تمایز ژنوتیپ های نخود حساس و مقاوم به بیماری برق زدگی با استفاده از سامانه تکثیر انتخابی جهش

نوع مقاله : علمی پژوهشی-فارسی

نویسندگان

1 استادیار، گروه گیاه‌پزشکی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

2 دانشیار، گروه گیاه‌پزشکی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

چکیده

بیماری برق‌زدگی نخود یکی از عوامل مخرب و محدود‌کننده زراعت نخود در کشورهای مختلف می باشد. شناسایی منابع مقاومت با استفاده از روش‌های مولکولی مبتنی بر ردیابی ژنومی صفت مقاومت می‌تواند در تسریع و افزایش راندمان مؤثر باشد. استفاده از جهش نقطه‌ای کارکردی و آلل‌های مرتبط با مقاومت جهت ردیابی منابع مقاومت با استفاده از روش سامانه تکثیر انتخابی جهش (ARMS) می تواند در پیشبرد برنامه‌های گزینش منابع مقاومت به بیماری برق‌زدگی بسیار مؤثر باشد. قطعه دربرگیرنده جهش نقطه‌ای جایگاه GSh118-2773 با استفاده از آغازگرهای اختصاصی در دو ژنوتیپ نخود مقاوم (MCC133) و حساس (ILC263) به بیماری برق‌زدگی تکثیر و توالی‌یابی شد و حضور آلل‌های مرتبط به مقاومت GSh118-2773C و حساسیت GSh118-2773g به‌ترتیب در نمونه‌های مقاوم و حساس تأیید شد. جفت آغازگرهای اختصاصی ردیاب آلل مقاومتPSh18-Fc و PSh18-Rتوانست روی ژنوتیپ مقاوم MCC133 قطعه 330 جفت بازی مورد انتظار را تکثیر نماید. نتایج تکثیر اختصاصی آلل‌ها را با استفاده از آغازگرهای ردیابی تأیید نمود. استفاده از جفت آغازگرهای اختصاصی آلل مقاومت می تواند در مطالعات گزینش مولکولی منابع نخود مقاوم به بیماری برق‌زدگی مورد توجه قرار گیرد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Differentiation of susceptible and resistant chickpea genotypes to Ascochyta blight using the ARMS molecular method

نویسندگان [English]

  • F. Shokouhifar 1
  • M. Mamarabadi 2
1 Assistant Professor, Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
2 Associate Professor, Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
چکیده [English]

Abstract

Background and Objectives

Chickpea, cultivated in rainfed and irrigated fields across many countries, faces biological stresses such as Ascochyta blight caused by the pathogenic fungus Ascochyta rabiei. This disease poses a severe threat to chickpea fields, resulting in substantial annual damage in various countries. Identifying resistance sources through molecular methods, specifically genomic tracking of the resistance trait, holds the potential to expedite and enhance control measures effectively. The use of the Amplification Refractory Mutation System (ARMS) method, involving functional point mutations and resistance-related alleles, proves highly efficient in advancing selection programs targeted at combating Ascochyta blight.

Materials and methods

The fragment harboring the SNP18-Pos57723 point mutation within the GSh118 gene sequence underwent amplification and sequencing using PSh118-F/R specific primers. This process utilized DNA extracted from both resistant (MCC113) and susceptible (ILC263) chickpea genotypes. The identification of alleles associated with resistance and sensitivity was established. Distinct differentiating primers were formulated for resistant (PSh118-Fc and PSh118-R) and sensitive (PSh18-Fg and PSh18-R) genotypes. The specific annealing temperature for each reaction was determined through a temperature gradient analysis. The efficacy of the designed primers in distinguishing between resistant and sensitive genotypes was assessed by conducting PCR and comparing their electrophoresis patterns.

Results

The presence of alleles linked to GSh18-2773C resistance and GSh18-2773g sensitivity was confirmed in resistant and sensitive chickpea genotypes, respectively. The specific primers PSh18-Fc and PSh18-R, designed for detecting the resistance allele, successfully amplified the expected 330 bp fragment in the resistant chickpea genotype MCC133. The study results affirmed the accurate amplification of alleles using these designated primers.

Discussion

In the present study, the ARMS method, known for its high sensitivity, specificity, rapidity, cost-effectiveness, multiplexing capabilities, compatibility with high throughput methods, and non-destructive nature, was employed to differentiate chickpea genotypes resistant to Ascochyta blight. The ARMS method's effectiveness in selecting resistance sources among various plant lines and cultivars has been demonstrated through allele tracking related to the resistance gene in prior studies. The results of this study indicated that the ARMS method accurately distinguishes chickpea genotypes resistant and sensitive to Ascochyta blight based on alleles associated with the GSh18-2773 position's point mutation. Thus, employing specific primers for the resistance allele is recommended in molecular selection studies of chickpea sources resistant to Ascochyta blight.



Keywords: Point mutation, allelic variation, molecular markers, resistance, Ascochyta rabiei

کلیدواژه‌ها [English]

  • Point mutation
  • allelic variation
  • molecular markers
  • resistance
  • Ascochyta rabiei
Al-Tanbouz, R., & Abu-Qauod, H. (2016). In vitro regeneration of chickpea (Cicer arietinum L.). Plant Cell Biotechnology and Molecular Biology17(1&2), 21-30.
Bui, M., & Liu, Z. (2009). Simple allele-discriminating PCR for cost-effective and rapid genotyping and mapping. Plant Methods, 5(1), 1-8. https://doi.org/10.1186/1746-4811-5-1  
Dar, A. A., Mahajan, R., & Sharma, S. (2019). Molecular markers for characterization and conservation of plant genetic resources. Indian Journal of Agricultural Sciences, 89(11), 1755-1763. https://doi.org/10.56093/ijas.v89i11.95286
Deokar, A., Sagi, M., & Tar’an, B. (2019). Genome-wide SNP discovery for development of high-density genetic map and QTL mapping of Ascochyta blight resistance in chickpea (Cicer arietinum L.). Theoretical and Applied Genetics, 132, 1861-1872. https://doi.org/10.1007/s00122-019-03322-3
Devasirvatham, V., Gaur, P. M., Mallikarjuna, N., Tokachichu, R. N., Trethowan, R. M., & Tan, D. K. (2012). Effect of high temperature on the reproductive development of chickpea genotypes under controlled environments. Functional Plant Biology, 39(12), 1009-1018. https://doi.org/10.1071/FP12033
Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical bulletin.
Duta-Cornescu, G.E.O.R.G.I.A.N.A., Simon-Gruita, A.L.E.X.A.N.D.R.A., Constantin, N., Stanciu, F., Dobre, M., Banica, D., Tuduce, R., Cristea, P., & Stoian, V. (2009). A comparative study of ARMS-PCR and RFLP-PCR as methods for rapid SNP identification. Romanian Biotechnological Letters, 14(6), 4845-4850.
FAO. (2016). Food and Agricultural Organization. www. fao. org. Accessed 10 May 2022. www. fao. Org.
Farahani, S., Talebi, R., Maleki, M., Mehrabi, R., & Kanouni, H. (2019). Pathogenic diversity of Ascochyta rabiei isolates and identification of resistance sources in core collection of chickpea germplasm. The plant pathology journal, 35(4), 321. https://doi.org/10.5423/PPJ.OA.12.2018.0299
Farahani, S., Talebi, R., Maleki, M., Mehrabi, R., & Kanouni, H. (2021). Mating type distribution, genetic diversity and population structure of Ascochyta rabiei, the cause of Ascochyta blight of chickpea in western iran. Phytopathologia Mediterranea, 60(1), 3-11. https://doi.org/10.36253/phyto-11616
Firouzmand, H., Toosi, S., Shokouhifar, F., & Mamarabadi, M. (2023). Resistance pattern of a cold tolerant chickpea cultivar (Saral) against different pathotypes of Ascochyta rabiei using an in vitro pathogenicity test method. Australasian Plant Pathology, 1-13. https://doi.org/10.1007/s13313-023-00920-0
Furtado, L. F. V., Alves, W. P., Moreira, T. B., Junior, L. M. C., Miranda, R. R. C., & Rabelo, É. M. L. (2016). Standardization and application of the tetraprimer ARMS-PCR technique for screening of the E198A SNP in the β-tubulin gene of hookworm populations in Brazil. Veterinary parasitology, 224, 65-67. https://doi.org/10.1016/j.vetpar.2016.05.013
Ghamghami, M., & Beiranvand, J. P. (2022). Rainfed crop yield response to climate change in Iran. Regional Environmental Change, 22, 1-17. https://doi.org/10.1007/s10113-021-01856-1
Li, Y., Ruperao, P., Batley, J., Edwards, D., Davidson, J., Hobson, K., & Sutton, T. (2017). Genome analysis identified novel candidate genes for Ascochyta blight resistance in chickpea using whole genome re-sequencing data. Frontiers in Plant Science, 8, 359. https://doi.org/10.3389/fpls.2017.00359
Little, S. (1995). Amplification‐refractory mutation system (ARMS) analysis of point mutations. Current protocols in human genetics, 7(1), 9-8. https://doi.org/10.1002/0471142905.hg0908s07
Liu, J., Huang, S., Sun, M., Liu, S., Liu, Y., Wang, W., Zhang, X., Wang, H., & Hua, W. (2012). An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods, 8, 34. https://doi.org/10.1186/1746-4811-8-34
Mahmoodi, F., & Banihashemi, Z. (2004). Distrbution of mating type, teleomorph formation, and genetic diversity in Didymella rabiei the causal agent of chickpea blight in fars province. Iranian Journal of Plant Pathology, 40, 15-30.
Mehta, G., Verma, P. K., & Ravi, M. (2015). Correlation studies in chickpea grown under rainfed and irrigated conditions in Northern Plains of India. Journal of Agroecology and Natural Resource Management, 2(5), 388-390.
Newman, T.E., Jacques, S., Grime, C., Kamphuis, F.L., Lee, R.C., Berger, J., & Kamphuis, L.G. (2021). Identification of novel sources of resistance to Ascochyta blight in a collection of wild Cicer accessions. Phytopathology, 111(2), 369-379. https://doi.org/10.1094/PHYTO-04-20-0137-R
Newton, C.R., Graham, A., Heptinstall, L.E., Powell, S.J., Summers, C., Kalsheker, N., Smith, J.C., & Markham, A. (1989). Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Research, 17(7), 2503-2516. https://doi.org/10.1093/nar/17.7.2503
Nourollahi, K., Javannikkhah, M., Naghavi, M.R., Lichtenzveig, J., Okhovat, S.M., Oliver, R.P., & Ellwood, S. R. (2011). Genetic diversity and population structure of Ascochyta rabiei from the western Iranian Ilam and Kermanshah provinces using MAT and SSR markers. Mycological progress, 10, 1-7.
Pande, S., Sharma, M., Gaur, P.M., Tripathi, S., Kaur, L., Basandrai, A., Khan, T., Gowda, C.L.L., & Siddique, K. H. M. (2011). Development of screening techniques and identification of new sources of resistance to Ascochyta blight disease of chickpea. Australasian Plant Pathology, 40, 149-156. http://dx.doi.org/10.1007/s13313-010-0024-8
Peever, T. L., Chen, W., Abdo, Z., & Kaiser, W. J. (2012). Genetics of virulence in Ascochyta rabiei. Plant Pathology, 61(4), 754-760. https://doi.org/10.1111/j.1365-3059.2011.02566.x
Ramezani Khozestani, F., Zaker Tavallaie, F., Shokouhifar, F., & Mamarabadi, M. (2023). Optimization of Ascochyta rabiei pathogenicity test on resistant and susceptible chickpea cultivars under in vitro condition. Iranian Journal of Pulses Research. https://doi.org/10.22067/ijpr.2023.77619.1039
Reddy, D. S., Bhatnagar-Mathur, P., Reddy, P. S., Sri Cindhuri, K., Sivaji Ganesh, A., & Sharma, K. K. (2016). Identification and validation of reference genes and their impact on normalized gene expression studies across cultivated and wild cicer species. PloS one, 11(2), e0148451. https://doi.org/10.1371/journal.pone.0148451
Rubio, M., Caranta, C., & Palloix, A. (2008). Functional markers for selection of potyvirus resistance alleles at the pvr2-eIF4E locus in pepper using tetra-primer ARMS–PCR. Genome, 51(9), 767-771. https://doi.org/10.1139/G08-056
Shokouhifar, F., Bagheri, A., & Rastegar, M. F. (2003a). Identification of genetic diversity in the Ascochyta blight pathogen of chickpea [Ascochyta rabiei (Pass.) Lab.] using RAPD markers. Isfahan University of Technology-Journal of Crop Production and Processing, 7(2), 193-204.
Shokouhifar, F., Bagheri, A., Falahat-Rastegar, M. and Malekzadeh-Shafaroodi, S. (2003b). Pathotyping of Ascochyta rabiei isolates in Iran. Jornal of Agricultural Sciences and Natural Resources, 37, 217-232.
Shokouhifar, F., Bagheri, A. and Falahati-Rastegar, M. (2006). Identification of resistant chickpea lines against pathotypes causing Ascochyta blight disease in Iran. Iranian Journal of Biology, 19, 29-42.
Singh, R., Kumar, K., Purayannur, S., Chen, W., & Verma, P. K. (2022). Ascochyta rabiei: A threat to global chickpea production. Molecular Plant Pathology, 23(9), 1241-1261. https://doi.org/10.1111/mpp.13235
Singh, R., Kumar, K., Purayannur, S., & Verma, P. K. (2023). Genomics-assisted genetics of complex regions from chickpea chromosome 4 reveals two candidate genes for Ascochyta blight resistance. Plant Science, 334, 111781. https://doi.org/10.1016/j.plantsci.2023.111781
Sudheesh, S., Kahrood, H. V., Braich, S., Dron, N., Hobson, K., Cogan, N. O., & Kaur, S. (2021). Application of genomics approaches for the improvement in Ascochyta blight resistance in chickpea. Agronomy, 11(10), 1937. https://doi.org/10.3390/agronomy11101937
Truong, H. T. H., Kim, K. T., Kim, S., Cho, M. C., Kim, H. R., & Woo, J. G. (2011). Development of gene-based markers for the Bs2 bacterial spot resistance gene for marker-assisted selection in pepper (Capsicum spp.). Horticulture, Environment, and Biotechnology, 52, 65-73. https://doi.org/10.1007/s13580-011-0142-4
Udoh, L. I., Obaseojei, W. P., & Uzoebo, C. (2021). Single nucleotide polymorphisms: a modern tool to screen plants for desirable traits. In Plant breeding-current and future views. IntechOpen. https://doi.org/10.5772/intechopen.94935
Zangene, K., Emamjomeh, A., Shokouhifar, F., Mamarabadi, M., & Mehdinezhad, N. (2022). Differentiation of an Iranian resistance chickpea line to Ascochyta blight from a susceptible line using a functional SNP. AMB Express, 12(1), 45. https://doi.org/10.1186/s13568-022-01385-y
Zhang, C., Liu, Y., Ring, B.Z., Nie, K., Yang, M., Wang, M., Shen, H., Wu, X., & Ma, X. (2013). A novel multiplex tetra-primer ARMS-PCR for the simultaneous genotyping of six single nucleotide polymorphisms associated with female cancers. PloS one, 8(4), e62126. https://doi.org/10.1371/journal.pone.0062126
 © 2023 by the authors. Licensee SCU, Ahvaz, Iran. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-Noncommercial 4.0 International (CC BY-NC 4.0 license) (http://creativecommons.org/licenses/by-nc/4.0/).