Epidemiological Characteristics and Transcriptome Analysis of Grape Infected by Grape Downy Mildew

doi:10.7668/hbnxb.20193053

Abstract

Abstract:

In order to explore the mechanism of grape defense response to grape downy mildew,the leaves of Muscat and Moldova were inoculated with grape downy mildew in vitro and cultured in light incubator.The development of downy mildews was observed from macroscopic and microscopic aspect.In macroscopic,it was showed that white mildew layer appeared on the back of leaves of Muscat four days after inoculation,and the area of disease spots expanded with time,meanwhile chlorosis yellow spots appeared on the opposite position of the front side of leaves.However,there were only a few black spots on leaves of Moldova at the inoculation point.Under electron microscope,it was found that the spores of the pathogen invaded the stomata and colonized,and germinated new sporangium stalks,which released the spores and propagated on the leaves of Muscat.But the number of sporangia was small on the leaves of Moldova,and most of the stomata was closed,only with spores,hyphae and tissue fragments scattered around the stomata.Furthermore,transcriptome sequencing and differentially expressed genes were analyzed by Illumina high-throughput sequencing.A total of 664.86 Mb of high-quality reads were obtained by RNA-seq.GO enrichment analysis showed that differentially expressed genes were mainly concentrated in biological pathways(metabolic process,cellular process,response to stimulus and biological regulation category),cell components(cell, membrane, membrane part and organelle),molecular functions(catalytic activity and binding,transporter activity and transcription regulator activity).KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in the plant pathogen interaction pathway and MAPK signal transduction pathway.Nine genes that may be related to grape disease resistance were screened and verified by Real time PCR analysis,and the results were consistent with the transcriptome sequencing results.

Key words: Downy mildew, Disease resistance gene, Electron microscopy(SEM), Transcriptome sequencing(RNA-seq)

CHEN Zhan, ZHAO Yanzhuo, NIU Zaozhu, XUAN Lifeng, NIU Shuaike, WEI Jianguo, YANG Lili. Epidemiological Characteristics and Transcriptome Analysis of Grape Infected by Grape Downy Mildew[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(S1): 324-332. doi: 10.7668/hbnxb.20193053.

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References 36

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引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
CaM4-F	CAGCAGGCCACTTAGCCAGAATG
CaM4-R	CTAGCTCCTCAGGACGCAGTCTC
CAT1-F	CGTCGCTCACCGTTGGATCAAG
CAT1-R	CTCTAGCATGGACCACACGTTCTG
EIX1-F	GAAGCGTGGACCAACGTCGATC
EIX1-R	AGGCCACTAGGAAGAGGCTTGTAC
PR1-F	CCACAAGACTTCCTCGATGCTCAC
PR1-R	GCGTAGGCAGCCACAGTGTTG
PAL-F	TCTCACACCACAACGGCAACG
PAL-R	GTCCGCCACCATTCTCTTCACC
E9-F	AACCAGTTCAGCAACGAGATTCCG
E9-R	CACACGCCTCCACAATCTTCCTAC
ABCB1-F	GTCAGGACTGTCAAGCCTTGGAAG
ABCB1-R	TCTTGTTCAGGACGCACGTATCG
F6H1-F	TCCAATCCAGTCAGGCTCTCCAC
F6H1-R	GGTGCAATACTCAGCCACATCCTC
IRAK4-F	TTCAGCAGCACTGGTCACTTCATG
IRAK4-R	GGCAGACAGGCGTGCAGTTG

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
CaM4-F	CAGCAGGCCACTTAGCCAGAATG
CaM4-R	CTAGCTCCTCAGGACGCAGTCTC
CAT1-F	CGTCGCTCACCGTTGGATCAAG
CAT1-R	CTCTAGCATGGACCACACGTTCTG
EIX1-F	GAAGCGTGGACCAACGTCGATC
EIX1-R	AGGCCACTAGGAAGAGGCTTGTAC
PR1-F	CCACAAGACTTCCTCGATGCTCAC
PR1-R	GCGTAGGCAGCCACAGTGTTG
PAL-F	TCTCACACCACAACGGCAACG
PAL-R	GTCCGCCACCATTCTCTTCACC
E9-F	AACCAGTTCAGCAACGAGATTCCG
E9-R	CACACGCCTCCACAATCTTCCTAC
ABCB1-F	GTCAGGACTGTCAAGCCTTGGAAG
ABCB1-R	TCTTGTTCAGGACGCACGTATCG
F6H1-F	TCCAATCCAGTCAGGCTCTCCAC
F6H1-R	GGTGCAATACTCAGCCACATCCTC
IRAK4-F	TTCAGCAGCACTGGTCACTTCATG
IRAK4-R	GGCAGACAGGCGTGCAGTTG

样本 Sample	RL1	RL2	RL3	RL5	RL6	SL1	SL2	SL3	SL5	SL6
全部原始数据/M Total raw reads	72.22	70.33	69.12	71.68	69.6	73.97	66.08	65.01	70.35	67.34
全部质控后数据/M Total clean reads	68.30	66.81	66.14	68.44	65.69	70.61	63.66	62.69	67.56	64.96
全部质控后碱基/Gb Total clean bases	6.83	6.68	6.61	6.84	6.57	7.06	6.37	6.27	6.76	6.50
碱基质量在20以上的数据/% Clean reads Q20	97.22	97.54	97.75	97.81	97.14	97.71	97.80	97.72	97.72	97.76
碱基质量在30以上的数据/% Clean reads Q30	88.39	89.44	90.12	90.35	88.15	89.93	90.47	90.19	90.18	90.31
质控后数据比例/% Clean reads ratio	94.58	94.99	95.69	95.49	94.39	95.46	96.34	96.42	96.04	96.47
能定位到基因组上的测序序列的数量统计/% Total mapping	87.63	88.58	87.09	84.99	87.56	91.87	91.69	90.24	90.48	90.18
有唯一对比位置的测序序列的数量统计/% Uniquely mapping	66.03	67.35	66.41	64.74	65.95	74.48	71.44	70.34	70.29	69.88

样本 Sample	RL1	RL2	RL3	RL5	RL6	SL1	SL2	SL3	SL5	SL6
全部原始数据/M Total raw reads	72.22	70.33	69.12	71.68	69.6	73.97	66.08	65.01	70.35	67.34
全部质控后数据/M Total clean reads	68.30	66.81	66.14	68.44	65.69	70.61	63.66	62.69	67.56	64.96
全部质控后碱基/Gb Total clean bases	6.83	6.68	6.61	6.84	6.57	7.06	6.37	6.27	6.76	6.50
碱基质量在20以上的数据/% Clean reads Q20	97.22	97.54	97.75	97.81	97.14	97.71	97.80	97.72	97.72	97.76
碱基质量在30以上的数据/% Clean reads Q30	88.39	89.44	90.12	90.35	88.15	89.93	90.47	90.19	90.18	90.31
质控后数据比例/% Clean reads ratio	94.58	94.99	95.69	95.49	94.39	95.46	96.34	96.42	96.04	96.47
能定位到基因组上的测序序列的数量统计/% Total mapping	87.63	88.58	87.09	84.99	87.56	91.87	91.69	90.24	90.48	90.18
有唯一对比位置的测序序列的数量统计/% Uniquely mapping	66.03	67.35	66.41	64.74	65.95	74.48	71.44	70.34	70.29	69.88

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