Background

Wheat Fusarium head blight, stripe rust, powdery mildew, and sharp eyespot are the four most important diseases in Hubei Province (Ao, 2002). In recent years, with the warming of climate, the improvement of fertilizer and water conditions, the plant height reduction of main recommended varieties, and the change of farming system such as straw returning, the occurrence and damage degree of wheat diseases in Hubei has been expanded. It has posed a great threat to grain production and food safety in Hubei Province. It has been proved that cultivation and large area planting of disease resistant varieties was the most economical and safe effective means to reduce the wheat disease. In addition to using phenotypic resistance characteristics of parents to undergo empirical routine disease resistance breeding, using marker-assisted selection (MAS) to polymerize major resistance genes and cultivate new wheat resistance varieties on the basis of molecular level detection of the major parents and major resistance gene of wheat varieties has been an important breeding strategy (Li et al., 2016).

So far, more than 200 resistance loci of Fusarium head blight have been located. These loci were distributed in all wheat chromosomes (Buer-stmayr et al., 2009; Cativelli et al., 2013; Garvin et al., 2015). The Fhb1 on 3BS and Fhb2 on 6BS have been applied in MAS breeding (Bernardo et al., 2014), and have achieved some success in commercial applications (Brown-guedira et al., 2008). In the research of stripe rust resistance genes, 74 major resistance genes to stripe rust had been officially named as Yr1~Yr74. These genes were distributed in all wheat chromosomes (Xue et al., 2016). In the research of powdery mildew resistance genes, more than 70 resistance genes to wheat powdery mildew had been located on 49 chromosomal loci. However, there was relatively little research on the sharp eyespot resistance QTL, and major was made by Chinese scholars. More than 20 resistances QTL to sharp eyespot had been distributed in other chromosomes except 3A, 7A, 1B, 4B, 5B, 1D, 4D and 6D chromosomes (Liu et al., 2015).

Due to fewer studies on the resistance gene (QTL) and resistance formation mechanism carried by the main varieties, the deep application of these varieties was restricted (Li et al., 2016). Genetic analysis of disease resistance genes (QTL) to potential varieties and its parents was analyzed by developed molecular markers of disease resistance genes. It could provide genetic information for using biotechnology to improve the disease resistance of wheat, and also help to understand the source of resistance genes and genetic rule, which might lay the foundation for the reasonable promotion in production and effective application in the breeding (Li et al., 2016).

Emai170 is a semi winter variety selected by Food Crop Institute, Hubei Academy of Agricultural Sciences, Institute of Cotton Research of CAAS and Crop Institute, Chinese Academy of Agricultural Sciences, with high yield, strong adaptability and good disease resistance characteristics, approved by Hubei Province in 2014. In the past two years in Hubei, it showed a great development potential in large area production. This study aimed at wheat Fusarium head blight, stripe rust, powdery mildew, and sharp eyespot, the four major diseases in Hubei province, selected resistance genes or molecular markers linked to the major QTL or co separation to identify and analyze disease resistance genes of Emai170 and its parents Yumai47 and Jimai19, which was to confirm the constituent and resistance formation mechanism of Emai170 resistance gene, and provide some references for the study on the genetic rule of disease resistance gene, variety popularization and application and as a backbone parent to cultivate new wheat resistance varieties by MAS.

1 Results and Analysis

1.1 Disease resistance performance in Hubei region trials and large area production demonstration

In Hubei region trials, the resistance of Emai170 to 4 main diseases was stable. For two consecutive years, stripe rust showed moderately resistant, while 3 diseases such as powdery mildew, Fusarium head blight and sharp eyespot were all moderately susceptible (Table 1). Under the condition of Natural infection in the field, resistance identification of large area cultivating Emai170 was carried out for two consecutive years in Wuhan and Suizhou. The results of two consecutive years of identification showed (Table 1) that the stripe rust occurred slightly for two years, between moderately resistant and highly resistant; powdery mildew occurred slightly, between moderately susceptible and moderately resistant; the incidence of sharp eyespot is lighter, almost was moderately susceptible; the incidence of Fusarium head blight was general, showed moderately susceptible in Wuhan and Suizhou for two years. In general, the resistance to stripe rust and powdery mildew of Emai170 was better, and the resistance to sharp eyespot and Fusarium head blight was common.

1.2 Genetic analysis of disease resistance genes of Fusarium head blight (QTL)

Emai170 and its parents Yumai47 and Jimai19, and two highly resistant to Fusarium head blight (Sumai3 and Wangshuibai) were made molecular detection by the 10 developed molecular markers. The results showed (Table 2) that the target bands could be amplified at 8 resistance loci of the highly resistant variety Sumai3 and Wangshuibai. As for Emai170, the target bands could be amplified at the resistance loci on 3BS (Fhb1), 5AS (Fhb5), and 7A, 7B, 7D. It was suggested that Emai170 might have these 3 resistance genes to Fusarium head blight. The resistance loci on 2A, 6BS (Fhb2) and 4BL, only Yumai47 could amplify the target band, while these target fragments were not detected in Emai170 and Jimai19.Maybe these resistance genes failed to transfer from Yumai47 to Emai170. At the resistance loci on 4AL, Yumai47 and Jimai19 could amplify the target bands, but they couldn’t be detected inEmai170. It was possible that the resistance genes could not be inherited from the parents to the Emai170, or the genetic distance from the target gene of the Xgwm160 marker was far away and the marker and the target gene were separated in the genetic process.

1.3 Genetic analysis of disease resistance genes of Stripe rust (QTL)

Emai170 and its parents Yumai47 and Jimai19 were made molecular detection by the 12 molecular markers closely associated with disease resistance genes of stripe rust (QTL).The results showed (Table 3) that the target bands could be amplified by the molecular markers of three resistant genesYr2, Yr5 and Yr26 in Emai170. And all four genes could be detected in parents. It was proved that Emai170 might have these 4 disease resistance genes of stripe rust. The Yr5was inherited from the parent Yumai47 to the Emai170, Yr26 was inherited from the parent Jimai19 to the Emai170, Yr2 gene was contained in two parents and Emai170. 8 genes were not detected in the Emai170, Yr10 and YrTp1could be detected in a parent, but they were not inherited to the Emai170. The other 6 genes were not detected in the Emai170 and 2 parents.

1.4 Genetic analysis of disease resistance genes of Powdery mildew (QTL)

Emai170 and its parents Yumai47 and Jimai19 were made molecular detection by the 15 molecular markers closely linked with disease resistance genes of powdery mildew (QTL). The results showed (Table 4) that the target bands could be amplified by the molecular markers of 8 resistant genes, which suggested that Emai170 might carry these 8 powdery mildew resistant genes. And 7 genes Pm2, Pm8, Pm21, Pm31, Pm33, Pm35 and Pm36could be detected in a parent or two parents, while Pm16could not be detected in two parents, but could be detected in the Emai170. It may be produced by gene recombination. 7 genes that cannot be detected in the Emai170, Pm32could be detected in the Yumai47, Pm34 could be detected in the Jimai19, but all of them cannot be inherited to the Emai170. The other 5 genes were not detected in the Emai170 and 2 parents.

1.5 Genetic analysis of disease resistance genes of Sharp eyespot (QTL)

Emai170 and its parents Yumai47 and Jimai19 were made molecular detection by the 15 molecular markers closely linked with disease resistance genes of sharp eyespot (QTL). The results showed (Table 5) that the target bands could be amplified in the Emai170 by the 6 resistance markers, which suggested that Emai170 might carry these 6 sharp eyespot (QTL) loci. And 5 markers Xwmc397, Xgwm212, Xgdm67, Xgwm644, and Xgwm526could amplify the target bands in a parent or two parents, while Xwmc497 could not amplify the target bands in two parents. It suggested that the resistance loci might be produced by gene recombination. 9 resistance loci that could not be detected in the Emai170, 2 loci marked withXwmc154 and Xwmc754 could be detected in Jimai19, but all of them were not inherited to the Emai170. 2 loci marked with Xbarc148 and Xbarc198 could amplify the target bands in two parents, but were not inherited to the Emai170. It suggested that these 2 resistance loci might be lost in the process of gene recombination. The other 5 genes were not detected in the Emai170 and 2 parents.

2 Discussion

Wheat resistance gene postulation by resistant strains was susceptible to genetic background, environment factors, and interaction between genes and environmental and between genes and genes. And there was also a circumstance that disease resistance genes lacked pathogenic strains. Therefore, it was difficult to infer whether there was a new disease resistance gene by gene postulation (Li et al., 2016). It could make up for the deficiency of gene postulation by the developed disease resistance genes molecular markers (Li et al., 2016). Meanwhile, with the continuous development and application of effective molecular markers for disease resistance, the identification of disease resistance genes had become more effective and rapid (Li and Xu, 2009; Li et al., 2014). Emai170 is a new semi winter wheat variety with comprehensive agronomic characters. Except for its high yield and strong adaptability, its outstanding feature is that the plant height was reduced (79 cm) and had good disease resistance at the same time. Therefore, detection method of disease resistance markers was used to explore resistance gene carried by Emai170 and their genetic rules, it was very important for the variety application and as a backbone parent by MAS to cultivate new better wheat resistant varieties.

Li et al. (2016) used 364 SSR and STS molecular markers that spread all over the wheat genome to analyze the association between 192 local varieties and commercial cultivars. The results showed that Xgwm160 on 4AL, Xgwm261 on 2D, Xcfa2263 on 2A, and Xwmc273 on 7A, 7B, 7D were significantly associated with Fusarium head blight resistance in more than 2 experimental environments. Each QTL could explain more than 11.7% of the phenotypic variation, indicating that these resistant QTLs were stable. And molecular markers linked with these resistant QTLs could be used to do Marker assisted selection (MAS). 2 resistance expansion major loci Fhb1on 3BS and Fhb2 on 6BS and 2 resistance infection major loci Fhb4 on 5AS and Fhb5 on 6BS have been identified and named by genetic population in wheat (Liu et al., 2016).In this study, resistance contrast Sumai3, Wangshuibai, Emai170 and its parents were detected by the molecular marker of the above - mentioned resistance loci. The results showed that Sumai3 could amplify target bands on the 8 markers; Wangshuibai could amplify target bands on the 7 markers (Table 2). It partially explained the molecular basis of high resistance to Fusarium head blight and stable resistance of these two varieties. As for Emai170, target bands could be amplified from the 4 markers; its parents Yumai47 and Jimai19, target bands could be amplified from the 7 and 4 markers, respectively. However, the identification of natural disease in the field at Wuchang experimental site for many years showed that the Fusarium head blight resistance of Emai170 was better than its parents Yumai47 and Jimai19. After analysis, we speculated that some Fusarium head blight resistance genes in Emai170 were not detected, and the better resistance of Emai170 than its parents might be related to heterobeltiosis (Liu et al., 2016).

At present, the rapid detection system of wheat disease resistance genes of stripe rust (QTL), such as Yr2, Yr5, Yr9, Yr10, Yr15, Yr17, Yr18, Yr26, Yr36, YrV23, and Yrt1, has been established (Li et al., 2016). In this study (Table 2), Emai170 with Yr2, Yr5, and Yr26had the characteristics of resistance to stripe rust. Yumai47 with Yr2, Yr5, and Yr10 also had the moderately resistant to stripe rust. And Jimai19 with Yr2, Yr26, and YrTp1 had the susceptible to stripe rust. It was indicated that Yr5 gene might play an important role in resistance to stripe rust in Emai170, which was similar to that previous studies (Wu et al., 2007).

The genes Pm12、Pm13、Pm16 and Pm21showed good resistance to wheat powdery mildew in China (Xue et al., 2010). And Pm1, Pm2, Pm3, Pm4, Pm5, Pm6, Pm7 and Pm8 have lost partial resistance and showed good resistance only in the state of polymerization (Li and Xu, 2009). In this study, Emai170 had 2 major resistance genes (Pm16, Pm21), Yumai47 and Jimai19 just had 1 major resistance gene (Pm21) (Table 4). In powdery mildew resistance, Jimai19 showed high resistance. Both Emai170 and Yumai47 showed moderately susceptible. It was indicated that the Pm21 gene had been shown to be susceptible, may be related to the emergence of a strain that was toxic to Pm21 (Zhao et al., 2010). And the high resistance to powdery mildew of Jimai19 may be associated with other non - detectable major genes or Polymerization of other micro effective genes.

Most of the varieties used in current production were susceptible sharp eyespot (Liu et al., 2015). Lack of resistant germplasm and difficulty in identification of resistance brought great difficulties to the improvement of resistance to sharp eyespot. At present, there were few reports on gene mapping and molecular marker development of wheat sharp eyespot resistance. In this study, Emai170 could amplify the target band in 6 markers, Yumai47 and Jimai19 could amplify the target band in 7 markers (Table 5). In sharp eyespot resistance, Yumai47 showed moderately resistant, both Emai170 and Jimai19 showed moderately susceptible. However, Yumai47 had no specific resistance loci compared with Emai170 and Jimai19 (Table 5). This might associate with the resistance loci of Yumai47 containing other undetected or unidentified development loci. Meanwhile, it was proved that the resistance loci were detected in this study could explain the low rate phenotypic variation (Liu et al., 2015). Therefore, it is necessary to strengthen the identification of Wheat Germplasm for resistance to sharp eyespot and to explore the excellent germplasm resources in the future of wheat breeding for resistance to sharp eyespot. On the other hand, it is necessary to accelerate the pace of application of molecular marker-assisted breeding, based on the localization of QTL and the development of its molecular markers.

3 Materials and Methods

3.1 Experimental materials

Emai170 and its parents Yumai47 and Jimai19 were used as research materials (Table 1). Sumai3 and Wangshuibai were used as disease-resistant control variety of molecular detection of disease resistance genes to Fusarium head blight (QTL). All varieties were preserved by this research group.

3.2 Evaluation of resistance to disease

In 2012-2014, Institute for Plant Protection and Soil Science, Hubei Academy of Agricultural Sciences made identification of disease resistance by artificial inoculation according to the requirements of document Examination (identification) Standard for Crop Varieties in Hubei Province (E Review Committee [2012] No.2).The test Puccinia striiformis and Erysiphe graminis are pathotypes, Gibberella saubinetii and Rhizoctonia solani vaccinated with strong pathogenicity strains. Strain vaccinated by stripe rust was a popular mixed colony of CY31, CY32 and CY33 race in China. The pathogen of powdery mildew inoculation is a mixture of 1,094 powdery mildew strains collected from 12 provinces (cities) in China. Gibberella saubinetii strain was Huanggang Ⅰ, Rhizoctonia solani strain was 64-1. The strains used for inoculation and identification were all preserved by the Hubei Key Laboratory of Crop Diseases, Insect Pests and Weeds Control. Each year was subjected to field inoculation and disease resistance evaluation according to Technical Specification for Evaluation of Wheat Resistance to Diseases and Insects (NY/T 1443-2007), and the disease was investigated after full onset of the control variety.

In order to evaluate the resistance disease performance of Emai170 in the demonstration of large area production more accurately, demonstration planting of 2.0 hm² was arranged respectively in Wuhan and Suizhou, Hubei Province, in 2014-2016. And field disease investigation was carried out according to the Forecast Technical Specification for Wheat Fusarium Head Blight (GB/T15796-2011), Forecast Technical Specification for Wheat Stripe Rust (GB/T157965-2011), Forecast Investigation Specification for Wheat Powdery Mildew (NY/T 613 - 2002), and Forecast Investigation Specification for Wheat Sharp Eyespot (NY/T614-2002). The reaction type is divided into highly resistant (HR), moderately resistant (MR), moderately susceptible (MS) and highly susceptible (HS). The disease survey was conducted randomly at 3 points per point each year, with the average value as the final disease resistance performance.

3.3 Molecular identification of disease resistance genes

Genomic DNA Extraction in wheat seedling by modified CTAB method. 1% agarose gel electrophoresis was used to detect the concentration and quality of DNA. 51 molecular markers related to disease resistance genes (QTL) of wheat Fusarium head blight, stripe rust, powdery mildew and sharp eyespot were selected for molecular identification (Table 2; Table 3; Table 4; Table 5). All Primers were compounded by Tianyi Huiyuan Bio Tech Ltd., Wuhan.

PCR reaction system was 20 μL, containing 16 μL ddH₂O, 2 μL 10 x PCR Buffer (Mg²⁺ Plus), 0.4 μL dNTPs (10 mmol/L each), 0.5 μL forward primer (10 μmol/L), 0.5 μL reverse primer (10 μmol/L), 0.2 μL Taq DNA polymerase (5 U/μL) and 1 μL DNA (50~100 ng), and it was reacted in a common PCR amplifier (ABI Veriti). The PCR products were analyzed by gel imaging system after 1% agarose gel electrophoresis. The amplified product was separated by 12% non-denatured polyacrylamide gel electrophoresis, stained by silver nitrate. Then analysis of the image appeared was made.

Authors’ contributions

LYK and GCB were the executors of experimental design and experimental research in this study, and also the main authors of this manuscript. ZZW and THW participated in the operation of the study, and analysis of data and test results. FCG, NFH, and CL partial participated in the operation of the study. ZJ, ZYQ and YLJ mainly completed the large-scale planting and disease investigation of Emai170. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by Science and Technology Major Project of Transgenic Organisms Breeding (2014ZX0800202B-003), Natural Science Foundation of Hubei Province of China (2016-CFB522), National Wheat Industry Technology System “Comprehensive Experimental Station of Wuhan" (CARS-03)", and Youth Foundation of Academy of Agriculture of Hubei Province of China (2016NK-YJJ03).

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