甜瓜KASP标记开发及指纹图谱构建

Development of melon KASP markers and construction of fingerprints

摘要

为丰富甜瓜分子标记类型，并为后续甜瓜种质、品种及种子纯度鉴定提供简便、高效的检测方法，分别在甜瓜每条染色体上挑选6个核心SNP位点，共72个标记，通过KASP技术对11份甜瓜种质进行基因分型，确定了45对具有较好分型效果的标记，标记设计成功率为62.5%。从45对分型较好的标记中挑选25个基因型分簇集中的最优标记，对31份甜瓜种质进行基因分型，明确了各种质在25个SNP位点的多态性，通过聚类分析，分成了3组，并构建了31份甜瓜种质的指纹图谱，为甜瓜种质资源利用和种子纯度鉴定提供了参考。

Abstract

To enrich spectrum of molecular markers in melon and offer a straightforward and efficient protocol for the subsequent identification of melon germplasm, variety, and seed purity, this investigation identified six pivotal single-nucleotide polymorphism (SNP) loci distributed across the melon genome, encompassing a total of 72 markers. Utilizing the KASP (kompetitive allele-specific PCR) assay, these markers were employed to genotype 11 melon germplasm accessions, resulting in the identification of 45 marker pairs exhibiting superior typing performance. The success rate of marker design reached 62.5%, and from these 45 pairs, the most optimal markers for the 25 identified genetic clusters were selected, and the polymorphisms of 31 muskmelon germplasm were divided into 3 groups, and the fingerprints of 31 muskmelon germplasm were constructed to provide reference for the utilization of muskmelon germplasm resources and seed purity identification.

关键词

甜瓜 / KASP / SNP / 指纹图谱

Key words

Melon / KASP / SNP / Fingerprints

参考文献

[1] 刘君璞, 马跃. 中国西瓜甜瓜发展70年暨科研生产协作60年回顾与展望[J]. 中国瓜菜, 2019, 32(8): 1-8.

[2] 张琳, 闫燊, 李琼华, 等. 2010-2020年中国甜瓜生产成本的县域定点观测数据集[J/OL]. 中国科学数据(中英文网络版), 2021, 6(4): 171-178. http://dx.doi.org/10.12205/A0017.20210901.20.ds0674.

[3] 胡建斌, 马双武, 简在海, 等. 中国甜瓜种质资源形态性状遗传多样性分析[J]. 植物遗传资源学报, 2013, 14(4): 612-619.

[4] 王倩, 任文斌, 赵智勇. 运旱系列小麦品种(系)品质功能基因KASP标记检测[J]. 中国粮油学报, 2024, 39(1): 47-53.

[5] BUROW G, CHOPRA R, HUGHES H, et al. Marker assisted selection in sorghum using KASP assay for yhe detection of single nucleotide polymorphism/insertion deletion[J]. Methods in Molecular Biology, 2019, 19(31): 75-84

[6] 赵勇, 刘晓冬, 赵洪锟, 等. 大豆SNP分型方法的比较[J]. 分子植物育种, 2017, 15(9): 3540-3546.

[7] 赵越, 孙宇峰, 徐磊, 等. KASP标记技术在作物基因定位中的应用进展[J]. 北方园艺, 2023(19): 122-127.

[8] 陆海燕, 周玲, 林峰, 等. 基于高通量测序开发玉米高效KASP分子标记[J]. 作物学报, 2019, 45(6): 872-878.

[9] MAKHOUL M, RAMBLA C, VOSS-FELS K P, et al. Overcoming polyploidy pitfalls:A user guide for effective SNP conversion into KASP markers in wheat[J]. Theoretical and Applied Genetics, 2020, 133(8): 2413-2430.

[10] STEELE K, TULLOCH M Q, BURNS M, et al. Developing KASP markers for identification of Basmati rice varieties[J]. Food Analytical Methods, 2021, 14(4): 663-673.

[11] CHEON K S, BAEK J, CHO Y, et al. Single nucleotide polymorphism (SNP) discovery and kompetitive allele-specific PCR (KASP) marker development with Korean Japonica rice varieties[J]. Plant Breeding and Biotechnology, 2018, 6(4): 391-403.

[12] CHEN Z J, TANG D G, NI J X, et al. Development of genic KASP-SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize[J]. BMC Plant Biology, 2021, 21(1): 157.

[13] 吴晶, 刘晓宇, 伏建国, 等. 濒危植物黑牡丹KASP鉴定方法的建立[J]. 安徽农业科学, 2024, 52(1): 98-100.

[14] GAO P, MA H Y, LUAN F S, et al. DNA fingerprinting of chinese melon provides evidentiary support of seed quality appraisal[J]. Plos One, 2012, 7(12): e52431.

[15] 王晋, 尤佳, 王溪桥, 等. 甜瓜品种SSR指纹图谱的构建[J]. 华北农学报, 2015, 30(增刊1): 66-68.

[16] 宋海斌, 崔喜波, 马鸿艳, 等. 基于SSR标记的甜瓜品种(系) DNA指纹图谱库的构建[J]. 中国农业科学, 2012, 45(13): 2676-2689.

[17] 王美荣, 许勇, 詹永乐, 等. 厚皮甜瓜品种组合SSR指纹图谱构建[J]. 中国农学通报, 2010, 26(20): 47-51.

[18] 刘欣, 程瑞, 徐兵划, 等. 基于KASP技术的SNP标记用于西瓜品种指纹图谱构建和种子纯度检测[J]. 江苏农业学报, 2022, 38(5): 1348-1356.

[19] LIU S, GAO P, ZHU Q L, et al. Resequencing of 297 melon accessions reveals the genomic history of improvement and loci related to fruit traits in melon[J]. Plant Biotechnology Journal, 2020, 18(12): 2545-2558.

[20] 林德佩. 中国栽培甜瓜植物的起源、分类及进化[J]. 中国瓜菜, 2010, 23(4): 34-36.

[21] PITRAT M, HANELT P, HAMMER K. Some comments on infraspecific class-sification of cultivars of melon[J]. Acta Horticulturae Sinica, 2000, 5(10): 29-36.

[22] PITRAT M. Melon[M]//RAJCAN I，VOLLMANN J. Handbook of plant breeding.Berlin:Springer, 2008, 283-315.

[23] 徐志红, 徐永阳, 刘君璞, 等. 甜瓜种质资源遗传多样性及亲缘关系研究[J]. 果树学报, 2008, 25(4): 552-558.

[24] 姚国新, 卢磊, 艾娜, 等. 遗传标记在甜瓜遗传育种中的应用[J]. 孝感学院学报, 2005, 25(6): 19-22.

[25] BERNARDO R. Molecular. markers and selection for complex traits in plants:Learning from the last 20 years[J]. Crop Science, 2008, 48(5), 1649-1664.

[26] TOAL T W, BURKART-WACO D, HOWELL T, et al. Indel group in genomes (IGG) molecular genetic markers[J]. Plant Physiology, 2016, 172(1), 38-61.

[27] HAO X P, YANG T, LIU R, et al. An RNA sequencing transcriptome analysis of grasspea (Lathyrus sativus L.) and development of SSR and KASP markers[J]. Frontiers in Plant Science, 2017, 8: 1873.

[28] 赵传超, 丛森, 梁思怡, 等. 水稻抗稻瘟病基因 Pi2的KASP标记开发与应用[J]. 华南农业大学学报, 2023, 44(5): 725-734.

[29] 王富强, 樊秀彩, 张颖, 等. SNP分子标记在作物品种鉴定中的应用和展望[J]. 植物遗传资源学报, 2020, 21(5): 1308-1320.

[30] 叶青静, 王荣青, 阮美颖, 等. 番茄枯萎病菌和颈腐根腐病菌KASP-SNP检测技术的建立[J]. 植物保护学报, 2022, 49(3): 879-889.

[31] 宋正峰, 刘树森, 夏连芹, 等. 甜瓜育种技术与方法研究进展[J]. 中国瓜菜, 2022, 35(6): 1-8.

基金资助

2023年西甜瓜良种联合攻关

宁夏农林科学院高质量发展和生态保护科技创新示范项目(NGSB-2021-7)