توسعه یک نشانگر مولکولی ناجفت پیوسته به ژن (های) مقاومت به ریزوکتونیا در چغندرقند

Extended abstract
Introduction
Root and crown rhizoctonia rot has been the most important fungal disease in sugar beet fields in the country. Therefore, the improvement of sugar beet varieties resistant to rhizoctonia is the most practical, safe and economical way to control this disease. Due to the fact that the classical evaluation methods of selection of resistance to this disease are phenotypic and depend on the environmental conditions and the uniformity of the inoculating agent and are carried out in a certain season of the year, hence, by using molecular methods, as a complementary or alternative method, it is possible to identify plants containing the resistance gene at the genotypic level. For genotypic screening of sugar beet breeding lines, molecular markers linked to resistance genes to rhizoctonia pathogen are needed. In this research, the aim was to develop and use a molecular marker linked to rhizoctonia resistance gene(s) in the genotypic screening of breeding materials of the Sugar Beet Seed Institute.
Materials and methods
In this research, the flanking sequences of a SNP molecular marker identified at the university of Padua, Italy were obtained and at the Sugar Beet Seed Institute, a pair of specific primers were designed and converted into a repulsion STS marker called SR2-r with normal PCR capability. This repulsion marker is able to identify homozygous resistant genotypes in the gene locus related to the marker. To check the reproducibility and confirm the repulsion marker SR2-r, firstly, several population and breeding lines of the existing germplasm of the institute were cultivated in microplots or in the greenhouse at different times. Then, the developed roots were inoculated with corn seeds infested with Rhizoctonia pathogen. Leaf samples were prepared from the inoculated plants and kept in a freezer at -70°C until use. After a few weeks of inoculation, single roots were taken from microplates or pots and the disease score of rhizoctonia in each of the roots was recorded and the disease index of each genotype was estimated and then the genotypes were grouped. PCR molecular test was performed with the primers related to the repulsion marker in the DNA of susceptible and resistant plants and the presence and absence of the marker in single plants was determined. Statistical analysis for the disease index was done in a completely randomized design with unequal replications using SAS software (2013) and comparing of the data mean with the LSD test method. Then, the percentage of homozygote resistant plants in the SR2-r repulsion marker locus was obtained by dividing the number of plants without the susceptible allele band of the marker by the total number of molecular tested plants. In the next step, the relationship between the marker results and the rhizoctonia infection score was investigated as a pairwise comparison in all the tested plants and various quantitative traits and the repulsion marker error were calculated.
Results and discussion
The molecular marker SR2-r developed in this research, which is designed from the SNP marker sequence related to susceptibility to rhizoctonia, is of the repulsion type, which is able to identify the susceptible allele to rhizoctonia in one of the genetic loci of resistance to rhizoctonia. In the sense that the presence of a marker band of 285 bp in a plant sample indicates the susceptible genotype or the possible heterozygosity of that plant, and the absence of the marker indicates the absence of a susceptible allele or, in other words, homozygous resistance in the mentioned locus. The molecular test with the aforementioned repulsion marker in the single plants of each of the selected genotypes harvested from the greenhouse and the infected microplot field showed that this marker has polymorphism in most genotypes and its presence percentage and its agreement with the results of resistance evaluation in genotypes is variable. Due to the oligogenic nature of resistance to rhizoctonia, it cannot be expected that one hundred percent of the phenotypic diversity of the trait can be covered by using only one repulsion marker linked to one of the resistance gene loci. However, with the SR2-r marker, about 48% of resistant plants can be selected from a breeding population, which is considered a very high genotypic screening power for a marker. In the next step, the relationship between the results of repulsion molecular marker and the disease score was checked in the form of pairwise comparison in all the tested plants. The results indicated a 13% error between the molecular marker and phenotypic resistance in greenhouse or microplot conditions. In this research, for the first time, various quantitative criteria for the superiority of a single molecular marker are presented. When several unique molecular markers are developed for a trait, the best markers can be selected and used for molecular screening based on the results of the above criteria. In order to correctly evaluate the results of different methods of sugar beet breeding for resistance to rhizoctonia, it is necessary to correctly identify resistant plants. For this purpose, two methods of phenotypic and genotypic evaluation can be used, which in general, genotypic evaluation has more advantages than phenotypic evaluation of resistance, including the selection of resistant homozygotes using repulsion markers or co-dominant markers, keeping the tested plant healthy for next use, application of plants at each stage of growth, the independence of the tested plant from the season or environmental conditions, Keeping the greenhouse and field clean from rhizoctonia pathogens, requires less cages, isolation and observation tests to find a specific number of resistant lines. Also, the presence percentage of the mentioned marker in a large number of pollinator lines, breeding populations, hybrids and susceptible and resistant commercial cultivars was investigated. According to the results of the molecular marker test in different genotypes, the percentage of presence of repulsion marker linked to rhizoctonia resistance gene(s) varied between zero and 100% and was mostly expected. The higher the proportion of susceptible plants in the primary population, the greater the selection gain with the help of the molecular marker in reducing the disease index. The overall agreement of the repulsion molecular marker with the rhizoctonia disease index in the greenhouse and microplot was 87%.
Conclusion
In this study, the reproducibility of a repulsion molecular marker (SR2-r) linked to rhizoctonia resistance genes, for which specific primer sequences were designed, was investigated. Thus, the overall agreement of the repulsion molecular marker with the rhizoctonia infection index in greenhouse and microplot was 87%. This repulsion marker was able to identify about 48% of rhizoctonia-resistant plants.
Key words: Disease, Molecular marker, Resistance evaluation, Rhizoctonia, Sugar beet.
References
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