Transcriptome Analysis and Resistance Gene Mining of Resistant and Susceptible Cauliflower Cultivars in Response to Black Rot Infection

doi:10.7668/hbnxb.20195488

Abstract

Abstract:

To investigate the transcriptome differences between resistant and susceptible varieties of cauliflower after inoculation with Xanthomonas campestris pv.campestris (Xcc),and to identify genes associated with cauliflower resistance to black rot disease,the susceptible variety Y1-2 and the resistant variety EC-247 of cauliflower were selected as the research subjects.Total RNA was extracted from cauliflower leaves at 0,1,3,and 5 days post-inoculation with Xcc,respectively.High-throughput parametric transcriptome sequencing was then conducted utilizing the Illumina RNA-Seq platform,followed by Real-time Quantitative PCR for validation of selected differentially expressed genes(DEGs).DEGs associated with disease resistance were screened and analyzed.The findings revealed that 6 355 genes exhibited significant differential expression between resistant and susceptible cultivars across the four time points.KEGG enrichment analysis focused on plant disease resistance pathways,identifying 47 genes involved in plant-pathogen interactions and 61 genes related to plant hormone signaling.Cluster analysis of these gene expression levels disclosed specific genes,including one CDPK,four CMLs,one PTK,one CaM,one RLK,and one SGT1 in the plant-pathogen interaction pathway,and three auxin-responsive protein genes,a TIFY gene,an indole-3-acetic acid-amido synthetase gene,two brassinazole-resistant protein genes,and a Shaggy-associated protein kinase zeta gene in the plant hormone signaling pathway.Notably,the expression of these genes was significantly higher in resistant varieties compared to susceptible ones,indicating their active response to pathogen infection at various time points.The results indicated that these differential genes might be related to disease resistance in cauliflower,which provided important genetic resources and scientific basis for molecular breeding of disease resistance in cauliflower.

Key words: Brassica oleracea L.var.botrytis, Xanthomonas campestris pv.campestris, Transcriptome, Differentially expressed genes, Disease-resistant type, Sensitive type

LI Ziyu, YAO Yurong, HAO Yongjuan, HUO Jianfei, BEN Haiyan, WANG Xudong, WANG Wanli, YAO Xingwei, LI Erfeng. Transcriptome Analysis and Resistance Gene Mining of Resistant and Susceptible Cauliflower Cultivars in Response to Black Rot Infection[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 161-172. doi: 10.7668/hbnxb.20195488.

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Figures/Tables 13

Tab.1 Primer sequence and fragment size of qRT-PCR

基因ID Gene ID	引物序列(5'—3') Primer sequence (5'—3')	退火温度/℃ Annealing temperature	片段大小/bp Product size
TRINITY_DN16231_c1_g2	F: AGATCGTTGGCTGGTTTGGT	60.0	166
	R: GGAGGGTGCTGCTGTTTGAA	62.0
TRINITY_DN16447_c0_g1	F: TGGACGAGTAGCCTTGAGAG	62.0	96
	R: GCTCCTATCTAGCCGCAC	58.0
TRINITY_DN11917_c3_g2	F: CTTTAGCCATCAAAGGCGGC	62.0	139
	R: GACTCCGGGGCTTTCCATTC	64.0
TRINITY_DN12966_c1_g11	F: GTTAGCACAAGCGCCTTCAC	62.0	131
	R: TCTTGTTCTGTGATTCTCTCCTT	54.8
TRINITY_DN10243_c0_g2	F: TAGCGTCGAAGGGAAACTGC	62.0	121
	R: GTCCGAATGAACAATACCGGAG	58.1
TRINITY_DN12323_c1_g1	F: AACCTGTGTGGGGAAAGAAGA	62.0	103
	R: AGAGTGAACCAACCATCTCCG	57.8
TRINITY_DN15781_c4_g7	F: CTCGTTTTTCTTCTCTCTCGCATC	58.6	132
	R: GAGGAGGTTGTTGTCGTTGGA	57.8
TRINITY_DN16687_c1_g5	F: CTCACATGCCCAGTTCCTCG	64.0	181
	R: CCCTAACTTTGCTTCGCCCG	64.0
TRINITY_DN15781_c4_g1	F: GATCACGATGCTCCAGTCGC	64.0	187
	R: TTGGTGCTCCTTCACTGCTC	62.0
ACTIN	F: GAGAGATAGATTTGTGTGTTGT	52.5	122
	R: AGATTAGTTGGAGTAGCAGAA	51.9
GAPDH	F: TCTTCACTCACTTGACTATTCT	52.5	136
	R: GATAACTCTCGCCACCAA	54.0

Fig.1 The appearance of cauliflower leaves inoculated with Xanthomonas campestris pv.campestris

Tab.2 Sequencing data statistics and quality assessment

样本 Sample	过滤后数据 Clean reads	过滤后碱基数 Clean bases	Q30碱基质量/% Q30 bases quality	GC含量/% GC content	比对率/% Mapped ratio
EC-247-0 d A	43 154 426	6 456 548 603	93.77	47.57	77.66
EC-247-0 d B	43 974 850	6 577 996 221	94.29	47.40	77.31
EC-247-0 d C	50 699 770	7 592 056 814	94.35	47.64	78.14
EC-247-1 d A	42 988 736	6 413 315 635	94.96	47.20	77.34
EC-247-1 d B	52 500 002	7 835 972 173	94.77	47.65	78.11
EC-247-1 d C	48 391 930	7 234 185 005	95.00	47.70	77.97
EC-247-3 d A	51 191 174	7 646 822 724	94.78	47.54	77.79
EC-247-3 d B	45 770 458	6 842 511 433	95.15	47.70	78.39
EC-247-3 d C	49 606 438	7 407 208 299	95.20	47.55	77.32
EC-247-5 d A	53 245 644	7 954 157 908	94.78	47.73	78.35
EC-247-5 d B	55 391 982	8 273 179 902	95.15	47.80	78.92
EC-247-5 d C	49 013 112	7 290 224 189	93.63	47.73	76.77
Y1-2-0 d A	47 286 472	7 063 573 512	93.51	47.93	77.51
Y1-2-0 d B	47 117 450	7 050 441 980	94.41	47.75	78.20
Y1-2-0 d C	52 893 978	7 918 048 749	94.43	47.79	77.68
Y1-2-1 d A	53 443 280	8 005 561 235	94.68	47.73	78.00
Y1-2-1 d B	48 226 630	7 223 351 270	94.30	47.85	78.03
Y1-2-1 d C	56 712 154	8 467 526 725	95.16	47.73	78.29
Y1-2-3 d A	50 350 874	7 526 585 702	95.00	47.59	78.79
Y1-2-3 d B	46 535 650	6 955 289 009	94.72	47.73	78.26
Y1-2-3 d C	45 238 288	6 763 421 485	95.25	47.54	77.68
Y1-2-5 d A	49 279 840	7 359 206 999	95.16	47.25	78.63
Y1-2-5 d B	50 816 334	7 595 400 207	95.16	47.15	78.94
Y1-2-5 d C	49 402 268	7 370 757 559	94.93	47.45	78.76

Fig.2 The cluster analysis among resistant and susceptible cauliflower samples

Fig.3 PCA analysis between samples

Fig.4 The Venn diagram of expression levels among samples

Fig.5 Venn diagram of differentially expressed genes between resistant and susceptible varieties at four time points

Fig.6 GO enrichment of differentially expressed genes between resistant and susceptible varieties at four time points 1.Cell wall;2.External encapsulating structure;3.Intrinsic component of membrane;4.Integral component of membrane;5.Extracellular region;6.Membrane part;7.Pectin catabolic process;8.ADP binding;9.Polysaccharide metabolic process;10.Drug catabolic process;11.Cell wall organization;12.Cell wall organization or biogenesis;13.Galacturonan metabolic process;14.Pectin metabolic process;15.Transmembrane transporter activity;16.Hydrolase activity,acting on glycosyl bonds;17.Polysaccharide catabolic process;18.External encapsulating structure organization;19.Hydrolase activity,hydrolyzing O-glycosyl compounds;20.Transporter activity.

Fig.7 KEGG functional enrichment of differentially expressed genes between resistant and susceptible varieties at four time point 1.Phenylpropanoid biosynthesis;2.Pentose and glucuronate interconversions;3.Stilbenoid,diarylheptanoid and gingerol biosynthesis;4.DNA replication;5.Limonene and pinene degradation;6.Alpha-linolenic acid metabolism;7.Starch and sucrose metabolism;8.Cutin,suberine and wax biosynthesis;9.Phenylalanine metabolism;10.Glucosinolate biosynthesis;11.Ubiquinone and other terpenoid-quinone biosynthesis;12.Homologous recombination;13.Mismatch repair;14.Linoleic acid metabolism.

Fig.8 KEGG functional enrichment of co-expressed up-and down-regulated differentially expressed genes between resistant and susceptible varieties at four time points A:1.Limonene and pinene degradation,2.Glucosinolate biosynthesis,3.Stilbenoid,diarylheptanoid and gingerol biosynthesis;B:1.Phenylpropanoid biosynthesis,2.Pentose and glucuronate interconversions,3.DNA replication,4.Starch and sucrose metabolism,5.Alpha-linolenic acid metabolism,6.Homologous recombination,7.Mismatch repair,8.Cyanoamino acid metabolism,9.Flavonoid biosynthesis,10.Cutin,suberine and wax biosynthesis,11.Phenylalanine metabolism.

Fig.9 Expression analysis of genes related to plant-pathogen interaction pathways

Fig.10 Expression analysis of genes related to plant hormone signal transduction pathways

Fig.11 qRT-PCR validation of nine differentially expressed genes

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