马铃薯育种材料PVX抗性基因的检测与鉴定

Detection and Identification of Potato Virus X (PVX) Resistance Genes in Potato Breeding Materials

摘要

马铃薯病毒是影响马铃薯品种退化的根本原因。鉴定国内外马铃薯育种材料中与PVX抗性基因相关的SCAR分子标记，对筛选评价出抗马铃薯X病毒（Potato virus X，PVX）的育种材料具有重要意义。以76个马铃薯品种（系）为参试材料，对马铃薯PVX抗性基因Rx1、Rx2和超敏基因Nb相关SCAR标记进行分析的同时，在受控条件下，人工接种马铃薯PVX，并通过酶联免疫吸附法（ELISA）检测确认病毒感染状况。分子标记检测结果发现，除在3个马铃薯材料的遗传物质中未检测到任何抗PVX基因的分子标记，其余所测马铃薯品种（系）均拥有1~4个与抗PVX基因有关的分子标记，带有Rx和Nb基因的马铃薯材料分别占比29%和15%；带有Rx和Nb复合基因的材料占比52%。对筛选后的23个马铃薯品种（系）人工接种PVX后并经ELISA检测法检测确认发现19个品种（系）表现出对PVX的高度抗性，具体表现为无表型症状以及ELISA试验结果呈阴性；‘Miras’‘Pamyati Kunaeva’和‘KC-4’表现出PVX耐受性，即在叶片组织中检测到PVX，但无表型症状。筛选出的对PVX具有高度抗性的品种（系）可纳入后续马铃薯新品种育种计划，为国内外培育抗PVX马铃薯新品种（系）提供分子标记辅助选择的理论依据。

Abstract

Potato viruses are the fundamental cause of potato variety degeneration. Identifying SCAR molecular markers associated with PVX resistance genes in potato breeding materials at home and abroad is crucial for screening and evaluating germplasm resistant to PVX. Utilizing 76 potato varieties (lines) as test materials, this study analyzed SCAR markers linked to PVX resistance genes Rx1, Rx2, and the hypersensitivity gene Nb. Concurrently, under controlled conditions, materials were artificially inoculated with PVX, and viral infection status was confirmed via enzyme-linked immunosorbent assay (ELISA). Molecular marker analysis revealed that except for three materials lacking detectable markers for any PVX resistance genes, all other tested varieties (lines) possessed one to four markers associated with PVX resistance genes. Materials carrying Rx genes and Nb gene accounted for 29% and 15% of the total, respectively, while 52% contained both Rx and Nb genes. Following screening, 23 potato varieties (lines) were artificially inoculated with PVX and confirmed by ELISA. Among these, 19 varieties (lines) exhibited high resistance to PVX, characterized by the absence of phenotypic symptoms and negative ELISA results. Three materials ('Miras', 'Pamyati Kunaeva' and 'KC-4') demonstrated tolerance to PVX, as PVX was detected in leaf tissues, yet no phenotypic symptoms were observed. The highly PVX-resistant varieties (lines) identified could be incorporated into subsequent potato breeding programs, providing a theoretical foundation for molecular marker-assisted selection in developing PVX-resistant potato varieties domestically and internationally.

关键词

马铃薯X病毒 / 马铃薯品系 / 分子标记 / 病毒侵染 / 抗病毒性

Key words

potato virus X / breeding line / molecular marker / infection / resistance

参考文献

[1] 王红艳. 烟草脉带花叶病毒和马铃薯X病毒的协生作用及二者侵染本氏烟的转录组学分析[D]. 泰安: 山东农业大学, 2017.

[2] 杨亚东, 杜娅婷, 杜歆仪, 等. 中国马铃薯农户种植意愿及其空间差异[J]. 中国农业资源与区划, 2022, 43(2): 220-230.

[3] 罗涛. PVY、PVX、PLRV马铃薯病毒数字PCR检测体系建立[D]. 武汉: 华中农业大学, 2023.

[4] Gulnazym O, Vadim K, Svetlana K, et al. Effect of infection of potato plants by potato virus Y (PVY), potato virus S (PVS), and potato virus M (PVM) on content and physicochemical properties of tuber starch[J]. Food Science and Nutrition, 2023, 11(7): 4002-4009.

[5] Liu J, Liu Y, Fang Y, et al. Evaluation of potato virus X resistance in potato cultivars and identification of an innate immunity-independent resistance phenotype[J]. Phytopathology Research, 2021, 3(1): 21-32.

[6] 卢瑶, 金鑫, 沈日敏, 等. 马铃薯抗晚疫病及病毒病种质资源分子标记辅助筛选[J]. 中国马铃薯, 2024, 38(4): 289-299.

[7] Boris K V, Kochieva E Z. NBS-LRR resistance genes to potato virus X[J]. Uspekhi Sovremennoi Biologii, 2013, 133(2): 124-132.

[8] Kohm B A, Goulden M G. A potato virus X resistance gene mediates an induced, nonspecific resistance in protoplasts[J]. Plant Cell, 1993, 5(8): 913-920.

[9] Smith J, Li J, Zheng J, et al. Improving detection of potato virus X resistance genes Rx1 and Rx2 in tetraploid potato using optimized PCR and KASP markers[J]. Plant Biotechnology Journal, 2023, 21(5): 1024-1035.

[10] 刘程, 王世尧, 史伟玲, 等. 马铃薯抗晚疫病基因R8、RB和抗病毒病基因Rx1、Ryadg的多重PCR检测[J]. 园艺学报, 2021, 48(2): 389-396.

[11] Kasai K, Morikawa Y, Sorri V A, et al. Development of SCAR markers to the PVY resistance gene Ryadg based on a common feature of plant disease resistance genes[J]. Genome, 2000, 43(1): 1-8.

[12] 谢芳茹, 毕恒翊, 傅欣雨, 等. 北方晚熟马铃薯病毒抗性鉴定及分子标记检测[J]. 中国马铃薯, 2023, 37(1): 26-35.

[13] Bonny S Q, Hossain M A M, Uddin S M K, et al. Current trends in polymerase chain reaction based detection of three major human pathogenic vibrios[J]. Critical Reviews in Food Science and Nutrition, 2020, 62(5): 1317-1335.

[14] Bendahmane A, Kanyuka K, Baulcombe D C. The Rx gene from potato controls separate virus resistance and cell death responses[J]. The Plant Cell, 1999, 11(5): 781-792.

[15] Ahmadvand R, Wolf I, Gorji M A, et al. Development of molecular tools for distinguishing between the highly similar Rx1 and Rx2 PVX extreme resistance genes in tetraploid potato[J]. Potato Research, 2013, 56(4): 277-291.

[16] 刘佳慧. 东北马铃薯主栽品种对PVX抗性鉴定及抗病机制分析[D]. 哈尔滨: 东北农业大学, 2020.

[17] Liu X, Cheng J, Mei Z, et al. SCAR marker for identification and discrimination of specific medicinal Lycium chinense Miller from Lycium species from ramp-PCR RAPD fragments[J]. 3 Biotech, 2020, 10(8): 328-334.

[18] Yoh H K, Yien S C Y, Ong J A, et al. Development of species-specific SCAR markers for identification and authentication of three rare Peninsular Malaysian endemic Coelogyne (Orchidaceae) orchids[J]. F1000 Research, 2021, 65(8): 1161-1171.

[19] Brigneti G, Garcia-Mas J, Baulcombe D C. Molecular mapping of the potato virus Y resistance gene Rysto in potato[J]. Theoretical and Applied Genetics, 1997, 94(1): 198-203.

[20] Szajko K, Strzelczyk-Żyta D, Marczewski W. Ny-1 and Ny-2 genes conferring hypersensitive response to potato virus Y (PVY) in cultivated potatoes: mapping and marker-assisted selection validation for PVY resistance in potato breeding[J]. Molecular Breeding, 2014, 34(1): 267-271.

[21] 张微. 酶联免疫吸附法(DAS-ELISA)检测主要马铃薯病毒[C]// 屈冬玉, 陈伊里. 马铃薯产业与脱贫攻坚. 哈尔滨: 哈尔滨地图出版社, 2018: 438-441.

[22] Nyalugwe E P, Wilson C R, Coutts B A, et al. Biological properties of potato virus X in potato: Effects of mixed infection with potato virus S and resistance phenotypes in cultivars from three continents[J]. Plant Disease, 2012, 9(1): 43-54.

[23] Anisimov B V, Belov G L, Varitsev Y A. Protection of potatoes from diseases, pests and weeds[J]. Potato Grower, 2009, 3(1): 272-278.

[24] Özkaynak E. Using molecular markers to improve potato lines resistant to pathogens[J]. Selcuk Journal of Agriculture and Food Sciences, 2024, 38(2): 193-204.

[25] Shaikhaldein H O, Hoffmann B, Alaraidh I A, et al. Evaluation of extreme resistance genes of potato virus X (Rx1 and Rx2) in different potato genotypes[J]. Journal of Plant Diseases and Protection, 2018, 125(3): 251-257.

[26] Xu Y, Liu F, Zhu S, et al. The maize NBS-LRR gene ZmNBS25 enhances disease resistance in rice and Arabidopsis[J]. Front Plant Science, 2018, 1033(9): 1-13.

基金资助

2023年内蒙古中哈合作“一带一路”广适马铃薯新品种选育项目(2023KJHZ0011)