基于BSA-seq和RNA-seq技术挖掘黄瓜白粉病抗性基因

doi:10.7668/hbnxb.20195359

摘要/Abstract

摘要：

黄瓜白粉病是危害黄瓜生产的主要病害之一,制约了黄瓜的安全生产。定位黄瓜抗白粉病相关基因,有助于理解黄瓜对白粉病抗性的遗传规律和分子机制,也为抗病育种提供了多样化的基因资源。以黄瓜抗病自交系QK和感病自交系QG为亲本,构建了QK×QG的 F₁、F₂ 群体。采用极端性状混池重测序(BSA-seq)的方法对黄瓜白粉病抗性基因进行初步定位,结合转录组数据和基因注释信息对抗病表型关联区间进行缩短,发掘序列变异,筛选关键基因。人工接种结果显示:F₁ 植株全部表现感病,说明白粉病杭性可能由隐性基因控制;F₂群体表现出从抗病到感病的连续正态分布。利用BSA-seq、SNP-Index 方法和QTG-seq法,分析结果的交集为5号染色体的19—21 Mb区段,该区段共有77个注释基因在样本间存在SNP差异,其中非同义突变共有33个。转录组测序(RNA-seq)结果显示,感病材料中共有309个上调基因,697个下调基因。在5号染色体的候选区段内有13个基因的表达量在接病后差异显著,基于差异表达基因和BSA分析结果将该区段内的候选基因缩减为3个,其中仅LOC101207011基因内检测到SNP突变。候选基因LOC101207011是由第656位的Val突变为了Leu,实时荧光PCR也验证该基因与黄瓜抗白粉病具有连锁关系。

关键词: 黄瓜, BSA-seq, RNA-seq, 白粉病, 定位

Abstract:

Cucumber powdery mildew is one of the main diseases that adversely impacts cucumber production,posing a significant challenge to its sustainable cultivation.Identifying genes related to cucumber resistance to powdery mildew can help understand the genetic principles and molecular mechanisms of cucumber resistance to powdery mildew,and provide diverse gene resources for disease resistant breeding.This study constructed F₁ and F₂ populations of QK×QG using cucumber resistant inbred line QK and susceptible inbred line QG as parents.Using the extreme trait mixed pool resequencing (BSA-seq) method,the genomic regions harboring the resistance genes of cucumber powdery mildew were preliminarily located.By integrating transcriptome data with gene annotation information,the association interval of the disease phenotype was narrowed,sequence variations were identified,and key genes were screened.The results showed that the resistance of powdery mildew may be controlled by recessive genes,and the population of F₂ showed a continuous normal distribution from resistant to susceptible.The BSA-seq analysis,combining the SNP-Index method and QTG(quantitative trait genomics)-seq method analysis highlighted the 19—21 Mb region of chromosome 5,where there were 77 annotated genes with SNP differences between samples,including 33 non-synonymous mutations.The transcriptome sequencing (RNA-seq) results showed that there were 309 upregulated genes and 697 downregulated genes in the susceptible material.The expression levels of 13 genes within the candidate segment of chromosome 5 showed significant differences after infection.Through a comprehensive analysis of differentially expressed genes and BSA,the candidate genes in this segment were narrowed to 3,and only SNP mutations were detected in the LOC101207011 gene.The candidate gene LOC101207011 was characterized by a mutation resulting in an amino acid change from Valine at position 656 to Leucine.This gene emerges as the primary focus of our investigation due to its potential role in conferring resistance to powdery mildew.

Key words: Cucumber, BSA-seq, RNA-seq, Powdery mildew, Mapping

秦垒, 张巧英, 孙雷, 石昊颖, 檀海斌, 石学萍. 基于BSA-seq和RNA-seq技术挖掘黄瓜白粉病抗性基因[J]. 华北农学报, 2025, 40(2): 153-160. doi: 10.7668/hbnxb.20195359.

QIN Lei, ZHANG Qiaoying, SUN Lei, SHI Haoying, TAN Haibin, SHI Xueping. Gene Mining of Resistance to Powdery Mildew Based on BSA-seq and RNA-seq in Cucumber[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 153-160. doi: 10.7668/hbnxb.20195359.

http://www.hbnxb.net/CN/Y2025/V40/I2/153

图/表 13

表1 遗传表型分析

Tab.1 Genetic phenotype analysis

子代 Generations	合计/株 Total	感病植株/株 Disease-type plants	抗病植株/株 Resistance-type plants	分离率 Segregation ratio	卡方(χ²) Chi-square(χ²)
P₁(QG)	50	50	0
P₂(QK)	50	0	50
F₁(P₁×P₂)	30	30	0
F₁(P₂×P₁)	30	30	0
BC₁(F₁×P₁)	60	60	0
BC₁(F₁×P₂)	90	46	44	1.05:1	0.04
F₂(F₁×F₁)	292	220	72	3.06:1	0.02

图1 F₂群体白粉病病情指数分布图

Fig.1 Distribution map of powdery mildew disease index in F₂

表2 质控后测序数据与参考基因组比对结果分析

Tab.2 Matching of quality control data with referene genome

子代 Generations	过滤后序列数/个 Clean reads	比对上数据/ 个 Mapping reads	比对率/ % Mapping rate	平均测序深度/X Average sequencing depth	覆盖度/% Coverage	4× 覆盖度/% Coverage ≥4×	10× 覆盖度/% Coverage ≥10×	20× 覆盖度/% Coverage ≥20×
感病亲本	61 968 587	61 783 729	99.70	33.23	98.74	97.52	92.08	48.84
Disease-susceptible parent (QG)
抗病亲本	57 041 607	56 758 890	99.50	30.38	98.65	97.13	87.18	29.11
Disease-resistant parent (QK)
高感子代池	109 767 621	109 159 849	99.45	55.80	99.10	98.42	96.78	87.57
Highly susceptible pool
高抗子代池	106 036 718	105 574 627	99.56	53.53	99.09	98.38	96.57	85.84
Highly resistant pool

图2 SNPs 在染色体上的密度分布图

Fig.2 Density distribution map of SNPs on chromosomes

图3 突变类型

Fig.3 Mutation type

表3 RNA质量评估

Tab.3 RNA quality assessment

样本 Sample	Reads 总数/ 个 Raw read number	碱基总数/ 个 Raw bases number	原始Q20 比例/% Raw Q20 rate	原始Q30 比例/% Raw Q30 rate
QG_1	44 228 470	6.68E+09	98.56	95.62
QG_2	42 921 534	6.48E+09	98.53	95.47
QG_3	46 708 516	7.05E+09	98.54	95.54
QK_1	45 138 140	6.82E+09	98.52	95.46
QK_2	41 316 264	6.24E+09	98.52	95.47
QK_3	43 105 000	6.51E+09	98.51	95.41

图4 RNA表达量PCA分析

Fig.4 PCA analysis of RNA expression levels

图5 RNA表达量相关性分析

Fig.5 Correlation analysis of RNA expression levels

图6 GO注释和富集 1.单加氧酶活性;2.谷胱甘肽转移酶活性;3.槲皮素7-O-葡糖基转移酶活性;4.槲皮素3-O-葡糖基转移酶活性;5.氧化还原酶活性;6.对外部刺激的响应;7.毒素代谢过程;8.对外部刺激的响应;9.对伤害的响应;10.毒素分解代谢过程;11.对刺激的响应;12.次生代谢产物合成过程;13.谷胱甘肽代谢过程;14.对激素的响应;15.激素代谢过程;16.对含氧化合物的响应;17.有毒物质的响应;18.化学响应;19.对酸性的化学响应;20.次生代谢过程。

Fig.6 GO annotation and enrichment 1.Monooxygenase activity;2.Glutathione transferase activity;3.Quercetin 7-O-glucosyltransferase activity;4.Quercetin 3-O-glucosyltransferase activity;5.Oxidoreductase activity;6.Response to external stimulus;7.Toxin metablic prosess;8.Response to external stimulus;9.Response to wounding;10.Toxin catabolic process;11.Response to stimulus;12.Secondary metabolite biosynthetic process;13.Glutathinone metabolic process;14.Response to hormone;15.Hormone metabolic process;16.Response to oxygen-containing compound;17.Response to toxic substance;18.Response to chemical;19.Response to acid chemical;20.Secondary metabolic process.

图7 KEGG 注释和富集 1.谷胱甘肽代谢;2.苯丙酸合成;3.倍半萜和三萜生物合成;4.α-亚麻酸代谢;5.异黄酮生物合成;6.细胞分裂素代谢;7.甘氨酸、丝氨酸和苏氨酸代谢;8.苯丙氨酸代谢;9.半乳糖代谢;10.二萜生物合成;11.牛磺酸和次牛磺酸代谢;12.色氨酸代谢;13.甾体类激素生物合成;14.亚油酸代谢;15.各种次生代谢物的生物合成-第三部分;16.芪类、二芳基庚烷和姜辣素生物合成;17.β-丙氨酸代谢;18.半胱氨酸和蛋氨酸代谢;19.植物激素信号传导;20.MAPK信号通路-植物。

Fig.7 KEGG annotation and enrichment 1.Glutathione metabolism; 2.Phenylpropanoid biosynthesis; 3.Sesquiterpenoid and triterpenoid biosynthesiss; 4.alpha-Linolenic acid metabolism; 5.Isoflavonoid biosynthesis; 6.Zeatin biosynthesis; 7.Glycine, serine and threonine metabolism; 8.Phenylalanine metabolism; 9.Galactose metabolism; 10.Diterpenoid biosynthesis; 11.Taurine and hypotaurine metabolism; 12.Tryptophan metabolism; 13.Brassinosteroid biosynthesis; 14.Linoleic acid metabolism; 15.Biosynthesis of various secondary metabolites-part 3; 16.Stilbenoid, diarylheptanoid and gingerol biosynthesis; 17.beta-Alanine metabolism; 18.Cysteine and methionine metabolism; 19.Plant hormone signal transduction; 20.MAPK signaling pathway-plant.

表4 候选区段内SNP-Index鉴定出的突变基因中的显著差异表达基因

Tab.4 DEGs among those identified as mutated by SNP-Index in the candidate region

基因名 Gene ID	log₂表达量的差异倍数 log₂ FC (FPKM+1)	基因表达调控 Gene regulation	染色体 Chromosome	开始位点/bp Start site	结束位点/ bp End site	方向 Direction	基因长度/ bp Gene length
LOC101218929	1.354 740	上调	NC_026659.2	28 700 918	28 705 562	-	2 404
LOC116403950	4.038 243	上调	NC_026659.2	24 775 752	24 779 012	+	978
LOC101207011	1.530 013	上调	NC_026659.2	20 323 009	20 327 115	+	2 837
LOC101210331	1.740 950	上调	NC_026659.2	27 697 071	27 698 402	+	742
LOC105434389	-1.462 570	下调	NC_026659.2	31 383 485	31 389 181	+	3 721
LOC101221769	-1.505 140	下调	NC_026659.2	28 803 145	28 807 150	-	1 422
LOC101215091	1.016 653	上调	NC_026659.2	27 039 556	27 041 811	-	2 086
LOC101211418	1.267 333	上调	NC_026659.2	30 843 570	30 846 580	+	3 011
LOC101213826	-1.055 710	下调	NC_026659.2	30 922 296	30 924 591	-	1 141
LOC101219640	-1.509 530	下调	NC_026659.2	30 805 088	30 808 349	+	1 832
LOC101213588	1.662 258	上调	NC_026659.2	30 915 439	30 918 702	-	2 462
LOC101218380	-3.564 630	下调	NC_026659.2	29 548 285	29 551 574	+	1 117
LOC101208991	-2.146 810	下调	NC_026659.2	21 305 912	21 309 524	-	1 510

表5 NC_026659.2染色体19—21 Mb区间内的显著差异表达基因

Tab.5 DEGs in the 19—21 Mb region of chromosome NC_026659.2

基因名 Gene ID	log₂表达量的差异倍数 log₂ FC (FPKM+1)	基因表达调控 Gene regulation	染色体 Chromosome	开始位点/ bp Start site	结束位点/ bp End site	方向 Direction	基因长度/ bp Gene length
LOC116403781	-2.254 640	下调	NC_026659.2	19 531 623	19 536 549	-	2 250
LOC101222479	-1.116 020	下调	NC_026659.2	19 852 884	19 853 703	+	820
LOC101207011	1.530 013	上调	NC_026659.2	20 323 009	20 327 115	+	2 837

图8 候选基因LOC101207011在4个样中的表达量 N.未接种白粉病菌;Y.接种白粉病菌。不同小写字母代表差异显著(P<0.05)。

Fig.8 Expression levels of the candidate gene LOC101207011 in four samples N.Not inoculated with powdery mildew; Y. Inoculated with powdery mildew. Different lowercase letters represent significant differences(P<0.05).

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