not enough to prevent the disease progress in this susceptible line. Through this study, a global view of differential genes expressed and metabolites accumulated during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize.     

玉米穗腐病抗性鉴定、遗传分析与分子机制*

玉米是我国主要农作物之一,其产量占全国谷物总产量三分之一左右,在国民生活中发挥了重要作用。玉米生长过程中受多种病害侵扰,其中穗腐病是一种由真菌导致的常见病害,目前已鉴定出40余种可诱发玉米穗腐病的病原菌。穗腐病不仅可造成玉米产量损失也导致籽粒品质严重下降,病原微生物所产生次生毒素更是危及人畜安全。目前穗腐病防治以化学方法为主,但是因此所造成的种植成本增加和环境污染问题日益突出,选育抗性品种成为防控玉米穗腐病最经济和安全有效的方法。玉米穗腐病抗性属于典型数量性状,国内外已有多个研究组通过建立玉米穗腐病抗性研究体系,开展玉米群体大规模穗腐病抗性鉴定工作,并筛选出一批抗病材料,这为玉米穗腐病抗性遗传改良奠定了材料基础。利用数量性状位点(quantitative trait locus, QTL)定位和全基因组关联分析(genome-wide association study, GWAS)等方法在玉米 1~10号染色体均检测到显著相关位点。尽管如此,玉米穗腐病抗性研究成果应用于生产的实例较少,生产上仍然缺乏综合性状优良且高抗穗腐病的玉米品种。这一方面是由于玉米穗腐病抗性遗传机制十分复杂、抗性基因克隆工作进展缓慢;另一方面为缺乏对玉米穗腐病抗性遗传研究进展进行系统总结,未有效开发分子标记用于分子育种所致。通过综述玉米穗腐病抗性遗传研究进展,汇总已定位QTL位点和显著关联SNP,构建一致性图谱和鉴定出定位热点区间,并进一步对比分析定位区间候选基因特征和转录组、代谢组研究进展,对促进玉米穗腐病抗性机制研究和玉米抗性育种均具有重要意义。     

Candidate gene association mapping of Sclerotinia stalk rot resistance in sunflower (Helianthus annuus L.) uncovers the importance of COI1 homologs

Key message

Functional markers for Sclerotinia basal stalk rot resistance in sunflower were obtained using gene-level information from the model species Arabidopsis thaliana.

Abstract

Sclerotinia stalk rot, caused by Sclerotinia sclerotiorum, is one of the most destructive diseases of sunflower (Helianthus annuus L.) worldwide. Markers for genes controlling resistance to S. sclerotiorum will enable efficient marker-assisted selection (MAS). We sequenced eight candidate genes homologous to Arabidopsis thaliana defense genes known to be associated with Sclerotinia disease resistance in a sunflower association mapping population evaluated for Sclerotinia stalk rot resistance. The total candidate gene sequence regions covered a concatenated length of 3,791 bp per individual. A total of 187 polymorphic sites were detected for all candidate gene sequences, 149 of which were single nucleotide polymorphisms (SNPs) and 38 were insertions/deletions. Eight SNPs in the coding regions led to changes in amino acid codons. Linkage disequilibrium decay throughout the candidate gene regions declined on average to an r ² = 0.2 for genetic intervals of 120 bp, but extended up to 350 bp with r ² = 0.1. A general linear model with modification to account for population structure was found the best fitting model for this population and was used for association mapping. Both HaCOI1-1 and HaCOI1-2 were found to be strongly associated with Sclerotinia stalk rot resistance and explained 7.4 % of phenotypic variation in this population. These SNP markers associated with Sclerotinia stalk rot resistance can potentially be applied to the selection of favorable genotypes, which will significantly improve the efficiency of MAS during the development of stalk rot resistant cultivars.     

Application of Genomic and Quantitative Genetic Tools to Identify Candidate Resistance Genes for Brown Rot Resistance in Peach

The availability of a complete peach genome assembly and three different peach genome sequences created by our group provide new opportunities for application of genomic data and can improve the power of the classical Quantitative Trait Loci (QTL) approaches to identify candidate genes for peach disease resistance. Brown rot caused by Monilinia spp., is the most important fungal disease of stone fruits worldwide. Improved levels of peach fruit rot resistance have been identified in some cultivars and advanced selections developed in the UC Davis and USDA breeding programs. Whole genome sequencing of the Pop-DF parents lead to discovery of high-quality SNP markers for QTL genome scanning in this experimental population. Pop-DF created by crossing a brown rot moderately resistant cultivar ‘Dr. Davis’ and a brown rot resistant introgression line, ‘F8,1–42’, derived from an initial almond × peach interspecific hybrid, was evaluated for brown rot resistance in fruit of harvest maturity over three seasons. Using the SNP linkage map of Pop-DF and phenotypic data collected with inoculated fruit, a genome scan for QTL identified several SNP markers associated with brown rot resistance. Two of these QTLs were placed on linkage group 1, covering a large (physical) region on chromosome 1. The genome scan for QTL and SNP effects predicted several candidate genes associated with disease resistance responses in other host-pathogen systems. Two potential candidate genes, ppa011763m and ppa026453m, may be the genes primarily responsible for M. fructicola recognition in peach, activating both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) responses. Our results provide a foundation for further genetic dissection, marker assisted breeding for brown rot resistance, and development of peach cultivars resistant to brown rot.     

Reprogrammed endophytic microbial community in maize stalk induced by Trichoderma asperellum biocontrol agent against Fusarium diseases and mycotoxin accumulation

Maize stalk rot and ear rot, caused by Fusarium graminearum and Fusarium verticillioides, respectively, are major diseases that threaten the sustainable production of maize. In this study, an artificial inoculation assay demonstrated that the control efficacy of maize stalk rot and ear rot by Trichoderma asperellum granules were 49.83 % and 39.63 %, respectively. By high-throughput sequencing of maize plants, a total of 76 196 ITS1 sequences and 887 226 V3V4 16S rRNA sequences were analyzed and were grouped into 2934 fungal and 24 248 bacterial operational taxonomic units (OTUs), respectively. It revealed a significantly higher endophytic microbial abundance in the stem tissue of plants grown in T. asperellum-treated soil than in those grown in the control, with the largest increase observed in the basal stem section. In addition, the endophytic microbial diversity and corresponding control effects all gradually decreased from the basal to apical parts of the stem in plants grown in Trichoderma-treated soil, indicating that Trichoderma stimulated a more significant effect on the defense system in the basal section of the stalk than in the apical parts of plants. Furthermore, the accumulation of deoxynivalenol (DON) and fumonisin B1 (FB1) decreased in the stem and ear of maize grown in T. asperellum-treated soil.     

Seed Treatment with Live or Dead Fusarium verticillioides Equivalently Reduces the Severity of Subsequent Stalk Rot

Fusarium verticillioides is a widely distributed fungus that can associate with maize as a deleterious pathogen and an advantageous endophyte. Here, we show that seed treatment with live F. verticillioides enhances maize resistance to secondary stalk rot infection and further demonstrate that dead F. verticillioides is sufficient to equivalently reduce F. verticillioides biomass. Seed treatment with live or dead F. verticillioides primes maize plants, and upon subsequent stalk infection, terpenoid phytoalexins accumulate faster than control‐treated plants. Seed treatment did not constitutively activate plant defences nor did it impact plant growth. These results suggest that seed treatment with dead F. verticillioides can be used as a ‘vaccination’ method to decrease the severity of stalk rot and potentially pathogen infection throughout the plant.     

Characterization of non-canonical G beta-like protein FvGbb2 and its relationship with heterotrimeric G proteins in Fusarium verticillioides

Fusarium verticillioides is a fungal pathogen that is responsible for maize ear rot and stalk rot diseases worldwide. The fungus also produces carcinogenic mycotoxins, fumonisins on infested maize. Unfortunately, we still lack clear understanding of how the pathogen responds to host and environmental stimuli to trigger fumonisin biosynthesis. The heterotrimeric G protein complex, consisting of canonical Gα, Gβ and Gγ subunits, is involved in transducing signals from external stimuli to regulate downstream signal transduction pathways. Previously, we demonstrated that Gβ protein FvGbb1 directly impacts fumonisin regulation but not other physiological aspects in F. verticillioides. In this study, we identified and characterized a RACK1 (Receptor for Activated C Kinase 1) homolog FvGbb2 as a putative Gβ-like protein in F. verticillioides. The mutant exhibited severe defects not only in fumonisin biosynthesis but also vegetative growth and conidiation. FvGbb2 was positively associated with carbon source utilization and stress agents but negatively regulated general amino acid control. While FvGbb2 does not interact with canonical G protein subunits, it may associate with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis and stress response in F. verticillioides.     

Biodiversity of Fusarium species in Mexico associated with ear rot in maize, and their identification using a phylogenetic approach

Fusarium proliferatum, F. subglutinans, and F. verticillioides are known causes of ear and kernel rot in maize worldwide. In Mexico, only F. verticillioides and F. subglutinans, have been reported previously as causal agents of this disease. However, Fusarium isolates with different morphological characteristics to the species that are known to cause this disease were obtained in the Highland-Valley region of this country from symptomatic and symptomless ears of native and commercial maize genotypes. Moreover, while the morphological studies were not sufficient to identify the correct taxonomic position at the species level, analyses based in the Internal Transcribed Spacer region and the Nuclear Large Subunit Ribosomal partial sequences allowed for the identification of F. subglutinans, F. solani, and F. verticillioides, as well as four species (F. chlamydosporum, F. napiforme, F. poae, and F. pseudonygamai) that had not previously been reported to be associated with ear rot. In addition, F. napiforme and F. solani were absent from symptomless kernels. Phylogenetic analysis showed genetic changes in F. napiforme, and F. pseudonygamai isolates because they were not true clones, and probably constitute separate sibling species. The results of this study suggest that the biodiversity of Fusarium species involved in ear rot in Mexico is greater than that reported previously in other places in the world. This new knowledge will permit a better understanding of the relationship between all the species involved in ear rot disease and their relationship with maize.     

Pathogenicity variation in Fusarium verticillioides populations isolated from maize in northern Italy

One hundred and eighty one strains were selected among Fusarium verticillioides populations isolated from maize samples collected in three fields located in northern Italy. All the isolates were tested for their pathogenicity on maize seeds by assessing the seed germination percentages and the percentage infection indexes concerning seed colonization, radicle decay and coleoptile rot. Fusarium verticillioides strains did not affect seed germination even in presence of high seed colonization, but showed a variable pathogenic behavior according to the maize growth stages. Seedborne F. verticillioides population as well as strains isolated at maturity was effective in seed colonization and in inducing coleoptile rot, not causing however serious radicle decay. Only populations isolated at seedling and pre-silking stages showed high radicle decay ability. These results provide baseline information on F. verticillioides pathogenicity. They constitute an important input for further investigation of F. verticillioides biology in order to define its evolutionary potential.     

Identification of charcoal rot resistance QTLs in sorghum using association and <Emphasis Type="Italic">in silico</Emphasis> analyses

Charcoal rot disease, a root and stem disease caused by the soil-borne fungus Macrophomina phaseolina (Tassi) Goid., is a major biotic stress that limits sorghum productivity worldwide. Charcoal rot resistance-related parameters, e.g., pre-emergence damping-off%, post-emergence damping-off%, charcoal rot disease severity, and plant survival rates, were measured in a structured sorghum population consisting of 107 landraces. Analysis of variance of charcoal rot resistance-related parameters revealed significant variations in the response to M. phaseolina infection within evaluated accessions. Continuous phenotypic variations for resistance-related parameters were observed indicating a quantitative inheritance of resistance. The population was genotyped using 181 simple sequence repeat (SSR) markers. Association analysis identified 13 markers significantly associated with quantitative trait genes (QTLs) conferring resistance to charcoal rot disease with an R² value ranging between 9.47 to 18.87%, nine of which are environment-specific loci. Several QTL-linked markers are significantly associated with more than one resistance-related parameter, suggesting that those QTLs might contain genes involved in the plant defense response. In silico analysis of four novel major QTLs identified 11 putative gene homologs that could be considered as candidate genes for resistance against charcoal rot disease. Cluster analysis using the genotypic data of 181 SSR markers from 107 sorghum accessions identified 12 main clusters. The results provide a basis for further functional characterization of charcoal rot disease resistance or defense genes in sorghum and for further dissection of their molecular mechanisms.     

The genetic architecture of complex traits in teosinte (Zea mays ssp. parviglumis): new evidence from association mapping

Previous association analyses showed that variation at major regulatory genes contributes to standing variation for complex traits in Balsas teosinte, the progenitor of maize. This study expands our previous association mapping effort in teosinte by testing 123 markers in 52 candidate genes for association with 31 traits in a population of 817 individuals. Thirty-three significant associations for markers from 15 candidate genes and 10 traits survive correction for multiple testing. Our analyses suggest several new putative causative relationships between specific genes and trait variation in teosinte. For example, two ramosa genes (ra1 and ra2) associate with ear structure, and the MADS-box gene, zagl1, associates with ear shattering. Since zagl1 was previously shown to be a target of selection during maize domestication, we suggest that this gene was under selection for its effect on the loss of ear shattering, a key domestication trait. All observed effects were relatively small in terms of the percentage of phenotypic variation explained (<10%). We also detected several epistatic interactions between markers in the same gene that associate with the same trait. Candidate-gene-based association mapping appears to be a promising method for investigating the inheritance of complex traits in teosinte.     

Covariation between line and testcross performance for reduced mycotoxin concentrations in European maize after silk channel inoculation of two <Emphasis Type="Italic">Fusarium</Emphasis> species

Fusarium spp. in maize can contaminate grain with mycotoxins harmful to humans and animals. Breeding and growing resistant varieties is one alternative to reduce contamination by mycotoxins. Little is known about the population parameters relevant to resistance breeding. The objectives of this study were to draw conclusions on breeding of reduced mycotoxin concentrations of deoxynivalenol, zearalenone and fumonisins, and resistance to ear rot after silk channel inoculation with F. graminearum or F. verticillioides, respectively. For that, variation and covariation of line and testcross performance and correlations between both species and between mycotoxin concentrations and ear rot resistance were calculated. Means of ear rot after infection with F. graminearum were higher than with F. verticillioides. Moderate phenotypic correlations (r = 0.46–0.65) between resistances to both Fusarium spp. implicate the need of separate testing. Analyses of variance revealed significant (P < 0.01) differences among lines in line and testcross performance for 30–60 entries per maturity group. Multi-environmental trials for accurate selection are necessary due to significant (P < 0.1) genotype × environment interactions. High genotypic correlations between ear rots and mycotoxins (r ≥ 0.90), and similar heritabilities of both traits, revealed the effectiveness of indirect selection for mycotoxin concentrations based on ear rot rating after inoculation. Moderate genotypic correlations between line and testcross performance were found (r = 0.64–0.83). The use of one moderately to highly susceptible tester is sufficient since genotypic correlations between testcrosses of different testers were high (r = 0.80–0.94). Indirect selection for testcross performance based on line performance is less effective than selection based on mycotoxin concentrations. Consequently, selection for resistance to ear rot and mycotoxin accumulation should be started among testcrosses tested first for general combining ability based on ear rot data in parallel with a negative selection for line per se performance.