番茄抗马铃薯孢囊线虫 HeroA 基因的分子检测

Molecular detection of the tomato HeroA gene conferring resistance to potato cyst nematodes

摘要

[目的]HeroA是一个对马铃薯孢囊线虫多个致病型具有广谱抗性的抗病基因,为了快速准确地鉴定不同番茄品种中是否携带该基因,从而为番茄抗性品种的合理布局及遗传育种提供指导,本文建立了一种基于SCAR-PCR的特异性分子检测技术。[方法]以马铃薯孢囊线虫抗性品系‘LA1792’和感病品种‘Moneymaker’的DNA样本为模板,扩增HeroA及其同源基因序列,设计并筛选特异性靶向HeroA的引物组合,优化PCR反应条件并测定其检测灵敏度。在此基础上进一步以30个不同番茄品种为检测对象,利用新建立的SCAR-PCR技术对HeroA进行检测,同时与已报道的方法进行比较。[结果]筛选获得的HERO-F9/HERO-R11引物组合,在62℃退火温度下,能够在马铃薯孢囊线虫抗性品系‘LA1792’样本中特异性扩增出约700bp的条带,而在感病品种‘Moneymaker’中无扩增条带,且检测灵敏度达到0.1ng·μL^-1DNA模板;对30个不同番茄品种的测试表明,SCAR-PCR检测结果与已报道的方法检测结果完全一致,其中‘红果’‘大红’‘黑金刚’等11个品种含有马铃薯孢囊线虫抗性基因HeroA,而其他19个品种均不含有该基因。[结论]本研究为马铃薯孢囊线虫抗性基因HeroA的分子检测提供更加准确的方法。

Abstract

[Objectives]HeroA is a broad-spectrum resistance gene against potato cyst nematodes. To rapidly and accurately identify tomato varieties that carry the HeroA gene, and to provide guidance for the rational deployment of varieties and genetic breeding programs, this study developed a specific molecular detection technique based on sequence characterized amplified region-polymerase chain reaction (SCAR-PCR). [Methods] Genomic DNA samples from the potato cyst nematode-resistant line ‘LA1792’ and susceptible variety ‘Moneymaker’ were used as templates to amplify and align the sequences of theHeroA gene and its homologs. Primer pairs specific to HeroA gene were then screened. The PCR reaction conditions were optimized and the detection sensitivity was also evaluated. The developed SCAR-PCR technique was subsequently applied to detect the HeroA gene in 30 different tomato varieties. The results were compared with those obtained from previously reported methods to assess the performance of this technique. [Results] The HERO-F9/HERO-R11 primer combination specifically amplified a 700-bp band from the DNA samples of LA1792 at an annealing temperature of 62℃, whereas no amplification was observed in the susceptible variety Moneymaker. The detection sensitivity of this assay reached up to 0.1 ng·μL^-1 of template DNA. Testing across 30 different tomato varieties demonstrated that the detection results of SCAR-PCR developed in this study were entirely consistent with those using previously reported methods. The results revealed that 11 varieties, including ‘Hongguo’, ‘Dahong’ and ‘Heijingang’, carry the potato cyst nematode resistance gene HeroA, while the remaining 19 varieties lacked this gene. [Conclusions] The present study provides a more accurate method for the molecular detection of the potato cyst nematode resistance geneHeroA in tomato plants.

关键词

马铃薯孢囊线虫 / 番茄 / 抗性基因 / HeroA基因 / 分子检测

Key words

potato cyst nematodes / tomato / resistance gene / HeroA / molecular detection

参考文献

[1] Price JA, Coyne D, Blok VC, et al. Potato cyst nematodes Globodera rostochiensis and G. pallida [J]. Molecular Plant Pathology, 2021, 22(5): 495-507.

[2] Moxnes JF, Hausken K. The population dynamics of potato cyst nematodes[J]. Ecological Modelling, 2007, 207(2/3/4): 339-348.

[3] Varandas R, Egas C, Conceição IL. Potato cyst nematodes: new solutions to an old problem[J]. Crop Protection, 2020, 137: 105303.

[4] 农业农村部.中华人民共和国农业农村部公告第351号[EB/OL]. (2020-11-04)[2022-06-25]. https://www.gov.cn/zhengce/zhengceku/2020-11/12/content_5560727.htm.

[5] Ministry of Agriculture and Rural Affairs. Announcement No. 351 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China. [EB/OL]. (2020-11-04)[2022-06-25]. https://www.gov.cn/zhengce/zhengceku/2020-11/12/content_5560727.htm(in Chinese).

[6] Orlando V, Boa E. Potato cyst nematodes: a persistent and fearsome foe[J]. Plant Pathology, 2023, 72(9): 1541-1556.

[7] Peng DL, Liu H, Peng H, et al. First detection of the potato cyst nematode (Globodera rostochiensis) in a major potato production region of China [J]. Plant Disease, 2022, 107(1): 233.

[8] Jiang R, Peng H, Li YQ, et al. First record of the golden potato nematode Globodera rostochiensis in Yunnan and Sichuan Provinces of China [J]. Journal of Integrative Agriculture, 2022, 21(3): 898-899.

[9] Jiang R, Li YQ, Huang LQ, et al. Association of Globodera rostochiensis (Nematoda) with stunted and chlorotic potato plants in Yunnan and Sichuan Provinces in China [J]. Plant Disease, 2023, 107(4): 1027-1034.

[10] 邓春菊, 李艳, 杨毅娟, 等. 云南省马铃薯孢囊线虫种类鉴定[J]. 云南农业大学学报(自然科学), 2023, 38(3): 400-408.

[11] Deng CJ, Li Y, Yang YJ, et al. Identification of potato cyst nematodes (Globodera rostochiensis) on potato in Yunnan Province [J]. Journal of Yunnan Agricultural University (Natural Science), 2023, 38(3): 400-408(in Chinese with English abstract).

[12] 顾建锋, 邵宝林, 方亦午, 等. 四川省马铃薯孢囊线虫的形态和分子鉴定[J]. 福建农业学报, 2022, 37(4): 520-528.

[13] Gu JF, Shao BL, Fang YW, et al. Morphological and molecular identification of potato cyst nematode from Sichuan[J]. Fujian Journal of Agricultural Sciences, 2022, 37(4): 520-528(in Chinese with English abstract).

[14] Sukhanova AA, Boyandin AN, Ertiletskaya NL. Potato cyst nematode management strategies: current situation and promising approaches[J]. ACS Agricultural Science & Technology, 2022, 2(3): 415-425.

[15] Bakker E, Achenbach U, Bakker J, et al. A high-resolution map of the H1 locus harbouring resistance to the potato cyst nematode Globodera rostochiensis [J]. Theoretical and Applied Genetics, 2004, 109: 146-152.

[16] Gartner U, Hein I, Brown LH, et al. Resisting potato cyst nematodes with resistance[J]. Frontiers in Plant Science, 2021, 12: 661194.

[17] Ellis PR, Maxon Smith JW. Inheritance of resistance to potato cyst-eelworm (Heterodera rostochiensis Woll.) in the genus Lycopersicon [J]. Euphytica, 1971, 20: 93-101.

[18] Ganal MW, Simon R, Brommonschenkel S, et al. Genetic mapping of a wide spectrum nematode resistance gene (Hero) against Globodera rostochiensis in tomato [J]. Molecular Plant-Microbe Interactions, 1995, 8(6): 886-891.

[19] Uehara T, Itou K, Narabu T. Differences in parasitism of potato cyst nematode, Globodera rostochiensis, to tomato varieties and strains of relative wild species [J]. Japanese Journal of Applied Entomology and Zoology, 2008, 52(3): 146-148.

[20] Cabrera PHL, Manzani lla López RH, Kanyuka K. Functionality of resistance gene Hero, which controls plant root-infecting potato cyst nematodes, in leaves of tomato [J]. Plant, Cell & Environment, 2006, 29(7): 1372-1378.

[21] Uehara T, Narabu T, Ito K, et al. Detection of the potato cyst nematode resistance gene HeroA in Japanese tomato cultivars by the PCR-RFLP method [J]. Nematological Research, 2015, 45(2): 115-120.

[22] 申恒, 刘思慧, 李跃, 等. 一种用于PCR的番茄DNA快速粗提方法[J]. 生物技术通报, 2022, 38(6): 74-80.

[23] Shen H, Liu SH, Li Y, et al. Rapid crude extraction of genomic DNA from Solanum lycopersicum for PCR [J]. Biotechnology Bulletin, 2022, 38 (6): 74-80(in Chinese with English abstract).

[24] Løvdal T, Lillo C. Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress[J]. Analytical Biochemistry, 2009, 387(2): 238-242.

[25] Benson DA, Cavanaugh M, Clark K, et al. GenBank[J]. Nucleic Acids Research, 2018, 46(1): 41-47.

[26] 黄骥, 张红生. 基于Windows的核酸序列分析软件的开发[J]. 生物信息学, 2004, 2(1): 13-17.

[27] Huang J, Zhang HS. Development of nucleotide sequence analysis software based on windows[J]. Bioinformatics, 2004, 2(1): 13-17 (in Chinese).

[28] van der Vossen EA, van der Voort JN, Kanyuka K, et al. Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode[J]. The Plant Journal, 2000, 23(5): 567-576.

[29] Paal J, Henselewski H, Muth J, et al. Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach [J]. The Plant Journal, 2004, 38(2): 285-297.

[30] Trifonova ZL, Sotirova V, Voulkova ZL. Resistance of wild tomato species to Globodera rostochiensis [J]. Nematologica, 1995, 41(1): 141-142.

[31] Dangl JL, Jones JD. Plant pathogens and integrated defence responses to infection[J]. Nature, 2001, 411(6839): 826-833.

[32] 刘云飞, 万红建, 李志邈, 等. 植物NBS-LRR抗病基因的结构、功能、进化起源及其应用[J]. 分子植物育种, 2014, 12(2): 377-389.

[33] Liu YF, Wan HJ, Li ZM, et al. Analysis of plant NBS-LRR resistance gene: structure, function, origin, evolution and their application[J]. Molecular Plant Breeding, 2014, 12(2): 377-389(in Chinese with English abstract).

[34] Ernst K, Kumar A, Kriseleit D, et al. The broad-spectrum potato cyst nematode resistance gene (Hero) from tomato is the only member of a large gene family of NBS-LRR genes with an unusual amino acid repeat in the LRR region [J]. The Plant Journal, 2002, 31(2): 127-136.

[35] 明会, 蒋伟, 刘太红, 等. 利用分子标记筛选马铃薯抗孢囊线虫资源[J]. 植物遗传资源学报, 2023, 24(4): 1194-1204.

[36] Ming H, Jiang W, Liu TH, et al. Molecular markers assisted identification of potato resources resistant to cyst nematode[J]. Journal of Plant Genetic Resources. 2023, 24(4): 1194-1204(in Chinese with English abstract).

[37] 黄立强, 江如, 朱波汁, 等. 马铃薯主栽品种抗马铃薯金线虫鉴定及抗性分子标记检测[J]. 中国农业科学, 2024, 57(8): 1506-1516.

[38] Huang LQ, Jiang R, Zhu BZ, et al. Identification and evaluation of major potato cultivars resistance to Globodera rostochiensis and detection of their H1 resistance gene marker [J]. Scientia Agricultura Sinica, 2024, 57(8): 1506-1516(in Chinese with English abstract).

基金资助

国家重点研发计划项目(2022YFD1100200)

国家自然科学基金项目(32272497)