| Maize (Zea mays L.) is the worldwide most important food and forage crop as well as a model plant for genetics and development biology, and plays a decisive role in agriculture. Maize rough dwarf disease (MRDD) is a viral disease that is widely distributed in the worldwide and causes great yield reduction of maize. In China, The pathogen of MRDD has been identified as rice black-streaked dwarf virus (RBSDV). RBSDV is mainly transmitted by a kind of planthopper (Laodelphax sreiatellus Fallen.) in a persistent manner among individuals. When the maize seedlings suffer the large scale migration of planthopper from wheat or other grasses in the field, it may cause the outbreak of MRDD. The MRDD has caused a great and large-scale yield reduction of maize in China for the poor resistance to MRDD of elite inbred lines and hybrids in recent years. The MRDD has become one of the hardest problems in maize production, and obstructs our grain production. The results of the assays of maize resistance to MRDD show that the maize resistance to MRDD take on the features of quantitative trait and is controlled by multiple genes. Meanwhile, Chinese breeders have selected a bulk of maize inbred lines with resistantance to MRDD, such as90110, Qi319, X178et al. To control the outbreak and transmission of MRDD and reduce the loss of maize yield, it is the key step to identify the resistant QTL in maize and improve the elite maize inbred and hybrid using these QTL.Recently, the researches of maize resistance to MRDD have been accelerated, but the breakthrough progresses are not made. The major reason is the conditions of MRDD are complicated that involve the correlations of the virus, vector-planthopper and maize. It is hard to accurately identify the individual phenotype and to map the QTL conferring resistance to MRDD. But, the phenotype identification is the foundation for mapping and cloning the resistant QTL that develop the appropriate scientific methods to identify maize resistance to MRDD accurately in the segregation populations. In this study, the improved method to identify the maize resistance to MRDD is developed and the QTL conferring resistance to MRDD are identified.1. The development of combined identification method based on the combination of natural infection and artificial inoculationIn this study, the planthoppers were used as the medium to transmit RBSDV and were cultured in the feeding buckets and fed on the diseased maize leaves from naturally infected field-grown plants with typical symptoms of MRDD in the Jinan area of Shandong province. Subsequently, five adult-planthoppers were randomly selected as a sample to detect RBSDV, and then the RBSDV specific primers were used to screen the samples from different feeding buckets.11out of14samples were positive. May1st in2010, the RILs and their parental lines (90110and Ye478) were germinated at plastic plates in the net house. When the seedlings grew to the1-leaf period, the planthoppers with RBSDV were dispersed into the net house. The average density of planthoppers was about500/m2. After a5-day inoculation, the insecticide was used to kill all of the planthoppers, and the seedlings were maintained to grow continually in the net house and then were transplanted to the experiment fields at the3-leaf stage in Jinan area. The seedlings were infected again by the planthoppers at the migratory flight period of planthoppers in field. The incidence of MRDD and the stability and repetitiveness of identification results were increased by the combination of the artificial inoculation and natural infection. And plenty of maize plants could be screened in a short time by using this method. The combined method has laid the foundation of identifying abundant examples in the segregation population.2. The genetic analysis of maize rough dwarf diseaseThe analysis was performed in common maize using a set of recombinant inbred lines (RILs) F7;9derived from ’Ye478×90110’.The RILs population, Ye478,90110and F1were grown continually at the area of Jinan and Laizhou respectively from2008to2010, and then were identified the resistance to MRDD through the method combining the artificial inoculation and natural infection. The phenotypes related to MRDD including the shortened superior internodes, enation, tassel type and disease severity index were evaluated at the flowering stage. Subsequently, the genetic analyses were performed using the3-year experimental data at two plots according to the disease grade and the level of traits related to MRDD. The broad sense heritability of resistance to MRDD is from0.71to0.94, that is to say, the resistance to MRDD was mainly determined by the genotype in the population of Ye478and90110, with minor influence of the environment and the interaction effect between the genotype and environment. The distribution analysis of traits related to MRDD showed that there were major QTL from90110conferring resistance to MRDD.3. Mapping of QTL conferring resistance to MRDDThe512SSR markers covering the whole maize genome were screened polymorphisms between the two parents Ye478and90110. And the polymorphism SSR markers with clear band were used for QTL mapping by using the RILs. Five additive QTL related to resistance were detected and were mapped on chromosome2,6,7,8and10respectively. The QTL were named as qMRD2, qMRD6, qMRD7, qMRD8and qMRD10, respectively. The qMRD8was a major QTL, that could be detected in all traits mapping, and explain the phenotypic variation of12-28.9%. QTL qMRD2, qMRD6and qMRD7also had good repetitiveness, which were detected3times at least through the whole mapping experiment. The qMRDIO was only detected once at the trait of shortened superior intemodes with relative small effect and need to be verified in subsequent experiment. All of these QTL could explain the phonotypical variance of41.43-50.84%. In this study, the QTL qMRD6, qMRD7and qMRD8were coincidence with the loci that were detected by Dr. Wang using SSR-BSA method, previously. The results suggested that these QTL and the loci nearby these QTL may contain the genes conferring resistance to MRDD. The major QTL detected in this study can be introduced into the elite inbred lines with little resistance to MRDD by MAS to accelerate the proceeding of maize breeding.In this research, the combined inoculation method including natural infection and artificial inoculation was used to study the resistance to MRDD for the first time, which could enhance the stability and validity of resistance to MRDD, and could increase the detecting amount of segregation population. Moreover, this study proved that the resistance to MRDD was mainly due to the genetic factors through the genetic analysis. There were5QTL conferring resistance to MRDD detected through the QTL mapping, and3of them were coincidence with the loci detected through SSR-BSA method. Finally, this study laid the foundation of MRDD resistance breeding in maize by the method of MAS. |
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