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توسعه یک نشانگر مولکولی ناجفت پیوسته به ژن (های) مقاومت به ریزوکتونیا در چغندرقند | ||
| چغندرقند | ||
| مقاله 3، دوره 40، شماره 1، شهریور 1403، صفحه 13-30 اصل مقاله (875.4 K) | ||
| نوع مقاله: کامل علمی - پژوهشی | ||
| شناسه دیجیتال (DOI): 10.22092/jsb.2024.365710.1360 | ||
| نویسندگان | ||
| پیمان نوروزی* 1؛ نعیمه ترک2؛ مژده کاکوئی نژاد3؛ سیدباقر محمودی1؛ مژده ملکی4 | ||
| 1دانشیار مؤسسه تحقیقات اصلاح و تهیه بذر چغندرقند- سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران | ||
| 2دانشجوی دکتری بیماری شناسی گیاهی، دانشگاه آزاد اسلامی، واحد ورامین- پیشوا. | ||
| 3استادیار مؤسسه تحقیقات اصلاح و تهیه بذر چغندرقند سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران. | ||
| 4استادیار گروه گیاه پزشکی، دانشکده کشاورزی، دانشگاه آزاد اسلامی واحد ورامین- پیشوا، ورامین، ایران | ||
| چکیده | ||
| بیماری پوسیدگی ریزوکتونیایی ریشه و طوقه مهمترین بیماری قارچی در مزارع چغندرقند کشور بوده و موثرترین روش مدیریت این بیماری، اصلاح رقمهای مقاوم به آن می باشد. برای غربال ژنوتیپی لاینهای اصلاحی چغندرقند نیاز به نشانگرهای مولکولی پیوسته به ژن های مقاومت به عامل بیمارگر ریزوکتونیا است. در این تحقیق توالیهای مجاور یک نشانگر مولکولی SNP شناسایی شده در دانشگاه پادوای ایتالیا دریافت و در مؤسسه تحقیقات اصلاح و تهیه بذر چغندرقند، جفت آغازگرهای اختصاصی طراحی و به یک نشانگر ناجفت STS موسوم به SR2-r با قابلیت PCR استاندارد تبدیل شد. این نشانگر ناجفت قادر به شناسایی ژنوتیپهای مقاوم خالص در جایگاه ژنی مرتبط با نشانگر میباشد. برای بررسی تکرارپذیری و تأیید نشانگر ناجفت SR2-r، ابتدا تودهها و لاینهای اصلاحی متعددی از ژرمپلاسم موجود مؤسسه در کرتهای آزمایشی میکروپلات یا در گلخانه در زمانهای مختلف کشت شدند. سپس ریشه-های توسعه یافته با دانههای ذرت آغشته شده به بیمارگر ریزوکتونیا تلقیح شدند. نمونههای برگی از بوتههای تلقیح شده تهیه و تا زمان استفاده در فریزر c◦70- نگهداری شدند. پس از گذشت چند هفته از زمان تلقیح، تک ریشهها از میکروپلات یا گلدانها برداشت و نمره آلودگی به بیماری ریزوکتونیا در هر یک از ریشهها یادداشتبرداری و شاخص آلودگی هر ژنوتیپ برآورد و سپس ژنوتیپها گروهبندی شدند. در مرحله بعد، بوتههای حساس و مقاوم انتخاب و از برگ های فریز شده آنها استخراج DNA انجام گرفت. آزمون مولکولی PCR با آغازگرهای مربوط به نشانگر ناجفت در DNA بوتههای حساس و مقاوم انجام گرفت و حضور و عدم حضور نشانگر در تک بوتهها مشخص شد. در مرحله بعد، ارتباط بین نتایج نشانگر مولکولی ناجفت با نمره آلودگی به بیماری به صورت مقایسه جفتی در تمام بوتههای آزمون شده بررسی شد. نتایج حاکی از توافق 87 درصدی بین نشانگر مولکولی با مقاومت فنوتیپی در شرایط گلخانه یا میکروپلات بود. این نشانگر ناجفت قادر به شناسایی 48 درصد از بوتههای مقاوم به ریزوکتونیا بود، اما با توجه به حضور غیرقابل انتظار باندهای بسیار ضعیف در برخی ژنوتیپ های هموزیگوت مقاوم، پیشنهاد می گردد در صورت امکان از Real-time PCR استفاده شود. | ||
| کلیدواژهها | ||
| ارزیابی مقاومت؛ بیماری؛ چغندرقند؛ ریزوکتونیا؛ نشانگر مولکولی | ||
| عنوان مقاله [English] | ||
| Development of a repulsion molecular marker linked to rhizoctonia resistance gene(s) in sugar beet | ||
| نویسندگان [English] | ||
| Peyman Norouzi1؛ Naime Tork2؛ Mozhdeh Kakueinezhad3؛ S.B. Mahmoudi1؛ Mojdeh Maleki4 | ||
| 1Associate Professor of Sugar Beet Seed Institute (SBSI) - Agricultural Research Education and Extension, Karaj,Iran | ||
| 2Ph.D. student of plant pathology, Varamin – Pishva Branch, Islamic Azad University. | ||
| 3Assistant Professor of Sugar Beet Seed Institute (SBSI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran | ||
| 4Department of Plant Protection, College of Agriculture, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran | ||
| چکیده [English] | ||
| 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 Buttner G, Pfähler B, Märländer B. Greenhouse and field techniques for testing sugar beet for resistance to Rhizoctonia root and crown rot. Plant Breeding, 2008; 123: 158-166. Doi:https://doi.org/10.1046/j.1439-0523. 2003. 00967.x. Mahmoudi SB, Mesbah M, Alizadeh A. Pathogenesis variation of rhizoctonia solani isolates of sugar beet. Iranian Journal of Plant Pathology. 2004; 40: 253-283. (in Persian). Scholten OE, Panella LW, De Bock TS, Lange W. A greenhouse test for screening sugar beet (Beta vulgaris) for resistance to Rhizoctonia solani. European journal of plant pathology, 2001; 107: 161-166. | ||
| کلیدواژهها [English] | ||
| Disease, molecular marker, resistance evaluation, rhizoctonia, sugar beet | ||
| مراجع | ||
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Buttner G, Pfähler B, Märländer B. Greenhouse and field techniques for testing sugar beet for resistance to Rhizoctonia root and crown rot. Plant Breeding, 2008; 123: 158-166. Doi:https://doi.org/10.1046/j.1439-0523. 2003. 00967.x. Dellaporta S L, Wood J, Hicks JB. A plant DNA minipreparation: version II. Plant Molecular Biology Reporter, 1983; 1: 19-21. Doi:https://doi.org/10.1007/BF02712670. Hassani M, Heidari B, Stevanato P, Campagna G, Broccanello C, Norouzi P, Concheri G, Panella L. A candidate single nucleotide polymorphism (SNP) marker linked to resistance to infection with rhizoctonia in sugar beet. 76th IIRB Congress, 2018; 5-7 June. Deauville, France. Herr LJ. Sugar beet diseases incited by Rhizoctonia species. pp. 341-350. In: Sneh B, Jabaji-Hare S, Neate S, Dijst G (Eds.). Rhizoctonia species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic Publishers. Dordrecht. Netherlands. 1996. Lein JC, Asbach K, Tian Y, Schulte D, Li C, Koch G, Jung C, Cai DG. Resistance gene analogues are clustered on chromosome 3 of sugar beet and co-segregate with QTL for rhizomania resistance. Genome, 2007; 50: 61–71. Doi:https://doi.org/10.1139/g06-131. Lein JC, Sagstetter CM, Schulte D, Thurau T, Varrelmann M, Saal B, Koch G, Borchardt DC, Jung C. Mapping of Rhizoctonia root rot resistance genes in sugar beet using pathogen response-related sequences as molecular markers. Plant Breeding, 2008; 127: 602–611. Doi:https://doi.org/10.1111/j.1439-0523. 2008.1525.x. Mahmoudi SB, Mesbah M, Alizadeh A. Pathogenesis variation of rhizoctonia solani isolates of sugar beet. Iranian Journal of Plant Pathology. 2004; 40: 253-283. (in Persian). Mahmoudi SB, Soltani J. Sugar beet root rot in Iran. Newsletter of Iranian Sugar Industries Research and Training Center, 2005; 16(178): 14-18. (in Persian). Nazari A. Identification of molecular markers linked to genes controlling rhizoctonia resistance in sugar beet. MSc thesis. Crop science and breeding department. AboureihanPardis. Tehran Univ. 2011.(in Persian). Norouzi P, Rahmani D, Oroojalian S, Mahmoudi B, Aghaeizadeh M, Kakuinejad M, Orazizadeh, MR, Vahedi S, Fathi MR. Confirmation of repulsion molecular markers linked to rhizomania resistance gene (Rz1) and evaluation of gene dose effect in sugar beet genotypes. Journal of Sugar Beet. 2013; 29: 133-145. (in Persian). Norouzi P, Sabzehzari M, Zeinali H. Efficiency of some molecular markers linked to rhizomania resistance gene (Rz1) for marker assisted selection in sugar beet. Journal of Crop Science and Biotechnology, 2015; 18 (5): 319-323. Doi:https://doi.org/10.1007/s12892-015-0033-9. Norouzi P, Rajabi A, Fasahat P. Molecular markers in sugar beet. Agricultural education publishing, Tehran. 2022; 275 p. (in Persian). Nouhi AA, Amiri R, Haghnazari A, Saba J, Mesbah M. Use of molecular marker for assay gene dosage resistant gene to rhizomania disease (Rz1) in sugar beet (Beta vulgaris L.). Asian Journal of Biotechnology. 2009; 1(1): 37-41. Doi:https://doi.org/10.3923/ajbkr.2009.37.41. Panella, LW, Ruppel, EG. Availibility of germplasm for resistance against Rhizoctonia spp. pp.515-527. In: Sneh B, Jabaji-Hare S, Neate S and Dijst G. (Eds.). Rhizoctonia species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic Publishers. Dordrecht. 1996. Rajabi A, Aghaeezadeh M. Sugar beet breeding. Training course in KWS company. Einbeck, Germany, June-July. 2012. (in Persian). Ravi S, Hassani M, Heidari B, Deb S, Orsini E, Li J, Richards C, Panella L, Srinivasan S, Campagna G, Concheri G, Squartini A and Stevanato P. Development of an SNP assay for marker-assisted selection of soil-borne Rhizoctonia solani AG-2-2-IIIB resistance in sugar beet. Biology. 2022; 11 (49): 1-17. Doi:https://doi.org/10.3390/biology11010049. Scholten OE, Panella LW, De Bock TS, Lange W. A greenhouse test for screening sugar beet (Beta vulgaris) for resistance to Rhizoctonia solani. European journal of plant pathology, 2001; 107: 161-166. Sheikholeslam M. Diagnosis of fungi of sugar beet root rot and determining of their dispersion in Kermanshah province. Final report of project. 2001; 17 p. (in Persian). Windels C, Panella L, Ruppel E, Sugar beet germplasm resistant to Rhizoctonia root and crown rot withstands disease caused by several pathogenic isolates of Rhizoctonia solani AG-2-2. Sugar beet. Research and Extension Reports, 1995; 26: 179-185. | ||
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