Development and verification of KASP marker for resistance to downy mildew in muskmelon

doi:10.6048/j.issn.1001-4330.2024.11.003

Xinjiang Agricultural Sciences ›› 2024, Vol. 61 ›› Issue (11): 2626-2634.DOI: 10.6048/j.issn.1001-4330.2024.11.003

• Germplasm Resources·Molecular Genetics·Physiology and Biochemistry·Microbes • Previous Articles Next Articles

Development and verification of KASP marker for resistance to downy mildew in muskmelon

SHEN Yue¹(), LING Yueming², DUAN Xiaoyu², YANG Wenli², LI Meihua², WANG Yirou², WANG Huilin¹(), ZHANG Xuejun²()

1. College of Horticulture,Xinjiang Agricultural University, Urumqi 830052, China
2. Hami Melon Research Center, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2024-04-19 Online:2024-11-20 Published:2025-01-08
Correspondence author: WANG Huilin, ZHANG Xuejun
Supported by:
Tianshan Innovation Team of Xinjiang Uygur Autonomous Region(2022D14015);Project of National Natural Science Foundation of China(32060689);MOF and MARA“China Agriculture Research System”(CARS-25);Hainan Provincial Academician Innovation Platform Scientific Research Special Project (Academician WU Mingzhu Team Innovation Center);Hainan Provincial Academician Innovation Platform Scientific Research Special Project(YSPTZX202141);Xinjiang Uygur Autonomous Region Key Research and Development Program for Special Projects(2022B02002)

甜瓜抗霜霉病KASP分子标记的开发与验证

沈悦¹(), 凌悦铭², 段晓宇², 杨文莉², 李寐华², 王懿柔², 王惠林¹(), 张学军²()

1.新疆农业大学园艺学院,乌鲁木齐 830052
2.新疆农业科学院哈密瓜研究中心,乌鲁木齐 830091

通讯作者: 王惠林,张学军
作者简介:沈悦(1999-),女,新疆人,硕士研究生,研究方向为甜瓜霜霉病抗性基因精细定位,(E-mail)shenyue052442@163.com
基金资助:
新疆维吾尔自治区天山创新团队(2022D14015);国家自然科学基金项目(32060689);财政部和农业农村部“国家现代农业产业技术体系资助”项目(CARS-25);海南省院士创新平台科研专项(吴明珠院士团队创新中心);海南省院士创新平台科研(YSPTZX202141);新疆维吾尔自治区重点研发任务专项(2022B02002)

Abstract

Abstract:

【Objective】 This study aims to develop and validate KASP molecular markers closely linked to melon downy mildew resistance genes, in the hope of providing a theoretical basis for downy mildew resistance molecular marker assisted breeding of downy mildew resistance in melons.【Methods】 The accession ‘PI 438685’ with high resistance of downy mildew, and the accession ‘Huangdanzi’ with high sensitivity of downy mildew were used as parents to construct a separate population.QTL mapping of downy mildew resistance genes in melon was drawn through a combination of BSA and whole genome sequencing.KASP molecular markers closely linked to downy mildew resistance genes were developed within this interval, and then screened and validated using F₂separate populations.【Results】 The downy mildew resistance gene was located on chromosome 9 through BSA analysis.We developed 66 pair of KASP molecular markers in the candidate region, screened 9 pairs of KASP molecular markers with high specificity.Subsequently, the 9 pairs of KASP molecular markers were validated, the KASP molecular marker (KASP-24832572), closely linked to downy mildew resistance genes was obtained, and its accuracy of distinguish genotype could reach 79%.【Conclusion】 One QTL related to downy mildew resistance has been located on chromosome 9 by BSA analysis, and a pair of KASP molecular markers closely linked to downy mildew resistance genes are developed in this interval, which has contributed to downy mildew resistance breeding in melons.

Key words: melon; downy mildew; BSA-seq; KASP molecular markers; molecular markers assisted breeding

摘要：

【目的】开发及验证与甜瓜霜霉病抗性基因紧密连锁的KASP分子标记,为甜瓜抗霜霉病分子标记辅助育种提供理论基础。【方法】以高抗霜霉病甜瓜品种“PI 438685”与高感霜霉病甜瓜品种“黄旦子”为亲本,构建F₂代分离群体。利用BSA-seq技术,对甜瓜霜霉病抗性基因进行QTL定位,根据SNP分布位置在定位区间内开发66对与甜瓜霜霉病抗性基因紧密连锁的KASP分子标记,并使用F₂代分离群体进行筛选验证。【结果】霜霉病抗性基因定位到9号染色体末端区域,筛选出9对在双亲间具有多态性的KASP分子标记。获得1对与甜瓜霜霉病抗性基因紧密连锁的KASP分子标记(KASP_24832572),其基因分型准确率为79%,准确度较高。【结论】在9号染色体上定位到1个与甜瓜霜霉病抗性相关的区间,在此区间内开发并筛选出1对与甜瓜霜霉病抗性基因紧密连锁的KASP分子标记,该标记有助于甜瓜抗霜霉病分子标记辅助育种。

关键词: 甜瓜, 霜霉病, BSA-seq, KASP分子标记, 分子标记辅助育种

CLC Number:

S436.5

SHEN Yue, LING Yueming, DUAN Xiaoyu, YANG Wenli, LI Meihua, WANG Yirou, WANG Huilin, ZHANG Xuejun. Development and verification of KASP marker for resistance to downy mildew in muskmelon[J]. Xinjiang Agricultural Sciences, 2024, 61(11): 2626-2634.

沈悦, 凌悦铭, 段晓宇, 杨文莉, 李寐华, 王懿柔, 王惠林, 张学军. 甜瓜抗霜霉病KASP分子标记的开发与验证[J]. 新疆农业科学, 2024, 61(11): 2626-2634.

Figures/Tables 8

References 33

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病情级别 Disease level	病情分级标准 Disease grading standards
0	无任何症状
1	病斑面积占整个叶片面积的1/10以下
2	病斑面积占整个叶片面积的1/10 ~ 1/4
3	病斑面积占整个叶片面积的1/4 ~ 2/4
4	病斑面积占整个叶片面积的2/4 ~ 3/4
5	病斑面积占整个叶片面积的3/4 以上或者叶片干枯

病情级别 Disease level	病情分级标准 Disease grading standards
0	无任何症状
1	病斑面积占整个叶片面积的1/10以下
2	病斑面积占整个叶片面积的1/10 ~ 1/4
3	病斑面积占整个叶片面积的1/4 ~ 2/4
4	病斑面积占整个叶片面积的2/4 ~ 3/4
5	病斑面积占整个叶片面积的3/4 以上或者叶片干枯

样品 Sample	原始数据量 Raw Base(bp)	有效数据量 Clean Base(bp)	Q20(%)	Q30(%)	GC总含量 GC Content(%)	比对率 Mapping rate(%)
huangdanzi	5 300 455 200	5 166 262 500	96.4	90.7	36.81	94.32
PI 438685	5 010 953 100	4 957 239 900	96.81	91.45	37.14	93.18
3-0	10 016 053 800	9 928 662 900	97.41	93.02	36.88	95.66
3-5	9 512 647 800	9 396 773 400	97.12	92.44	37.45	95.86

样品 Sample	原始数据量 Raw Base(bp)	有效数据量 Clean Base(bp)	Q20(%)	Q30(%)	GC总含量 GC Content(%)	比对率 Mapping rate(%)
huangdanzi	5 300 455 200	5 166 262 500	96.4	90.7	36.81	94.32
PI 438685	5 010 953 100	4 957 239 900	96.81	91.45	37.14	93.18
3-0	10 016 053 800	9 928 662 900	97.41	93.02	36.88	95.66
3-5	9 512 647 800	9 396 773 400	97.12	92.44	37.45	95.86

引物名称 Primer name	序列(5’-3’) Sequence (5’-3’)			是否具有特异性 Is it polymorphic
KASP_23055782	GAAGGTGACCAAGTTCATGCTAAATTATTCAAGTTGCCAAACTCCTTGATA			是
KASP_23063365	GAAGGTGACCAAGTTCATGCTCTTAGACAAAACTCTCCCACCTC			否
KASP_23067310	GAAGGTGACCAAGTTCATGCTGAAAATTTTGGGGAGATTGAGAAGAGA			是
KASP_23070054	GAAGGTGACCAAGTTCATGCTATTTCCTTAGTTTTACAACGGACCATG			是
KASP_23071460	GAAGGTGACCAAGTTCATGCTGGAAAGGATCTTTTAATATACTTTAGACCT			是
KASP_23080398	GAAGGTGACCAAGTTCATGCTAATAAAGAAAAGAAAGAGGAAGAGGATTGA			是
KASP_23092852	GAAGGTGACCAAGTTCATGCTAGTGTTTTGTGACAAACATCATACCGA			否
KASP_23093210	GAAGGTGACCAAGTTCATGCTGGCAACATAGTCCTACTCTAACTCA			是
KASP_23094651	GAAGGTGACCAAGTTCATGCTACAATTGGGGTAATAAGGAAGTGGA			否
KASP_23112033	GAAGGTGACCAAGTTCATGCTCACACACACACACGGTTTAGAGAA			是
KASP_23120772	GAAGGTGACCAAGTTCATGCTCATCGATACCAAATTTGTAAGCAATCATAAT			是
KASP_23247274	GAAGGTGACCAAGTTCATGCTGACGAGCAAAATCCAGGGTGG			是
KASP_23299420	GAAGGTGACCAAGTTCATGCTTTGTCTACCTCTTCTCCTCT			否
KASP_23308722	GAAGGTGACCAAGTTCATGCTCATGGAAACGTGATCAATGAAGCGA			是
KASP_23399433	GAAGGTGACCAAGTTCATGCTGTGTTCATTATGCTTTAAGGCTATTGC			是
KASP_23399886	GAAGGTGACCAAGTTCATGCTGAAAGTTCTATGGTTCTCTGAGCC			是
KASP_23401162	GAAGGTGACCAAGTTCATGCTGGTAGAAATGCTTAACAATCATCTTCCA			是
KASP_23401360	GAAGGTGACCAAGTTCATGCTAAGCACAGCAAAGAGTTCAGATTCC			否
KASP_23402938	GAAGGTGACCAAGTTCATGCTGCAATGCAATTCCTTAAAGGTGCTG			否
KASP_23404381	GAAGGTGACCAAGTTCATGCTATGTCTTCCGCTTTTATGAGTTGAAAC			否
KASP_23406473	GAAGGTGACCAAGTTCATGCTAATTTAATTTCACTTCCCTTTTATTTCACCAT			是
KASP_23408092	GAAGGTGACCAAGTTCATGCTATTTAGTACCTTCTAAAGATGGTGCATTAT			是
KASP_23420662	GAAGGTGACCAAGTTCATGCTATGAATCTACTTCGCATAAGTTCCATC			是
KASP_23470437	GAAGGTGACCAAGTTCATGCTCGAAGAAATCTGCACCGTCCTG			是
KASP_23470767	GAAGGTGACCAAGTTCATGCTTTGCCTACGTTTGTATGGAAGCTG			否
KASP_23470860	GAAGGTGACCAAGTTCATGCTCGAAGCTATTCTCTGGCAACTCA			否
KASP_23473896	GAAGGTGACCAAGTTCATGCTATTTCATGGGAAATGGAAGTCAAATGC			是
KASP_23476195	GAAGGTGACCAAGTTCATGCTAACCTGCAACCCATTTCAACATTGTA			是
KASP_23517447	GAAGGTGACCAAGTTCATGCTCCAAAAATAGGTCCTTGTTAGTGCAC			否
KASP_23518588	GAAGGTGACCAAGTTCATGCTCAAATTTCAATTTAAGTGATTCTAGCAAATAC			否
KASP_23518973	GAAGGTGACCAAGTTCATGCTGACATTGCGACTACTACCAATGGTT			否
KASP_23526537	GAAGGTGACCAAGTTCATGCTGGAGGCAAGAACTCCTAGACACA			否
KASP_23527871	GAAGGTGACCAAGTTCATGCTTAGACTTCTCAAAACCAACGGCA			否
KASP_23530097	GAAGGTGACCAAGTTCATGCTCGTGAATGTGAAAAAGAAAGACAAAAAAG			否
KASP_23530714	GAAGGTGACCAAGTTCATGCTACCAACTTTTAGAGACACTTCACGC			是
KASP_23554586	GAAGGTGACCAAGTTCATGCTAGACTAGTTGAATTATGCTCGAGTTCA			是
KASP_23592188	GAAGGTGACCAAGTTCATGCTTTAATGTGATGAACAACAACCTCCAC			否
KASP_23623803	GAAGGTGACCAAGTTCATGCTAGAAGTCTAAGCAACACATACAGAG			否
KASP_23630472	GAAGGTGACCAAGTTCATGCTGTTTGTTGAAATAGACACCAAAGGAAG			是
KASP_23693029	GAAGGTGACCAAGTTCATGCTGAACTTTGGAATTGTAATTGCTTGCTTAATT			否
KASP_23757315	GAAGGTGACCAAGTTCATGCTAGGTGAGGAATAAGATGTGCTAGC			是
KASP_23774894	GAAGGTGACCAAGTTCATGCTCAGGACTCTGTAATTAGCAAGAGAAC			否
KASP_23779883	GAAGGTGACCAAGTTCATGCTGGTGGGGAAGTGGGTTAGGC			否
KASP_23789334		GAAGGTGACCAAGTTCATGCTAAACGCTCCCCTAATAAATAGACCC	否
KASP_23789363		GAAGGTGACCAAGTTCATGCTTGTAGACAGGTAGACAAGATTCAACA	是
KASP_23789561		GAAGGTGACCAAGTTCATGCTTCACCCCTTGGAGTCATTGGG	是
KASP_23794137		GAAGGTGACCAAGTTCATGCTATCTTTCAGTGTTCAAAAGGACTGGAA	否
KASP_23796506		GAAGGTGACCAAGTTCATGCTCTCCTTAACCTTTTTCCCAAATTTTCT	是
KASP_23797202		GAAGGTGACCAAGTTCATGCTCTCTTTTATGATGTTCGTTCTCTA	否
KASP_23797386		GAAGGTGACCAAGTTCATGCTCCTCGCAATGAGTCAAAACAGATG	否
KASP_23797740		GAAGGTGACCAAGTTCATGCTGGAAAGGAAGATAACGATTCAGATAAGTT	是
KASP_23798623		GAAGGTGACCAAGTTCATGCTAAGCCTAGACATATTGGTACAATAACG	是
KASP_23799658		GAAGGTGACCAAGTTCATGCTAACGATAATGCAGAAGATCATGTTGAC	是
KASP_23855524		GAAGGTGACCAAGTTCATGCTTAGAAGAGAATTAAATGTTGGCGGCA	否
KASP_23891190		GAAGGTGACCAAGTTCATGCTAAATGAATAAAATACTCCACGTTTAGAGG	否
KASP_23898936		GAAGGTGACCAAGTTCATGCTAAATGTGCTCTAAATATGTTTCTTCACATG	否
KASP_23903385		GAAGGTGACCAAGTTCATGCTAAATTGACGAGTTTTTGATCATTTTATGGG	否
KASP_23907245		GAAGGTGACCAAGTTCATGCTAAGAAAATGGCGAGGAAGAAAGAGAAA	否
KASP_23934209		GAAGGTGACCAAGTTCATGCTCATACCCTCTATAGCCACAAGTACAA	是
KASP_23940394		GAAGGTGACCAAGTTCATGCTAAGAAAAGAAATGAATGGACACAC	是
KASP_23999679		GAAGGTGACCAAGTTCATGCTCAAATTTTTGTGTATAGGAACTGCGC	否
KASP_24032929		GAAGGTGACCAAGTTCATGCTATGCATTTCTCTTTCATATGTAATCTCTTG	否
KASP_24060858		GAAGGTGACCAAGTTCATGCTGTTCAGCCCAAAAGTCCATCAAGTA	是
KASP_24065711		GAAGGTGACCAAGTTCATGCTCTCCTTGATGTTCTTTCTCACCA	否
KASP_24066737		GAAGGTGACCAAGTTCATGCTCATGTTATGAATGTGGAGGGAGCT	否
KASP_24832572		GAAGGTGACCAAGTTCATGCT CTCCACTGGTTTAGGAGGAAGA	是

Development and verification of KASP marker for resistance to downy mildew in muskmelon

甜瓜抗霜霉病KASP分子标记的开发与验证

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