Molecular Mapping Of Three Loci Conferring Resistance To Maize (Zea Mays L.) Rough Dwarf Disease

Maize (Zea mays L.) is an important food, forage and energy crop as well as a model plant for genetics in the world. In China, the maize yield is only next below the rice yield and plays an important role in the national economy. Maize rough dwarf disease (MRDD) is a viral disease that is widely distributed in the world and causes great losses in grain yield. The pathogen of MRDD has been identified as rice black-streaked dwarf virus in China and is transmitted by the insect vector Laodelphax striatellus in a persistent manner. Disease resistance breeding could potentially reduce crop losses with minimal efforts by growers, in an environmentally safe, cost-effective manner. Marker-assisted selection (MAS) may greatly increase the efficiency in plant breeding for resistance to MRDD compared to conventional breeding. However it must identify the molecular markers that are tightly linked with the target genes or QTLs. There have no reports about the mapping of genes conferring resistance to MRDD for the difficulty in the resistance evaluation of plants and the lacking of the appropriate mapping populations.In this study, the planthoppers were used as the vector to transmit RBSDV and were fed on the maize plants with RBSDV that were naturally infected field-grown plants with typical symptoms of MRDD in the Jinan area. After the rearing of virus acquisition, some planthoppers were randomly separated into three groups. Each sample from group 1, 2 and 3 respectively contained one, five or 10 planthoppers. The RBSDV content of each sample was detected using indirect enzyme-linked ummunosorbent assay (ELISA) to determine how many planthoppers were infected with RBSDV. Three of the 12 samples of group 1 were positive, which showed that 25% of planthoppers were viruliferous. The results from the samples consisting of five planthoppers indicated that viruliferous rate was 13.3%. All three samples with 10 planthoppers were positive. So a maize seedling should be inoculated with 10 planthoppers, in which there must be at least one viruliferous planthopper. The effects of maize plant age, insect density and inoculation duration on the success rate of inoculation were respectively examined by inoculating susceptible maize line Ye478 seedlings. The results indicated that a higher disease incidence could be obtained when the younger maize plants were inoculated; the disease incidence was higher when the plants were inoculated with 15 and 10 planthoppers per plant with 100% and 96% diseased plants, respectively; the longer the inoculation duration, the greater the incidence when the two-leaf seedlings were inoculated with 10 planthoppers per plant. Based on these results and the livability of the seedlings at different inoculation conditions, the appropriate inoculation conditions were two-leaf plants and 15 planthoppers per plant for five days.To determine the resistance to MRDD of different genotype maize plants, 10 inbred lines were inspected after inoculation with the appropriate conditions. As the results of inoculation, 90110, P138, 178 and F022411 were MRDD resistant lines, Ye478 was the highly susceptible line and Ye515, F112132, Luyuan92 and 8112 were moderately susceptible to MRDD. The inoculation results of these inbred lines were consistent with those of field assessments for three years, but the incidences of field assessments were lower than those of the inoculations and showed great fluctuations between different years.Using indirect ELISA and real-time quantitative RT-PCR, the RBSDV content in the inoculated plants of Ye478, 90110, F112132 and F022411 was detected. Detected by ELISA, all the plants scoring 3 were positive. The healthy plants could not be significantly distinguished from the slightly diseased plants with a score of 1 or 2. However, using real-time quantitative RT-PCR, RBSDV could be detected at levels as low as 1×10³ copies in 100 ng total RNA from the infected plants, and the differences in the average viral content among plants with different disease levels were significant. The plants infected with RBSDV did not all present the MRDD symptoms. But in general, the viral content was higher as the disease level rose in susceptible maize inbred lines, and the plants with 1×10³ viral copies in 100 ng total RNA belonged to susceptible individuals and the plants with 1×10⁶ viral copies in 100 ng total RNA belonged to highly susceptible individuals.Combined with artificial inoculation, plants in a segregate population of MRDD resistance could be accurately identified as diseased, or not, by using real-time quantitative RT-PCR. This will make a contribution to the study of localization of MRDD resistance genes. Maize inbred line Ye478 is an elite line with many excellent agricultural traits and has accounted much for the maize production in China. However, Ye478 is highly susceptible to MRDD. Maize inbred line 90110 is highly resistant to MRDD. A molecular marker linkage map was constructed using a Ye478×90110 F₂ population consisting of 150 individuals. The polymorphisms between the parental lines were detected with 86 RFLP and 456 SSR markers. Out of these markers, 347 (66.0%) markers including 59 RFLP and 288 SSR markers showed polymorphisms between the two parents. Twenty-six RFLP and 252 SSR markers, which exhibited good hybridization or amplification quality, were used to genotype the F₂ individuals. For a codominant marker, the homozygote individuals with the allele from 90110 at the locus were scored A; the homozygote individuals with the allele from Ye478 at the locus were scored B and the heterozygote individuals with both parents alleles at the locus were scored H. For a dominant marker, the individuals that were not a homozygote for allele from 90110 at the locus were scored C and the individuals that were not a homozygote for allele from Ye478 at the locus were scored D. The individuals with missing data at a locus were scored "-". The chi-square test was used to test each marker for the expected 1:2:1 segregation ratio for codominant markers, or 3:1 for dominant markers in the F₂ population. Based on the segregation data of RFLP and SSR markers, a maize molecular marker linkage map was constructed with MAPMAKER/EXP Version 3.0. Total 278 markers were used to construct molecular linkage map and presented four unlinked SSR markers: umc1144, bnlg1779, umc1018 and bnlg1446, and three unlinked RFLP markers: umc67/EcoR I, umc97/EcoR I and umc63/EcoR I. Ten linkage groups were obtained. The total map length was 2164.3 cM and the average distance between the markers was 7.98 cM. The molecular marker linkage map could be used as the frame molecular linkage map of Ye478×90110 and used in the primary mapping of major genes and QTLs. The map provided the basis for the mapping of genes conferring resistance to MRDD and could also be used in the genetic improvement of the two parents and the inbred lines those derived from Ye478.The two parental lines and their F₂ population, BC₁ population and some recombinant inbred (RI) lines were evaluated for their resistance to MRDD by both inoculation assays and field assessments. The results of inoculation assays were in accordance with those of field assessments in these populations. In the F₂ population, 14.6% and 13.3% plants were diseased plants in the field tests of Laizhou area; 14.0% and 12.7% plants were diseased plants in the field tests of Jinan area and the incidence of inoculation tests was 14.0%, 14.0% and 18.0%, respectively for each replication. In the BC₁ population, the natural incidence was 36.0% and 35.3% in the field tests of Laizhou area, 33.3% and 31.3% in the field tests of Jinan area and 36.0%, 40.0% and 35.0% in the inoculation tests for each replication. The numbers of resistant plants and susceptible plants were counted in the F₂ and BC₁ populations, respectively. The results of Chi-square tests of the ratio of resistant plants to susceptible plants indicated that the resistance to MRDD in the combination of Ye478 × 90110 did not follow the simple genetic model controlled by one or two genes. The inoculation results of the 37 RI lines were in accordance with the results of the three-year field assessments. These not only confirmed the MRDD resistance of these RI lines, but also represented the repeatability of the inoculation assays.For bulk segregant analysis, equal amounts of DNA from each of 15 resistant F₂ plants and 10 severely diseased F₂ plants were pooled respectively to form the F₂ resistant bulk (B_RF₂) and susceptible bulk (B_sF₂). The BC₁ resistant bulk (B_rBC₁) and susceptible bulk (B_sBC₁) were prepared by mixing equal amounts of DNA from each of 15 resistant BC₁ plants and 15 severely diseased BC₁ plants, respectively. The SSR markers on the molecular linkage map were used to genotype the two parents and the two F₂ DNA pools. The markers which exhibited polymorphisms between the two F₂ pools were further used to detect the two BC₁ pools. The markers, which exhibited polymorphisms between the two BC₁ pools according with those between the two F₂ pools, were identified as the potential markers linked to the resistant loci. Four SSR markers possibly linked to the MRDD resistant loci were identified, which were umc1656 (bin6.02), umc1401 (bin7.02), bnlg1823 (bin8.07) and umc1268 (bin8.07). Thus there were probably three loci conferring resistance to MRDD in 90110 for the four markers were respectively located on chromosome 6, 7 and 8. The three loci were denoted as Mrdd1, Mrdd2 and Mrdd3, respectively.Based on the molecular marker linkage map, Five SSR markers umc1656, umc1006, umc1083, bnlg2191 and umc1595 from chromosome 6, four SSR markers umc1401, umc1695, umc1666 and umc2142 from chromosome 7 and five SSR markers bnlg1823, umc1268, umc1728, umc2014 and umc1032 from chromosome 8 were used to genotype an F₂ population consisting of 150 investigated individuals. The cosegregation analysis was independently performed in the F₂ population for each resistant locus. The SSR markers umc1656 was the closest marker linked to Mrdd1 locus. The Mrdd1 locus was mapped to a 4.5 cM region between the SSR markers umcl656 and bnlg2191. Among the four SSR markers from chromosome 7, umc1401 was the closest marker linked to Mrdd2 locus. The Mrdd2 locus was mapped to an 11.1 cM region between the SSR markers umc1401 and umc1666. Among the five SSR markers from chromosome 8, bnlg1823 was the closest marker linked to Mrdd3 locus. The Mrdd3 locus was mapped to a 5.8 cM region between the SSR markers bnlg1823 and umc1268.Eighteen resistant RI lines and 19 susceptible RI lines were genotyped with nine SSR markers umc1083, umc1656, bnlg2191, umc1695, umc1401, umc1666, umc2014, bnlgl823 and umcl268. The cosegregarion analysis was also performed using the data of the RI lines for each resistant locus independently. In the RI lines population, the Mrdd1 locus was mapped between the SSR markers umc1656 and bnlg2191, the Mrdd2 locus was mapped between umc1401 and umc1666 and the Mrdd3 locus was mapped between bnlg1823 and umc1268. These results were accordance with those in F₂ population and confirmed that Mrdd1, Mrdd2 and Mrdd3 conferred resistance to MRDD and derived from the resistant parent line 90110. In addition, the results from the data of RI lines implicated a line that inherited at least two resistant loci from resistant parent line 90110 could be resistant to MRDD.The primary mapping results indicate that the MRDD resistance is controlled by at least three genes at molecular level in the cross of Ye478 and 90110. The results lay the foundation for the fine mapping of resistant loci. The six SSR markers, which are on the two sides of the three resistant loci respectively, can be used in molecular assisted selection breeding for the resistance to MRDD.