CN101138313B - Breeding of Maize Inbred Lines Resistant to Rough Dwarf Disease Using Molecular Markers - Google Patents

Abstract

The invention discloses a method for breeding corn inbred lines resistant to rough dwarf disease by using molecular markers of corn rough dwarf disease resistant loci. Markers are used in maize rough dwarf resistance breeding and gene aggregation breeding. Using corn inbred lines resistant to rough dwarf disease and susceptible to rough dwarf disease as materials, segregating populations were constructed, and molecular markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823 and umc1268 etc. conducted assisted selection, and used plants with 3 or 2 disease resistance loci as selected materials for backcrossing and selfing, combined with comprehensive trait selection and selfing homozygosity, and obtained high-quality maize with high resistance to rough dwarf disease inbred line.

Description

Breeding of Maize Inbred Lines Resistant to Rough Dwarf Disease Using Molecular Markers

technical field

The invention belongs to the fields of corn breeding and plant bioengineering, and in particular relates to a method for breeding corn inbred lines resistant to rough dwarf disease by using molecular markers of corn rough dwarf disease resistance loci. the

Background technique

Research progress of maize rough dwarf disease:

1. Hazards and causes of rough dwarf disease of corn

Maize Rough Dwarf Disease (MRDD) is a viral disease that seriously endangers corn production. Symptoms of rough dwarf disease of corn are small transparent dotted dots between the fine veins on both sides of the midrib of the young leaves, and the transparent dots gradually increase, waxy white protrusions of different thicknesses appear on the veins on the back of the leaves, and there is obvious roughness when touched Infected plants are dwarfed, with shortened internodes; leaves are dark green; leaves are short, wide and thick, stiff and erect, often wrinkled; male and female ears are underdeveloped, and in severe cases, they cannot bear fruit. the

Corn rough dwarf disease was first discovered in Italy in 1949, and has since occurred to varying degrees in European countries such as France and South American countries such as Argentina. In 1954, rough dwarf disease of maize was first discovered in southern Xinjiang and western Gansu of my country. Since the 1970s, maize rough dwarf disease has caused large-scale destruction or extinction in parts of North China, Northwest and Northeast China, and has become an important disease of maize production. Up to now, there are 13 provinces and cities where maize rough dwarf disease has been reported in our country. According to incomplete statistics in 1996, the incidence area of maize rough dwarf disease in China was 2.33 million hm ² , with 40,000 hm ² of total crop failure. The general diseased plant rate in severe disease areas is about 40%, with an average of 10% to 30%. In 2006, rough dwarf disease of corn occurred in large areas in Shandong, Hebei and other provinces, causing great losses.

Maize rough dwarf virus (MRDV), Malde Rio Cuarto virus (MRCV) and rice black-streaked dwarf virus (RBSDV) are the three reported so far that can cause maize rough dwarf disease. pathogen. Among them, maize rough dwarf virus and MaldeRio Cuarto virus are the pathogens of maize rough dwarf disease in Europe and South America, respectively. All three viruses belong to group II of the genus Fijivirus in the Reoviridae family. The three are very similar in terms of virion morphology, host range, transmission media, serology and genome electrophoresis and sequence. They are all transmitted by planthoppers and are all confined to the phloem after infestation. In my country, there was a debate on the pathogen of maize rough dwarf disease. In 1981, Gong Zuxun observed with an electron microscope that the shape and size of the pathogen of maize rough dwarf disease in Baoding, Hebei Province were similar to the rice black-streaked dwarf virus that occurred in East China in the 1960s. The diameter of the complete virus was 70-75nm, and the diameter of the virus was 65nm without the shell. In 2001, according to the genome sequence information of virus isolates, Chinese scholars successively reported that the pathogen causing maize rough dwarf disease in China was rice black-streaked dwarf virus instead of maize rough dwarf virus. According to the biological and molecular biology comparison and identification results of the isolates of corn rough dwarf disease in Baoding, Hebei and Wuhan and the isolates of rice black-streaked dwarf disease in Zhejiang under the same conditions, it shows that their host range and relationship, main host symptoms, mediators Insects, disease-transmitting characteristics, and pathogenic forms are all the same, and the nucleotide and amino acid sequences of the three genome segments S7-S10 share 94.0%-99.0% and 96.3%-100% homology, respectively, and are closely related to Japanese RBSDV. Close, slightly distant relationship with the Italian MRDV. In 2003, Wang Chaohui and others completed the complete genome sequence determination and functional prediction analysis of RBSDV Hubei maize isolates, further proving that the pathogens of maize rough dwarf disease and rice black-streaked dwarf disease in my country are both RBSDV. the

In my country, RBSDV is mainly transmitted by the homopteran insect, Laodelphax striatellus Fallen. RBSDV is not transmitted by soil, seeds, pollen, grafting and friction of branches and leaves. SBPH is a temperate pest and is widely distributed in my country. It occurs more frequently in the winter wheat region of North China and the rice region of the middle and lower reaches of the Yangtze River. The optimum temperature for growth and development is 15-28°C. Its generation generation is restricted by geography and climate, and the number of generation algebra in a year gradually increases from 3 to 8 generations in my country from north to south. The hosts of SBPH are mainly grasses, and the most suitable hosts are rice, wheat and so on. Corn, millet, sorghum, etc. are unsuitable hosts, on which adults feed, but rarely lay eggs and hatch. The non-toxic insects of SBPH obtained the virus by sucking the juice on the plants infected with RBSDV, and the time required for obtaining the virus was the shortest 1 hour, and most of them were 24-48 hours. The minimum temperature required for poisoning is about 8°C. The circulation period of the virus in the worm is generally 3 to 35 days. Under optimum temperature conditions, the cycle period shortens as the temperature rises. When the average temperature is 24°C, the virus can be transmitted in the shortest 2 hours, and the virus can be fully transmitted in 48 hours. The avirulent SBPH can transmit the virus for life after being infected, but it cannot be passed on to the next generation through eggs. In the "wheat-corn" planting area, the first generation of SBPH on wheat plants is the main source of infection causing the epidemic of maize rough dwarf disease. The disease cycle process is as follows: in spring, the first generation of SBPH adults feeds on the overwintering host and acquires poison, then migrates to corn plants, and a migration peak is formed during wheat harvest, and corn rough dwarf disease occurs about 21 days after the migration peak peak. The 2nd, 3rd, and 4th generations of SBPH migrate to wheat fields and fieldside weeds, pass the virus and overwinter in wheat seedlings or grass. the

Large-scale planting of non-resistant or poorly resistant hybrids is an important reason for the prevalence of maize rough dwarf disease. Liu Zhizeng et al. identified the resistance to rough dwarf disease of 96 maize inbred lines and 136 hybrid combinations composed of these inbred lines. The results showed that most of the identified inbred lines were susceptible or moderately resistant, only 6 inbred lines were highly resistant, and no immune type was found. In the hybrid combination, most of the hybrids used for production show moderate or high sensitivity, such as Yedan 2, Yedan 12, Yedan 13, Yedan 19 and Xiyu 3, etc., which were born in the 1990s. The main cultivars in areas with a high incidence of corn rough dwarf disease in the 1990s. the

2. Identification of corn rough dwarf disease resistance and genetic analysis of resistance

There are many reports on the grading of corn rough dwarf disease severity, but the standards are not consistent, and there is a lack of unified disease severity grading standards and disease resistance evaluation standards, which reflects the complexity of corn rough dwarf disease symptoms. This has caused difficulties in the research on the mapping of disease resistance genes. the

In 1986, Chen Xunzhen et al. divided the severity of rough dwarf disease of maize into 0-4 grades based on plant height, male and female ear shape, loose powder condition, and leaf symptoms. In 1996, Liu Zhizeng and others divided the diseased plants into grades 0-3 based on plant height and leaf symptoms. In 1998, Zhang Huikong et al. conducted sample analysis on 256 corn rough dwarf diseased plants, and formulated a 6-level grading standard based on the loss of grain weight per plant. In 2005, Miao Hongqin formulated a 5-level grading standard based on the typical symptoms of the disease. In 2006, Lu Yingui measured the plant height and yield per plant of three maize varieties including Nongda 108, Zhengdan 958 and Deyu 18 infected with maize rough dwarf disease, and established the Maize rough dwarf severity grading standard, that is, when the ratio of the plant height of the detected plant to the plant height of the healthy plant is 1, 4/5, 2/3, 1/2, 1/3 respectively, the corresponding plant disease grades are as follows: 0, 1, 2, 3, 4 grades, and the yield loss rate of 0-4 grade plants were 0, 25%, 50%, 75% and 100% in turn. the

In recent years, some scholars have successively carried out the work of identifying the resistance of maize varieties to rough dwarf disease. These works are mainly identified through natural disease in the field, and there are also a small number of reports on artificial inoculation identification. Combined with the identification of rough dwarf disease resistance, research on the genetic law of maize rough dwarf disease resistance is also carried out at the same time. In 1995, Guo Qitang et al. identified the resistance to rough dwarf disease of maize in the field and found that there were obvious differences in resistance between varieties and inbred lines. No diseased plants were found in Taidanzao 12, 4.7% in Tai9101×Tai9102, 9.6% in Jinsui 47×D358, 11.8% in Jingdan 901, and 17% in A343×Dan340. 185 inbred lines were observed, among which 9 were highly resistant (incidence rate less than 10%): Ji 8, Zong 3, Zhonghuang 64, GY237, etc., and found that the disease resistance of selected lines from different germplasm materials The difference is very large, the disease-resistant lines of the descendants of the pioneer group and the subtropical group are 70% and 55% respectively, and the high-resistant materials account for more than 30%; %, while only 1 of the 19 derived lines of 5003 was disease-resistant. Through the analysis of the disease resistance of the hybrid combination prepared by the inbred line, it is concluded that the disease resistance of the inbred line has significant heritability to the offspring. Basso et al. analyzed the combining ability of maize rough dwarf disease resistance in four types of natural disease areas, and detected that the dominant effect and additive effect significantly restricted the performance of resistance, and the general combining ability effect of inbred lines accounted for a large proportion of genetic factors. Larger specific gravity. In 1996, Liu Zhizeng et al. identified 96 inbred lines and 136 hybrid combinations for rough dwarf resistance. The results showed that most of the inbred lines were susceptible or moderately resistant, and only 6 were highly resistant. The disease resistance of foreign germplasm is generally better than that of domestic germplasm. There are few disease-resistant varieties of hybrids for production, and most of them show moderate or high sensitivity. The disease resistance of hybrids was closely related to the disease resistance of their parents. Most of the disease resistance of the hybrids was between the parents, and there were few super-parents. It was speculated that the resistance to maize rough dwarf disease was a quantitative trait. Lu Yinggui et al. conducted a natural identification of rough dwarf disease resistance in Baoding, Hebei Province on 78 corn rough dwarf resistant recombinant inbred lines imported from the United States and 69 domestically bred maize inbred lines. The results showed that among the imported materials, 2 samples showed high resistance, 6 samples showed high resistance, and 5 samples showed moderate resistance, showing good resistance to rough dwarf disease. There were no high-resistance materials among the 69 domestic inbred lines; 178, P138, 901141, 9138, Shen 137 were resistant, Qi 319, 87-1, Zong 3, Ji 35, Huotang and 2379 were moderately resistant. The test results also showed that the inbred line selected from the American 78599 line performed well in resistance. Wang Anle et al. analyzed the effect of directional recurrent selection on the improvement of maize resistance to rough dwarf disease by using 6 inbred lines. large proportion. the

For viral diseases such as maize rough dwarf disease, the occurrence of the disease is obviously different in different years and different regions, and it is difficult to naturally identify the disease resistance of plants. Therefore, it is necessary to establish artificial inoculation identification technology for maize rough dwarf disease. Reliable artificial inoculation technology can ensure the continuity and repeatability of the identification of the pathogen and disease resistance of maize rough dwarf disease. In 1998, Wang Anle et al. used net sheds to control the virus transmission of SBPH, and identified the resistance of 50 corn inbred lines to rough dwarf disease. The disease incidence of each material was investigated with the plant height as a parameter, and the disease resistance index was calculated. The result is more stable and reliable than natural disease identification. In 2005, Di Dianping and others carried out group inoculation of cages with poisonous SBPH. The results show that this inoculation method combined with plant transplantation can realize large-scale and large-scale artificial inoculation. the

The use of disease-resistant varieties is a time-saving, labor-saving, cost-effective and efficient method for preventing and controlling viral diseases, and avoids the pollution of the environment by pesticides. Therefore, it is of great significance to screen and breed inbred lines and hybrids with high resistance to maize rough dwarf disease from maize germplasm resources. In recent years, maize breeders in my country have carried out resistance source screening and identified some inbred lines with high resistance to maize rough dwarf disease, such as P20, P138, Qi 319 and so on. Breeders from the Hebei Academy of Agricultural Sciences used 78599 from the United States as a material to breed an inbred line 90110 with high resistance to corn rough dwarf disease. Utilizing the existing disease-resistant germplasm resources to cultivate high-yield and disease-resistant inbred lines and hybrids is an urgent problem in agricultural production in my country. However, the traditional breeding method not only takes a long time, requires a lot of manpower and material resources, but also is subject to factors such as difficulties in identifying disease resistance, and the results are slow. Molecular marker positioning of disease-resistant genes by means of molecular biology can not only deeply study the genetic rules and mechanism of disease resistance of rough dwarf disease, lay the foundation for the cloning of disease-resistant genes, but also greatly benefit from molecular marker-assisted selection. Shorten the breeding time and improve the breeding efficiency. the

3. Mapping of maize rough dwarf disease resistance genes

Maize disease is an important factor affecting maize production. In the past ten years, a lot of work has been done on the use of molecular markers to mark and locate disease-resistant genes. At present, most of the resistance genes or QTL mapping studies of important foreign corn diseases have been reported, such as large leaf spot, small leaf spot, wilt, gray leaf spot, common rust, southern rust, ear rot, and head smut. corn mosaic, sugarcane mosaic, corn mosaic, corn stripe, corn yellow dwarf, corn spot and high altitude virus. Chinese scholars have located a number of disease-resistant genes using domestic maize materials, such as maize dwarf mosaic disease, large spot disease, sheath blight, southern rust and stem rot, etc., but research on maize rough dwarf disease resistance genes There are no reports at home and abroad. the

According to the report on the mapping of disease resistance genes, some problems can be found: 1) different mapping results may be obtained by using different hybrid combinations; 2) different positioning results may be obtained when identifying disease resistance in different environments; 3) different The QTL mapping population type may affect the QTL mapping results; 4) The identification of disease resistance is a crucial factor affecting gene mapping. It is difficult to accurately identify the resistance of maize plants to rough dwarf disease. The artificial inoculation identification system of maize rough dwarf disease established by the applicant's laboratory and the fluorescent quantitative RT-PCR detection technology of the pathogen can accurately and reliably identify whether the plants in the isolated population are infected or not. the

In our laboratory, the F ₂ population, BC ₁ population and partial recombinant inbred lines obtained by crossing the high resistance inbred line 90110 and the high sensitivity inbred line Ye 478 were tested by means of artificial inoculation identification and field natural identification. The population was identified for disease resistance, and the identification results with good repeatability were obtained, which laid a solid foundation for the positioning of resistance genes. In order to quickly find the molecular markers linked to the maize rough dwarf disease resistance gene, the applicant carried out SSR-BSA analysis. The DNA of 15 F2 disease-resistant plants is equally mixed to form the F2 resistant pool, and the DNA of 10 typical F2 susceptible plants is equally mixed to form the F2 susceptible pool; the DNA of 15 BC ₁ disease-resistant plants is equally mixed to form the BC ₁ resistant pool, the DNA of 15 typical susceptible plants of BC ₁ was mixed in equal amounts to form the BC ₁ susceptible pool. The F ₂ resistant and F ₂ sensitive pools were detected with SSR markers to construct molecular marker linkage maps, and then the BC ₁ resistant and sensitive pools were detected with SSR markers showing polymorphism between the F ₂ resistant and F ₂ sensitive pools. As for the polymorphism between the resistant pool and the sensitive pool of BC ₁ which was consistent with the polymorphism between the resistant pool and the sensitive pool of F ₂ , it was preliminarily determined that the marker might be linked with the resistance gene. Results There were 4 SSR markers showing polymorphisms between the resistant and sensitive pools of F ₂ and between the resistant and sensitive pools of BC ₁ , and the polymorphism between the resistant and sensitive pools of BC ₁ was different from that in The polymorphism between the resistant pool and the sensitive pool of ₂ is consistent. These four markers are: umc1656 (bin6.02 region) on chromosome 6, umc1401 (bin7.02 region) on chromosome 7, bnlg1823 (bin8.07 region) and umc1268 (bin8.07 region) on chromosome 8 region), where bnlg1823 and umc1268 are adjacent markers. That is, the maize rough dwarf disease resistance locus (gene) Mrdd1 was mapped to the bin6.02 region, the Mrdd2 locus was positioned to the bin7.02 region, and the Mrdd3 was positioned to the bin8.07 region. Since the four markers were located on chromosomes 6, 7, and 8, further analysis showed that at least three maize rough dwarf resistance loci, Mrdd1, Mrdd2, and Mrdd3, might exist in the inbred line 90110.

On the basis of the above work, the genotypes of 150 F ₂ individual plants identified for disease resistance were detected. By comparing the co-segregation ratio between the resistance loci and each SSR marker, the three resistance loci were mapped to the chromosome. When locating the Mrdd1 locus, the genotypes of the 21 _F2 individual plants at the Mrdd1 locus did not match the plant phenotypes (that is, the plants should be judged to be susceptible based on the genotypes at this locus, but the plants showed obvious resistance. disease resistance, this is because the resistance to maize rough dwarf disease is determined by polygenes, and it is likely that the resistance genes at other sites determine the disease resistance of the plant) were eliminated, and the data of a total of 129 F ₂ single plants were used for Co-segregation analysis of the Mrdd1 locus and its nearby markers (Table 1). Among the five SSR markers selected on chromosome 6, umc1656 was the closest marker to Mrdd1, and the co-segregation ratio between them was 98.1%. According to the co-segregation ratio between the Mrdd1 locus and the five SSR markers, the Mrdd1 locus was located between the markers umc1656 and bnlg2191, and the genetic distance between umc1656 and bnlg2191 in the _F2 molecular marker linkage map was 4.5cM.

Table 1. Co-segregation ^analysis of Mrdd1 locus in _F2 populationa

a Five SSR markers near the Mrdd1 site were ^selected to detect 150 F ₂ individuals, and the data of 129 F ₂ individuals were used for co-segregation analysis.

When locating the Mrdd2 site, the data of 24 F ₂ individual plants were eliminated because their genotypes at the Mrdd2 site did not match the plant phenotype, and a total of 126 F ₂ individual plant data were used for the markers at the Mrdd2 site and its vicinity Co-segregation analysis (Table 2). Among the four selected SSR markers on chromosome 7, umc1401 was the closest marker to Mrdd2, and the co-segregation ratio between them was 94.5%. According to the co-segregation ratio between the Mrdd2 locus and these four SSR markers, the Mrdd2 locus was located between the markers umc1401 and umc1666, and the genetic distance between umc1401 and umc1666 in the _F2 molecular marker linkage map was 11.1cM.

Table 2. Co-segregation ^analysis of Mrdd2 locus in _F2 populationa

^a Four SSR markers near the Mrdd2 site were selected to detect 150 F ₂ individuals, and the data of 126 F ₂ individuals were used for co-segregation analysis.

When locating the Mrdd3 site, the data of 24 F ₂ individual plants were eliminated because their genotype at the Mrdd3 site did not match the plant phenotype. The data of a total of 126 F ₂ individuals were used for the co-segregation analysis of the Mrdd3 locus and its nearby markers (Table 3). Among the five selected SSR markers on chromosome 8, bnlg1823 was the closest marker to Mrdd3, and the co-segregation ratio between them was 97.8%. According to the co-segregation ratio between the Mrdd3 locus and these five SSR markers, the Mrdd3 locus was located between the markers bnlg1823 and umc1268, and the genetic distance between bnlg1823 and umc1268 in the _F2 molecular marker linkage map was 5.8cM.

Table 3. Co-segregation analysis of Mrdd3 locus in _F2 population ^a

a Five SSR markers near the Mrdd3 site were ^selected to detect 150 F ₂ individuals, and the data of 126 F ₂ individuals were used for co-segregation analysis.

4. Research status of crop molecular marker-assisted breeding

In crop breeding, the most effective selection method is to select directly based on the genotype of the plant, and the emergence of molecular markers provides the possibility for this direct selection. Molecular marker-assisted selection (MAS) can accurately and stably select the target gene in the early generation, so as to accelerate the breeding process and improve the breeding efficiency. The key to this technology is the discovery and high-throughput detection of DNA molecular markers closely linked to important agronomic traits. In recent years, scholars from various countries have identified a series of molecular markers of agronomic traits of rice, wheat, corn, cotton, soybean and other crops, which laid the foundation for MAS. Wu Weiren et al., Moreau et al., Berloo et al., and Hospital et al. published several research papers respectively explaining the methods and influencing factors of MAS, which provided theoretical support for the implementation of MAS. MAS includes the tracking of target genes, that is, foreground selection (foreground selection) and the selection of genetic background (background selection). The reliability of foreground selection mainly depends on the degree of linkage tightness between the marker and the target gene. If only one marker is used to select the target gene, the recombination rate between the marker and the target gene should be less than 5% when the correct rate of selection is required to reach more than 90%. If the target gene is tracked and selected with adjacent markers on both sides, the correct rate of selection can be greatly improved. Background selection can speed up the recovery of genetic background, shorten the breeding period and reduce linkage burden. Through computer simulation analysis, Tanksley et al. concluded that it only takes 3 generations to restore the hybrid to the genotype close to the recurrent parent by using MAS. At present, MAS has been applied in the breeding practice of rice, wheat, corn and other major crops and has made great progress, mainly including gene transfer, gene aggregation and quantitative trait MAS. The International Rice Research Institute used four near-isogenic lines containing different rice bacterial blight resistance genes (xa4, xa5, xa13, xa21) to carry out aggregation of resistance genes, and the resistance genes accumulated in the lines produced were more than those containing single resistance genes. Lines with sex genes have a higher level of resistance and a broader spectrum of resistance to pathogenic bacteria. Using IRBB21 as a donor material, Chen Sheng et al. used 4 molecular markers closely linked to Xa21 to carry out molecular marker-assisted improvement of 'Minghui 63', and obtained 'Minghui 63' resistant to bacterial blight. Xue Qingzhong et al., Huang Tingyou et al., Peng Yingcai et al., Tong Haijun et al., Cao Liyong et al., Luo Yanchang et al. introduced the gene into different rice parents through hybridization and backcrossing, combined with molecular marker-assisted selection, and bred a batch of resistant rice parents. The new restorer line and sterile line of bacterial blight were further combined to breed new disease-resistant hybrid rice combinations such as Xieyou 218, Zhongyou 218, IIyou 8220, and Guodao 1. In addition, some progress has been made in rice insect resistance and rice blast resistance. Relatively little work has been done on molecular marker-assisted selection in maize, but there has been considerable success. American scholars transferred some yield-related quantitative trait loci of maize to inbred lines such as B73 and Mo17, and the yield of hybrids assembled from these inbred lines increased by more than 15% compared with control hybrids. Zhang Shihuang et al. used molecular markers closely linked to the opaque-2 gene to carry out molecular marker-assisted selection of high-quality protein maize, and established a new economical and practical molecular marker-assisted breeding system for high-quality protein maize. A large number of theoretical studies and breeding practices have proved that molecular marker-assisted selection is the development direction of crop breeding technology. the

Generally speaking, the effect of molecular marker-assisted selection breeding on those complex traits controlled by multiple genes and heavily influenced by the environment is more obvious than the selection effect on quality traits controlled by single genes that are less affected by environmental factors. Maize rough dwarf disease is a complex trait that is difficult to identify for disease resistance. Conventional breeding methods, whether through natural disease identification in the field or through artificial inoculation to identify disease resistance, are important in breeding rough dwarf disease-resistant inbred lines and hybrids. It is time-consuming and labor-intensive with poor results. The application of molecular marker-assisted selection is expected to improve the efficiency of rough dwarf disease resistance breeding. So far, there is no report on the breeding of maize inbred lines resistant to rough dwarf disease by molecular marker-assisted selection. the

Contents of the invention

The present invention relies on 2 basic research works: 1) The Chinese corn inbred line Ye 478, which is highly susceptible to corn rough dwarf disease, is used as the female parent, and the inbred line 90110, which is highly resistant to corn rough dwarf disease, is used as the male parent to construct the map For the population, 150 individual plants of F ₂ were selected to construct a molecular marker linkage map. Among the 278 polymorphic markers, 271 markers were divided into 10 linkage groups. The map covers the maize genome (total length) 2164.3cM, and the average distance between markers is 7.98cM, forming a molecular marker linkage frame map of 478×90110 inbred lines, which is suitable for the mapping of major genes and QTL in maize. In this work, in order to accurately judge the resistance of rough dwarf disease of maize plants, the developmental period (leaf age), inoculated population density and inoculation duration of maize were studied respectively by using the net room population inoculation method to identify the rough dwarf disease artificial inoculation. The pathogen was identified by ELISA and real-time RT-PCR, the virus content in different plants was analyzed, and a stable artificial inoculation identification system for corn rough dwarf disease was established. Combined with the natural identification results in the field, the mapping population The disease resistance of the F ₂ single plant was reliably identified. 2) Using the F ₂ population and BC ₁ population constructed by crossing the inbred line Ye 478 and the inbred line 90110 as the mapping population, the disease resistance locus Mrdd1 locus was located in the 4.5cM region between umc1656 and bnlg2191 of bin6.02 Inside, the Mrdd2 site was located in the 11.1cM region between umc1401 and umc1666 in bin7.02, and the Mrdd3 site was located in the 5.8cM region between bnlg1823 and umc1268 in bin8.07, and the recombinant inbred line population was used verified. In addition, through association analysis, it was determined that the genetic locus of resistance to rough dwarf disease in the population of "Ye 478×90110" came from 90110.

Plant material of the present invention

Maize rough dwarf high resistance inbred line 90110, susceptible inbred lines Ye 478, 488 and their derived inbred lines, DH4866, DH9942, Ye 502, Ye 515, Ji 853, Zheng 58, etc. are susceptible to corn rough dwarf disease Excellent corn inbred lines and intermediate breeding materials, F _2-6 plants and F _2-4 BC _1-3 plants constructed from corn inbred lines susceptible to rough dwarf disease and inbred line 90110. F _2-6 plants represent the plants of the corn inbred line susceptible to rough dwarf disease and the high resistance inbred line 90110 after selfing for 2 to 6 generations, and the plants of F _2-4 BC _1-3 represent the corn inbred line susceptible to rough dwarf disease After crossing with the high-resistant inbred line 90110, the number of self-generations is 2-4, and the plants are backcrossed with the inbred line susceptible to rough dwarf disease (recurrent parent) for 1-3 generations. When obtaining hybrid F1, both 90110 and the inbred line with rough dwarfism can be used as female parent or male parent. In backcrossing, the inbred line with rough shrinkage disease can be used as the male parent or the female parent.

Identification of plant disease resistance

The identification of plant disease resistance adopts the strategy of combining artificial inoculation identification and natural disease identification in the field. The method reported by Wang Fei et al. (2006) was used for manual inoculation identification, that is, 15 SBPHs per plant were inoculated into corn plants at the second-leaf stage, and the inoculation period was 5 days. The source of the virus was a typical corn rough dwarf diseased plant in the field. Fifteen strains were inoculated and identified for each genotype, with 3 replicates. The disease was investigated during the flowering stage of the plants. Natural disease identification materials are generally sown twice in the first and middle days of May, and there are many weeds around the experimental site. In the identification of disease resistance, the inbred line 90110 was used as the control for disease resistance, and Ye 478 was used as the control for susceptibility to rough dwarf disease. In field natural identification, generally 30 plants of each genotype are planted for each repetition. Set up the test area according to random block arrangement. The row length is 4.0m, 15 plants are planted, and the row spacing is 75cm. No pesticides are used in the test area, weeds are not removed, and water and fertilizer management is carried out as usual. Incidence was investigated during the flowering period. The lines with disease incidence between 0% and 10.0% are resistant materials, and the inbred lines with disease incidence between 10.1% and 100% are susceptible inbred lines. Real-time RT-PCR can be used to identify pathogens, analyze the virus content of different plants, and determine the identification results. Fluorescent quantitative RT-PCR was used to detect the virus content, and the RNA was extracted from the uppermost fully developed leaf of the plant, which was extracted with TRIZOL kit. Reverse transcription and quantitative RT-PCR were performed according to the procedures and methods reported by Wang Fei et al. (Wang Fei et al., 2006). the

SSR markers and primer sequences of maize rough dwarf resistance loci

The SSR markers of Mrdd1 on chromosome 6 were umc1656, bnlg2191 and umc1656, the SSR markers of Mrdd2 on chromosome 7 were umc1401 and umc1666, and the SSR markers of Mrdd3 on chromosome 8 were bnlg1823 and umc1268. the

Table 4. SSR marker primer sequences used in the detection of maize rough dwarf disease resistance loci

SSR mark Primer sequence umc1656 AGTTTTGACCGCGCAAAAGTTAGTACGAGCAGGCCATTAACCC bnlg2191 CAGGTGGTGCAGAGTTTCACAT AAGGTGGAGGATGACTCCAAGAT

umc1595 GCTGCTGGTCTACAACCTCTTGTTCGCTTGAAATGGAAAGGTAGAAAG umc1401 CTCTGGTCCATCCTCATCGACT TCTCTTGATCACATATCGATCCCA umc1666 TTATTGCCCTCCCTGTTCTTGTTACCTTGACGCAGCAATCCTC bnlg1823 TGTGACTCCATACCGCACATCTCATCATGTTGTACATGGCG umc1268 ACGAACAAACCTAGCACAGTCCTAAACAAGGCGGTTACCAAGTTTACATC

SSR primers were synthesized according to the primer sequences provided by MaizeGDB (Table 4). the

Molecular marker detection

Take an appropriate amount of leaves from plants at the 3-leaf stage or the filling stage, and use the CTAB method to extract DNA. the

The SSR reaction system is: 10×PCR buffer (without Mg ²⁺ ) 1.0μl, MgCl ₂ (25mM) 0.6μl, primer I (50ng/μl) 0.7μl, primer II (50ng/μl) 0.7μl, dNTP ( 10mM each) 0.1μl, Taq DNA polymerase (5U/μl) 0.06μl, DNA template (20ng/μl) 2.5μl, sterile double distilled water 4.34μl, total 10μl.

SSR reaction conditions: The PCR reaction was carried out on a Biometra 96-well gradient PCR instrument, and the program was: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing at 65°C for 30 seconds; extension at 72°C for 1 minute; Each cycle annealing temperature decreased by 1°C (down to 55°C), a total of 11 cycles; followed by: denaturation at 95°C, 30 seconds; annealing at 55°C, 30 seconds; extension at 72°C, 1 minute; 30 cycles; overextension at 72°C ,8 minutes. the

The SSR amplification products were separated by electrophoresis on 6% polyacrylamide gel (acrylamide:methylenebisacrylamide=19:1), and detected by silver staining. the

Selection of rough dwarf disease resistant individuals from segregating populations

The selection of target genes is called prospect selection. The reliability of foreground selection mainly depends on the tight linkage between the marker and the target gene. If only one marker is used to select the target gene, the linkage between the marker and the target gene must be very close to achieve a high accuracy rate. Assuming that a marker locus (M/m) is linked to a target gene locus (Q/q), the recombination rate is r. The probability of obtaining the target genotype Q/Q (that is, the correct rate of single plant selection) by selecting the marker genotype M/M in the F ₂ generation is: p=(1-r) ² . According to this formula, the correct rate of selection decreases rapidly with the increase of recombination rate. If the correct rate of selection is required to reach more than 90%, the recombination rate between the molecular marker and the target gene should be less than or equal to 5%. If two adjacent markers are used to select the target gene at the same time, the correct rate of selection can be greatly improved. Assuming that there are two marker loci (M1/ml and M2/m2) located on both sides of the target gene locus (Q/q), the recombination rates with the target gene are r1 and r2, respectively, and the genotype of the F1 generation is M1QM2/m1qm2 . Then, there are two types of gametes produced by F1 with the marker genotype M1M2, one contains the target allele (M1QM2), which is the parental type, and the other contains the non-target allele type (M1qM2), which is the double crossover type . Due to the low probability of double crossover, the proportion of double crossover gametes is small. Therefore, the correct rate of selecting the target allele Q by simultaneously tracking M1 and M2 in the offspring must be high. In the case of no interference between single crossovers, the probability of obtaining the target genotype Q/Q by selecting the marker genotype M1M2/M1M2 in the F2 generation is: p=(1-r ₁ ) ² (1-r ₂ ) ² / [(1-r ₁ )(1-r ₂ )+r ₁ r ₂ ]. From this formula, it can be known that when the distance between the two markers is fixed, r ₁ =r ₂ (that is, the target gene is exactly located at the midpoint between the two markers) is the worst case, and the selection accuracy at this time is minimum. In the present invention, the distance between the SSR markers umc1656 and bnlg2191 on both sides of Mrdd1 is 4.5 cM; the distance between umc1401 and umc1666 on both sides of Mrdd2 is 11.1 cM; between bnlg1823 and umc1268 on both sides of Mrdd3 The image distance is 5.8cM. Use these 6 markers to select Mrdd1, Mrdd2 and Mrdd3 respectively, and the respective minimum selection correct rates of obtaining disease-resistant homozygous genotypes are about 95.5%, 88.9% and 94.2%; obtaining two disease-resistant loci is pure The minimum selection correct rates of the syngenotypes were 84.9% (Mrdd1 and Mrdd2), 90.0% (Mrdd1 and Mrdd3) and 83.7% (Mrdd2 and Mrdd3). In practice, there is always mutual interference between single crossovers, which makes the probability of double crossovers smaller, so the correct rate of double marker selection is higher than the above minimum theoretical expectation. In addition, recombinant inbred lines or plants that inherit any two resistance loci from 90110 exhibit resistance to maize rough dwarf disease under normal disease conditions.

In the present invention, 3 rough dwarf resistance sites of maize are located on chromosome 6, 7, and 8 respectively, and the individual ratio with 3 or 2 resistance sites in the segregation population varies due to the type of the segregation population. Variations, therefore, in the use of molecular marker-assisted selection in breeding maize inbred lines resistant to rough dwarf disease also depend on the technical route chosen. According to the law of genetic segregation, if one only wants to improve the rough dwarf disease resistance of an elite inbred line, the BC ₁ F ₁ population can be used as the initial selection generation, and the proportion of individuals with three heterozygous resistance loci is 12.5%, and the proportion of individuals with 2 heterozygous resistance loci was 37.5%. Then continuously backcross for 3 generations, if each generation uses individuals with 3 heterozygous resistance sites as parents, and uses 6 SSR markers on both sides of the 3 resistance sites to detect simultaneously, the BC ₄ F ₁ plants It has been basically the same as the recurrent parent (contains 96.9% of the genetic material of the recurrent parent), and then selfed for 1 generation, and the BC ₄ F ₂ plants were strictly identified for disease resistance, and 3 rough dwarf disease resistance loci were selected from them A high combining ability inbred line with excellent comprehensive traits. If you want to improve other traits of an excellent inbred line besides improving the resistance to rough dwarf disease, you can choose F ₂ as the initial selection generation, and there are 3 homozygous or heterozygous in the F ₂ population. Individuals with resistant loci were 42%. Then, using individuals with 3 resistance loci as parents, backcross continuously for 2 to 3 generations, and then self-cross for 1 to 2 generations to obtain new excellent inbred lines that meet the breeding objectives. In this process, the individuals with 3 resistance sites in the BC ₁ F ₂ population>12.5%; the individuals with 3 resistance sites in the BC ₃ F ₂ population were also>12.5%, and the selected plants The average proportion of genetic material of recurrent parents was 87.5%; in the BC ₄ F ₂ population, individuals with 3 resistance loci were also >12.5%, and the average proportion of genetic material of recurrent parents of selected plants was 93.8%. In order to maintain the high combining ability of the recurrent parents, 3 to 4 generations of backcrossing were generally carried out between the hybrid progenies of the susceptible inbred line and 90110, and each generation was complemented by molecular marker-assisted selection.

Since there are 3 resistance loci to maize rough dwarf disease, two procedures can be used for molecular marker-assisted selection: 1) Only individuals with 3 resistance loci can be selected; 2) individuals with 2 resistance loci can be selected Individuals at the locus crossed different selected materials when the plant traits were close to the recurrent parents, so as to obtain rough dwarf resistant materials with 3 resistance sites. the

Generally speaking, by using these 6 SSR markers for genotype selection, excellent individuals with high resistance to rough dwarf disease (with 3 resistance loci) can be selected in each generation, and the workload is not large. the

Basic operation process:

1) Take the excellent maize inbred line susceptible to rough dwarf disease as the recurrent parent, 90110 as the donor parent of the resistance gene, cross and backcross or self-cross, and establish segregation populations (such as BC ₁ F ₁ , BC ₁ F ₂ , BC ₁ F ₃ , BC ₂ F ₂ , BC ₁ F ₁ ×BC ₁ F ₁ , BC ₁ F ₁ ×BC ₁ F ₂ etc.).

2) Plant the seeds of the segregated population. When the seedlings are at the 3-leaf stage, extract the leaf DNA of a single plant as a template, and use the primers of SSR markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823 and umc1268 to carry out PCR amplification, and the reaction products are subjected to 6 % polyacrylamide gel electrophoresis separation, silver staining method to detect the band pattern of the amplified product, and select individuals with 3 disease-resistant sites or 2 disease-resistant sites to be retained.

3) After the selected plants grow up, carry out bagging backcrossing or selfing according to the breeding program, and optimize according to the comprehensive traits of the plants. The selected plant seeds are sown in pots or fields for next-generation molecular marker-assisted selection and plant selection. the

4) Continue planting and molecular marker-assisted selection and plant optimization for the offspring of the optimized plants until the plant traits are no longer significantly separated. Then self-cross for 1-2 generations to obtain excellent inbred lines with high resistance to rough dwarf disease. the

5) For selected materials with 2 disease-resistant loci, if one of them is different, 3 disease-resistant loci can be aggregated by mutual hybridization. Then, individuals with 3 disease-resistant loci were obtained from the hybrid offspring by molecular marker-assisted selection, and then the latter were selfed for 1 to 2 generations to obtain an excellent inbred line with high resistance to rough dwarf disease. the

6) Carry out artificial inoculation and natural disease identification in the field for lines with basically stable traits, optimize agronomic traits and measure combining ability, and determine the application value of excellent inbred lines with high resistance to corn rough dwarf disease. the

If the selected target inbred line has the same traits as the recurrent inbred line (susceptible excellent inbred line) except for the resistance to corn rough dwarf disease, the continuous backcross breeding strategy can be adopted from the BC ₁ F ₁ population Improved inbred lines. Molecular marker-assisted selection was performed in each backcross generation. In this way, starting from hybridization, the expected goal can be achieved generally through 5 to 6 generations.

The genetic background of different cross combinations will have a certain impact on the effect of molecular marker-assisted selection. In different cross combinations, that is, the molecular markers of genes under different genetic backgrounds will be different. Therefore, when multiple cross combinations are formed, it is advisable that the donors of the target gene are all selected from the inbred line 90110. In this way, the molecular markers that have been found are generally closely linked to the target gene, and can be applied to select different segregated populations of the same target gene. In small segregating populations, molecular markers near these SSR markers can be used if there is a change in the availability of some molecular markers. In the present invention, the operation scheme is determined on the premise of maximally ensuring the availability of existing molecular markers. the

Description of drawings

Figure 1 Phenotype of susceptible maize plants. the

Figure 2 BSA analysis of SSR markers umc1656, umc1401, bnlg1823 and umc1268. the

The samples in lanes 1-9 are: 90110, Tuck 478, F ₁ , F ₂ resistant pool, F ₂ sensitive pool, 90110, Tuck 478, BC ₁ resistant pool and BC ₁ sensitive pool.

Detailed ways

Example 1: Breeding Ye 478 excellent improved line resistant to corn rough dwarf disease

Using Ye 478 or its improved line as the recipient and recurrent parent, and the inbred line 90110 as the donor of the disease resistance gene locus, excellent improved lines were obtained mainly through the prospect selection of the target gene. the

Procedure one:

1) Hybridization was carried out with Ye 478 as the female parent and 90110 as the male parent to produce F ₁ plants. This step can be completed in the greenhouse or in Hainan from January to April of the first year.

2) F ₁ plants selfed to produce F ₂ seeds. This step is completed in northern my country from May to August of the first year.

3) F2 seeds were sown in flower pots, leaf DNA was extracted at the 3-leaf stage of the plants, PCR amplification was performed with primers of SSR markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823 and umc1268 respectively, and the reaction products were subjected to 6% polyacrylamide Gel electrophoresis separation, silver staining method to detect the band pattern of the amplified product, and 20-30 individuals with 3 disease-resistant loci were selected and transplanted into the field. This step can be completed in northern my country or Hainan in September to October of the first year. the

4) The selected plants were bagged and backcrossed with Ye 478 after tasseling, and field observation and records were strictly carried out, and inferior plants were eliminated according to the comprehensive traits of the plants before harvesting. After the ears are harvested, the seeds are tested and optimized. Generally keep 10 to 15 ears. This step can be done in the greenhouse or Hainan from October of the first year to January of the second year. the

5) Sow the remaining different BC ₁ F ₂ seeds in flowerpots for molecular marker detection (same as the F ₂ generation), and then transplant the selected plants into the field for growth. After the plants tassel, they are bagged and backcrossed with Ye 478 to produce BC ₂ F ₂ seeds. Strict selection of plants and ears. Generally, 4 to 5 fruit ears of plants with 3 disease-resistant sites are kept, or 10 fruit ears with 3 or 2 disease-resistant sites are reserved. If there are many types of plants, you can choose to keep more fruit ears. This step is completed in northern my country from March to June of the second year. The plants at the seedling stage are best grown in a greenhouse, and transplanted to a plastic greenhouse after testing.

6) Sowing BC ₂ F ₂ seeds in flower pots for molecular marker-assisted selection (same as F ₂ generation) and plant selection. The selected plants were backcrossed with Tuck 478 bagging to produce BC ₃ F ₂ seeds. Generally, 4 to 5 fruit ears of plants with 3 disease-resistant sites are kept, or 10 fruit ears with 3 or 2 disease-resistant sites are reserved. This step can be completed in northern my country from July to October of the second year.

7) The BC ₃ F ₂ seeds were sown in the field to continue molecular marker-assisted selection (same as the F ₂ generation), and the comprehensive traits of the selected lines were selected, and after the plants tasseled, they were bagged and backcrossed with Ye 478 to produce BC ₄ F ₂ seeds. Generally, the number of retained ears is similar to that of the previous generation. The traits of the plants in the lines of this generation are neat, but there may be obvious differences among the lines. At the same time, some plants of the selected lines were crossed with excellent inbred lines (including inbred lines with excellent heterosis crossed with recurrent parents), and hybrid seeds were prepared for the determination of combining ability. This step can be completed in the greenhouse or Hainan from November to March of the second year.

8) When performing step 6) or 7), the selected materials with 2 disease resistance sites can be hybridized. The selected plants used for crossing are different in one disease resistance site, and the traits of each other are preferably not much different. Three disease-resistant loci can be aggregated into one genotype through hybridization, and the plant traits change slightly. Then, individuals with 3 disease-resistant loci were obtained from the hybrid offspring by molecular marker-assisted selection. the

9) Selfing the plants with 3 disease-resistant loci for 1 to 2 generations to obtain inbred lines homozygous for the disease-resistant loci, and conducting strict identification of disease resistance, selection of comprehensive traits and determination of combining ability on the plants, An excellent inbred line with high resistance to rough dwarf disease was bred in line with the breeding objective. This step is carried out in northern my country from April to September in the third year, and can also be repeated in northern my country from April to September in the fourth year. the

In this program, multigeneration is carried out through greenhouse and Hainan planting, and comprehensive trait selection and combining ability determination are the same as conventional breeding. The selected inbred lines are highly resistant to rough dwarf disease, and may be different from recurrent parents in some traits. the

Procedure two:

1) F ₁ plants were produced by crossing Ye 478 as the female parent and 90110 as the male parent. This step can be completed in the greenhouse or in Hainan from January to April of the first year.

2) F ₁ plants were backcrossed with Ye 478 to produce BC ₁ F ₁ seeds. This step is completed in northern my country from May to August of the first year.

3) BC1F1 seeds were sown in flowerpots, leaf DNA was extracted at the 3-leaf stage of the plants, PCR amplification was performed with primers of SSR markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823 and umc1268, and the reaction products were subjected to 6% polyacrylamide Gel electrophoresis separation, silver staining method to detect the band pattern of the amplified product, and 6 individuals with 3 disease-resistant loci were selected and transplanted into the field. This step can be completed in northern my country from September to October of the first year. the

4) The selected plants were bagged and backcrossed with Ye 478 after tasseling, and strictly observed and recorded in the field, and the inferior plants were eliminated according to the comprehensive traits of the plants before harvesting. After the ears are harvested, the seeds are tested and optimized. Generally keep about 3 ears. This step can be done in the greenhouse or Hainan from October of the first year to February of the second year.

5) The selected BC ₂ F ₁ seeds from different ears were sown in flower pots for molecular marker-assisted selection (same as BC1F1 generation), and then the selected plants (about 5-8 plants) were transplanted to the field for growth, and the plants tasseled Then backcross with Tuck 478 to produce BC ₃ F ₁ seeds. And carry out plant and ear selection. Generally, 3 to 5 ears of plants with 3 disease-resistant sites are reserved. This step is completed in northern my country from March to June of the second year. The plants at the seedling stage grow best in the greenhouse, and then transplanted in the plastic greenhouse.

6) Sowing BC ₃ F ₁ seeds in flowerpots for molecular marker-assisted selection (same as BC1F1 generation) and plant selection. The selected plants were backcrossed with Ye 478 to produce BC ₄ F ₁ seeds. The plant traits of the same line in this generation are basically uniform, and seeds for combining ability determination can be prepared at the same time (the selected inbred lines and recurrent parents have excellent heterosis). Generally, 3 fruit ears of plants with 3 disease-resistant loci are selected and retained. This step is completed in northern my country from June to October of the second year.

7) The BC ₄ F ₁ seeds were sown in the field to continue molecular marker-assisted selection (same as BC1F1 generation), and then the plants with 3 disease-resistant loci were selfed. Generally, 3 to 5 ears are selected. This step can be done in the greenhouse or in Hainan from November of the second year to March of the third year. .

8) The BC ₄ F ₂ plants (plants with 3 disease resistance loci) were selfed for one more generation to obtain an inbred line homozygous for rough dwarf disease resistance loci. At the same time, the combining ability was measured, and the plants were strictly identified for disease resistance and comprehensive traits were selected to obtain an excellent inbred line with high resistance to rough dwarf disease that met the breeding goal. The inbred line is basically the same as the recurrent parent.

Example 2: Breeding an excellent improved line of Ye 502 resistant to corn rough dwarf disease

Using Ye 502 or its improved line as the recipient and recurrent parent, and the inbred line 90110 as the donor of the disease resistance gene locus, excellent improved lines were obtained mainly through the prospect selection of the target gene. Because Ye 502 has poor disease resistance, it is the best plan to improve multiple traits. the

Procedure one:

1) F ₁ plants were produced by crossing Ye 502 as the female parent and 90110 as the male parent. This step can be completed in the greenhouse or in Hainan from January to April of the first year.

2) F ₁ plants selfed to produce F ₂ seeds. This step is completed in northern my country from May to August of the first year.

3) F2 seeds were sown in flower pots, leaf DNA was extracted at the 3-leaf stage of the plants, PCR amplification was performed with primers of SSR markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823 and umc1268 respectively, and the reaction products were subjected to 6% polyacrylamide Gel electrophoresis separation, silver staining method to detect the band pattern of the amplified product, and 30-50 individuals with 3 disease-resistant loci were selected and transplanted into the field. This step can be completed in northern my country or Hainan in September to October of the first year. the

4) The selected plants were bagged and backcrossed with Ye 502 after tasseling, and field observation and records were strictly carried out, and inferior plants were eliminated according to the comprehensive traits of the plants before harvesting. After the ears are harvested, the seeds are tested and optimized. Generally, about 20 ears are selected and left. This step can be done in the greenhouse or Hainan from October of the first year to January of the second year. the

5) The BC ₁ F ₂ seeds with retained fruit ears were sown in pots for molecular marker-assisted selection (same as the F ₂ generation), and then the selected plants were transplanted to the field for growth. After the plants tasseled, they were backcrossed with Ye 502 to produce BC _{2 F2} seed. And carry out plant and ear selection. A total of 40 fruit ears with 3 or 2 disease-resistant sites are generally reserved. If there are many types of plants, you can choose to keep more fruit ears. This step is completed in northern my country from March to June of the second year. The plants at the seedling stage are best grown in a greenhouse and then transplanted in a plastic greenhouse.

6) Sow BC ₂ F ₂ seeds in the field for molecular marker-assisted selection (same as F ₂ generation). The selected plants were backcrossed with Ye 502 to produce BC ₃ F ₂ seeds. Simultaneously carry out plant and fruit ear selection, generally keep 20 fruit ears with 3 or 2 disease resistance sites. This step is completed in northern my country from July to October of the second year.

7) The BC ₃ F ₂ seeds were sown in the field to continue molecular marker-assisted selection (same as the F ₂ generation), and the comprehensive traits of the selected lines were selected. The plant traits of the same line in this generation are relatively neat. However, there may be significant differences between strains. The selected plants were backcrossed with Ye 502 after tasseling to produce BC ₄ F ₂ seeds. Generally, the number of retained ears is similar to that of the previous generation. At the same time, some plants of the selected lines were crossed with excellent inbred lines (including inbred lines with excellent heterosis with recurrent parents) to prepare hybrid seeds for the determination of combining ability. This step can be completed in the greenhouse or Hainan from November to March of the second year.

8) When performing step 6) or 7), the selected materials with 2 disease resistance sites can be hybridized. The selected plants for hybridization should be different in one disease-resistant locus, and have small differences in traits among each other. Three disease-resistant loci can be aggregated into one genotype through hybridization. Then the plants with 3 disease-resistant loci were obtained from the hybrid offspring by molecular marker-assisted selection. the

9) Selfing the plants with 3 disease-resistant loci for 1 to 2 generations to obtain inbred lines homozygous for the disease-resistant loci, and conducting strict identification of disease resistance, selection of comprehensive traits and determination of combining ability on the plants, An excellent inbred line with high resistance to rough dwarf disease was bred in line with the breeding objective. This step is carried out in northern my country from April to September in the third and fourth years. the

Procedure two:

1) F ₁ plants were produced by crossing Ye 502 as the female parent and 90110 as the male parent. This step can be completed in the greenhouse or in Hainan from January to April of the first year.

2) F ₁ plants selfed to produce F ₂ seeds. This step is completed in northern my country from May to August of the first year.

3) F ₂ plants are selfed to generate F ₃ plants. This step can be done in the greenhouse or Hainan from October of the first year to February of the second year.

4) _F3 seeds are sown in the field, and the seedling and flowering stages are strictly eliminated according to the agronomic characters. After the plants were pollinated, leaf DNA was extracted, and PCR amplification was carried out with primers of SSR markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823 and umc1268, and the reaction products were separated by 6% polyacrylamide gel electrophoresis, and the amplification was detected by silver staining For the band pattern of the product, 50-60 individual plants with 3 disease-resistant loci or 2 disease-resistant loci were selected and backcrossed with Ye 502. Before harvesting, the inferior plants were eliminated according to the comprehensive traits of the plants, and then the seeds were tested, and about 30 ears were selected to remain. This step can be completed in northern my country from March to June of the second year.

4) The BC ₁ F ₃ seeds with retained fruit ears were sown in the field for molecular marker-assisted selection (same as the F ₃ generation), and then the selected plants were transplanted into the field for growth. After the plants tasseled, they were bagged and backcrossed with Ye 502 to produce BC ₂ F ₃ seeds. And carry out plant and ear selection. Generally, 8 to 10 ears of plants with 3 disease-resistant sites are reserved, or 10 to 15 ears of plants with 3 or 2 disease-resistant sites are reserved. If there are many types of plants, you can choose to keep more fruit ears. This step can be completed in northern my country from July to October of the second year.

6) Sowing BC ₂ F ₃ seeds in the field for molecular marker-assisted selection and plant selection (same as F ₃ generation). The selected plants were backcrossed with Ye 502 to produce BC ₃ F ₃ seeds. Generally, 8 to 10 ears of plants with 3 disease-resistant sites are reserved, or 10 to 15 ears of plants with 3 or 2 disease-resistant sites are reserved. This step is completed in the greenhouse or Hainan from November of the second year to March of the third year.

7) The BC ₃ F ₃ seeds were sown in the field to continue molecular marker-assisted selection (same as the F ₃ generation), and the comprehensive traits of the selected lines were selected, and the BC ₄ F ₃ seeds were produced by backcrossing with Ye 502 after the plants tasseled . Generally, the number of ears left is similar to that of the previous generation. The plant traits of the same line in this generation are relatively neat, but there may be obvious differences among the lines. At the same time, some plants of the selected lines were crossed with excellent inbred lines (including inbred lines with excellent heterosis with recurrent parents) to prepare hybrid seeds for the determination of combining ability. This step can be completed in northern my country from April to September of the third year.

8) When performing step 6) or 7), the selected materials with 2 disease resistance sites can be hybridized. The selected plants for hybridization should be different in one disease-resistant locus, and the traits of each other are less different. The three disease-resistant loci can be aggregated into one genotype by crossing, and then the plants with the three disease-resistant loci can be obtained from the offspring of the cross by molecular marker-assisted selection. the

9) The plants with 3 disease resistance loci are selfed for one generation to make part or all of the rough dwarf resistance loci homozygous. This step can be completed in the greenhouse or Hainan from November of the third year to February of the fourth year. the

10) The plants with 3 disease-resistant loci were selfed for one generation to obtain an inbred line homozygous for the disease-resistant loci, and the plants were subjected to strict identification of disease resistance, selection of comprehensive traits and determination of combining ability, and selected An excellent inbred line with high resistance to rough dwarf disease was bred in line with the breeding objective. This step is carried out in northern my country from April to September of the fourth year. the

Example 3: Breeding excellent inbred lines resistant to corn rough dwarf disease

With excellent inbred lines DH4866 and Zheng 58 as recipients and recurrent parents, and inbred line 90110 as donor of disease resistance genes, excellent improved lines with different characteristics were obtained mainly through prospect selection of target genes. Proceed as follows:

1) Crossing DH4866 and Zheng 58 as the father and mother respectively to produce F ₁ seeds. This step can be completed in the greenhouse or in Hainan from January to April of the first year.

2) F ₁ plants are selfed to produce F ₂ generation seeds, and F ₂ plants are selectively selfed to produce F ₃ generation seeds, generally controlled at 30-40 ears. This step is completed in northern my country and/or Hainan from May to December of the first year.

3) F ₃ plants were selectively selfed to produce F ₄ generation seeds, and 40-60 ears were harvested. This step is completed in the greenhouse or in Hainan from January to April of the second year.

4) Sow the seeds of the F _4th generation in the field, strictly observe the field, divide the plants into DH4866-like, Zheng 58-like and intermediate types, and select 20-30 excellent plants to cross with 90110 respectively to produce a series of "F _{4- n} ×90110" seeds (F _4-n represents F ₄ individual number n, n can be 1, 2, 3-----X). This step will be completed in northern my country from May to September of the second year.

5) The "F _4-n × 90110" seeds are sown in the field, and the offspring of DH4866-like and Zheng 58-like plants are respectively selected to be superior to F _4-n (the n value is the same, that is, a specific "F _4-n × 90110 "The F _4-n selfed progeny of the combined parent is the recurrent parent) backcrossed to produce a series of BC ₁ "F _4-n × 90110" seeds. The progeny of intermedium plants are self-crossed to produce F ₂ "F _4-n ×90110" seeds. Each combination backcrossed 3 panicles or selfed 20 panicles. This step is completed in the greenhouse or Hainan from October of the second year to March of the third year.

6) Sow BC ₁ "F _4-n ×90110" and F ₂ "F _4-n ×90110" seeds in flowerpots, extract leaf DNA of 3-leaf stage plants, and use SSR to mark umc1656, bnlg2191umc1401, umc1666, bnlg1823 and The primers of umc1268 were amplified by PCR, and the reaction products were separated by 6% polyacrylamide gel electrophoresis, and the band pattern of the amplified products was detected by silver staining method, and 3 disease-resistant loci were selected from each backcross population 5-10 individuals from each selfing population, 30-40 individuals with 3 disease-resistant loci were selected from each selfing population, and then transplanted to the field for growth and eliminated inferior plants. The selected plants were backcrossed with their parental _F4 self-bred offspring, and the inferior plants were eliminated according to the comprehensive traits of the plants before harvesting. After the ears are harvested, the seeds are tested and optimized. Generally, 3 to 5 ears of backcrossed plants were selected for each BC ₁ "F _4-n ×90110" or F ₂ "F _4-n ×90110" population. This step is completed in northern my country from April to September of the third year.

7) The seeds of the selected ears are sown in flower pots to continue molecular marker-assisted selection (same as step 6), and then the selected plants are transplanted to grow in the field. Fruit ears are preferred. Generally, 2 to 5 ears of backcrossed progeny of the selected plants of each BC ₂ "F _4-n ×90110" or BC ₁ F ₂ "F _4-n ×90110" population are selected and retained. If there are many types of plants, you can choose to keep more fruit ears. This step is completed in the greenhouse or in Hainan from October of the third year to February of the fourth year.

8) Sowing the seeds of the ears selected from the previous generation in the field for molecular marker-assisted selection (same as step 6). Selected plants are backcrossed with recurrent parents, and plants and ears _are selected at the same time. _{Generally , 3 or} 2 resistant There are 2-5 fruit ears at the disease site. This step is completed in northern my country from April to September of the fourth year.

9) Sow the seeds of the remaining ears selected from the previous generation in the field to continue molecular marker-assisted selection (same as step 6), evaluate the comprehensive traits of the selected lines, and carry out strict disease resistance identification of the plants. The selected plants with 3 rough dwarf loci were self-fertile. Generally, the number of ears left is similar to that of the previous generation. At the same time, some plants of the selected lines were crossed with excellent inbred lines (including inbred lines with excellent heterosis with DH4866 or Zheng 58) to prepare seeds for the determination of combining ability. The plant traits of the same line in this generation are relatively neat. However, there are significant differences between strains. This step can be completed in Hainan from October of the fourth year to February of the fifth year. the

10) When performing step 8), the selected materials with 2 disease resistance sites can be hybridized. The selected plants for hybridization should be different in one disease-resistant locus and have small differences in traits from each other. The three disease-resistant loci were aggregated into one genotype by crossing, and then individuals with three disease-resistant loci were obtained from the offspring of the cross by molecular marker-assisted selection. the

11) The plants with 3 disease-resistant loci are selfed for 1 to 2 generations to obtain homozygous inbred lines. Combining ability determination, disease resistance identification, and comprehensive trait optimization are carried out at the same time, and finally the breeding target is selected. Excellent inbred lines with high resistance to rough dwarf disease, some of which have excellent characteristics of DH4866 or Zheng 58 and high resistance to rough dwarf disease. This step is completed in northern my country between April and September of the fifth and sixth years.

Claims (8)

1. A method for selecting a rough dwarf resistant maize inbred line using a molecular marker of a corn rough dwarf resistance locus, characterized in that: the rough dwarf resistant maize inbred line 90110 and the rough dwarf inbred line susceptible to rough dwarf are used Cross-lines were used to construct segregating populations, and molecular markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823, and umc1268 closely linked to maize rough dwarf resistance loci were used for assisted selection. The plants at the disease-resistant loci were backcrossed and selfed, and through several generations of selection, an excellent inbred line of maize with high resistance to rough dwarf disease was obtained. 2. The method according to claim 1, characterized in that: the corn inbred line resistant to rough dwarf disease is 90110; the inbred line susceptible to rough dwarf disease includes Ye 478, DH4866, Ye 502, and Zheng 58 excellent inbred lines . 3. The method according to claim 1, characterized in that: the isolated populations include the F _2-6 population produced by the continuous selfing of the hybrid progeny of the inbred line 90110 resistant to rough dwarf disease and the inbred line susceptible to rough dwarf disease, the hybrid progeny self F _2-4 BC _1-3 population generated by cross-crossing and backcrossing, and BC ₁ F ₁ , BC ₁ F ₁ ×BC ₁ F ₁ , BC ₁ F ₁ ×BC ₁ F ₂ population; where: F _2-6 refers to 2 to 6 generations of selfing; F _2-4 BC _1-3 refers to 2 to 4 generations of selfing, and 1 to 3 generations of backcrossing with recurrent parents. 4. The method according to claim 1, characterized in that: the SSR markers used for assisted selection of maize rough dwarf resistance loci have umc1656 and bnlg2191 molecular markers on chromosome 6, umc1401 and bnlg2191 on chromosome 7 umc1666 molecular marker, bnlg1823 and umc1268 molecular markers on chromosome 8. 5. The method according to claim 1, characterized in that: the selection of corn rough dwarf resistance gene loci is based on prospect selection, that is, the selection of target gene loci is combined with the selection of comprehensive traits; The target gene is selected by two markers adjacent to both sides of the target gene at the same time, or only one closely linked marker is used to select the target gene. 6. The method according to claim 1, 2 or 3, characterized in that: when the hybrid F1 is obtained, the rough dwarf disease-resistant 90110 plant and the rough dwarf disease inbred line plant can be used as the female parent or the male parent; When carrying out backcrossing, the inbred line susceptible to crude dwarfism was used as the male parent or the female parent. 7. The method according to claim 1, 4 or 5, characterized in that: the corn inbred line Ye 478 which is susceptible to rough dwarf disease is used as the recipient and the recurrent parent, and the corn inbred line 90110 which is highly resistant to rough dwarf disease As the donor parents of the resistance gene, an segregation population was established; the primers of SSR markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823 and umc1268 were used to perform PCR amplification to detect seedlings at the 3-leaf stage, and plants with 3 disease-resistant loci were selected Carry out backcrossing, and optimize according to the comprehensive traits of the plants, select the seeds of excellent plants for next-generation molecular marker-assisted selection and plant selection, backcross for 3 consecutive generations, and then self-cross for 1 to 2 generations to obtain maize with high resistance to rough dwarf disease Excellent inbred line. 8. The method according to claim 1, 4 or 5, characterized in that: Zheng 58, an excellent maize inbred line susceptible to rough dwarf disease, is used as the recipient and recurrent parent, and the inbred line 90110 with high resistance to rough dwarf disease is The resistance gene donor parents were used to establish segregation populations; the primers of SSR markers umc1656, bnlg2191, umc1401, umc1666, bnlg1823 and umc1268 were used to perform PCR amplification to detect seedlings at the 3-leaf stage, and plants with 3 disease-resistant loci were selected for testing. Selfing or backcrossing, and selection based on the comprehensive traits of the plants, selecting the seeds of excellent plants for next-generation molecular marker-assisted selection and plant selection, through selfing for 2 to 3 generations, continuous backcrossing for 1 to 3 generations, and then selfing Excellent inbred lines with high resistance to rough dwarf disease were obtained in 1-2 generations.

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