ردیابی ژن‌های rdg1a و mlo در جو برای مقاومت به بیماری‌های لکه قهوه‌ای نواری و سفیدک پودری

بیماری های سفیدک پودری  (blumeria graminis f.sp. hordei)و لکه قهوه ای نواری  (pyrenophora graminea)از جمله بیماری‌های مهم قارچی جو در ایران می باشند و شناسایی ارقام مقاوم یکی از اقتصادی ترین و کارآمدترین روش ها برای کنترل این بیماری ها می باشد. این تحقیق، با هدف ردیابی الل های مقاومت rdg1a و mlo11 در جمعیت f2 حاصل از تلاقی والد مقاوم ril27 با رقم حساس یوسف انجام شد. ابتدا ارزیابی ژنوتیپی با استفاده از نشانگر hvcsg برای الل مقاومت rdg1a انجام گردید و به ترتیب در نمونه های مقاوم و حساس قطعاتی به اندازه‌ی 705 و 480 جفت باز تکثیر شدند همچنین جهت بررسی الل مقاومت mlo از نشانگر های mlo6 و mlo10 استفاده شد این نشانگرها قطعاتی به اندازه ی440 جفت باز از ژن mlo (الل مقاومت) و380 جفت باز از ژن mlo (الل حساسیت) را تکثیر کردند. نتیجه ی واکنش والدین و جمعیت f2 در برابر آلودگی به بیمارگر لکه قهوه ای نواری نشان داد که شدت بیماری در نمونه های مقاوم دارای الل مقاومت rdg1a خیلی پایین است. در بررسی واکنش این نمونه ها به بیمارگر سفیدک پودری جو،  لاین l94 به دلیل حضور الل مقاومت mlo11فاقد علایم بیماری بود که احتمالاً ناشی از تاثیر این الل در ایجاد مقاومت است. به طور کلی نتایج حاصل از ارزیابی ژنوتیپی به پتانسیل نشانگر های mlo6 و mlo10 برای شناسایی الل مقاومت mlo11 در لاین های انتخابی اشاره می کند. نشانگر hvcsg از کارایی پایینی برخوردار بود که لازم است با طراحی نشانگر مولکولی مناسب ژنوتیپ های مقاوم را شناسایی کرد.

Detection of Rdg1a and mlo Genes for Resistance to Leaf stripe and Powdery Mildew Diseases in Barley

Introduction: Powdery mildew and Leaf stripe diseases are the most important of barley fungus diseases in Iran. Identification of new genetic resources and breeding for resistance is one of the most economical and adaptive methods for controlling these diseases. Therefore, the aim of this study was an identification effective molecular marker for detection Rdg1a and mlo resistance alleles for Marker Assisted Selection against two diseases Leaf stripe and powdery mildew.Materials and Methods: This study was conducted on F2 population derived from crosses the RIL27 that has Rdg1a, mlo11 resistance alleles and Yousef which has susceptible alleles (rdg1a, MLO). The RIL27 line is one of the lines of the recombinant inbred line population that derived from a cross between the VADA cultivar and the L94 Ethiopian line. 60 samples of F2 population were divided into two populations of 30 for genotyping and phenotyping against two diseases. We used HVCSG and mlo6 and mlo10 markers for the presence detection Rdg1a and mlo11 respectively. Also Manchuria cultivar, which is susceptible to powdery mildew was used to produce inoculum powdery mildew. Genomic DNA of the plants was extracted from noninfected leaves in a twoleaf stage by using the CTAB method. Then, the quantity and quality of extracting DNA were studied by using (Thermoscientific (USA)) and Agarose gel (1%). The HVCSG, mlo6 and mlo10 markers amplified fragments 705, 440 and 380 base pairs respectively. Phenotyping evaluation against P.‌graminea was performed by using the sandwich method and for phenotyping evaluation against B. graminis was done using Aghnoum and et al method. And then the percentage of infect plants were counted.Results and Discussion: In this study at first, HVCSG marker was used to distinguish Rdg1a resistance allele in parents and F2 population. This marker amplified the 705 base pair band that the result obtained was corresponding to what Biselli and et al showed in 2010. Biselli et al. Developed the HVCSG molecular marker to identify the Rdg1a resistance allele in the RIL population from the VADA × L94 crosses by rice EST sequence and they said, this marker amplifies the region from 4500 to 5025 sequences encoding the Shalcone synthase gene. But in this study, the results of using this marker in the F2 population are completely inconsistent with what Biselli and et al have stated. The results show that the HVCSG marker has a low efficiency. Second to check and confirm the presence of mlo11 allele of mlo6 and mlo10 markers were used. The size of amplified regions (440 of the mlo11 gene and 380 base pairs of the MLO gene) was corresponding to the results of Reinstadlr and et al. showed that these markers amplified 380 and 440 base pair fragments. After of inoculation test and the appearance of the symptoms of the diseases the percentage of infect plant for phenotyping against leaf stripe disease was counted. The Yousef cultivar, which was infected with P. graminea as a susceptible parent, showed success in the inoculation test. In other samples, 24 days after planting, symptoms of leaf stripe disease appeared in the five leaf stage. First, on the infected leaves, a yellow strip appeared, and most of the leaves that were later formed showed signs of the disease. Then the yellow strips on the leaves infected joined each other and caused the death of the leaf. Two resistant parents, RIL27 and VADA showed very low symptoms of leaf stripe disease. The results of inoculation test in this study was corresponding with studies from Arru and et al (2002) and Biselli and et al (2010). Arru et al showed that VADA was showing very little about 7% of the symptoms disease due to the presence of the resistance gene. Biselli and et al (2010) also reported that the percentage of infections in VADA parent is 2%. For phenotyping evaluation against powdery mildew disease after infecting the seedlings with B. graminis fungus, in the Yousef parent, symptoms were observed as white fluffy dots on the leaf surface. In the VADA variety, there were necrotic points. L94 and RIL27, which had mlo11 resistance gene, did not show any symptoms of the disease. L94 is a native Ethiopian cultivar that allele carries mlo resistance, so it is resistant to powdery mildew. The F2 population showed different signs based on the presence and absence of allele resistance. The samples with resistance allele, there were appeared mildew cholestasis and necrosis symptoms, and those that did not have resistance alleles were appeared necrotic and fluffy symptoms. In mlo11 resistance alleles, the necrotic symptoms can be due to the presence of the Ml (La) resistance gene and also the pleiotropy effects of the molecular gene. Xintian et al reported that the mlo genes were not without pleiotropic effects, and necrotic symptoms on leaves of plants with mlo11 resistance gene could be due to the effect Overlapping of the mlo gene with other QTLs. Conventional plant breeding methods are based on phenotypic selection of superior genotypes in segregation generation. Phenotyping methods are often costly and timeconsuming for specific traits, but Marker Assisted Selection (MAS) is one of the methods developed to prevent of common problems in conventional plant breeding techniques. In some studies, the researchers pointed to use of molecular markers for facilitating of plant breeding programs.Conclusion: Molecular markers are used as a new tool for increasing the efficiency of breeding programs to identify genetic resources. In addition, shortening the duration of breeding programs and the selection of recessive alleles, the molecular markers helps to facilitate the pyramiding of resistance genes to provide a broad and durable resistance. In general, the development of efficient molecular markers and the identification of different genetic resources against plant diseases and the pyramiding of resistance resources are preventing of the increasing use of chemical pesticides and fertilizers to control the pathogen.