| Cabbage(Brassica oleracea var.capitata)is a kind of leafy vegetables widely cultivated all over the world.In recent years,due to the increasing of multiple cropping indexes,unreasonable cultivation methods and the introduction of a large number of varieties,black rot has been prevalent and aggravating in most of cabbage production areas in China and aggravating year by year,affecting the quality and yield of cabbage seriously and causing huge economic losses.Understanding the genetic mechanism and breeding resistant varieties are the most economical and effective methods to control black rot of cabbage.In this study,the genetic map of B.oleracea was constructed using the recombinant inbred lines(RILs)developed from R4-P1 and R2-P2.A set of cabbage chromosome segment substitution lines(CSSLs)with R2-P2 as the background and R4-P1 as the donor were constructed by continuous multi-generation backcrossing and self-crossing strategies combined with molecular marker-assisted selection.Black rot resistance QTL mapping was performed using the constructed RILs and CSSLs,9 candidate genes in the major stable resistance region were sequencing and comparison,expression levels and transient expression were analyzed at different time periods after Xcc inoculation.The identification and genetic analysis of the whole genome NBS-LRR genes of cabbage were also performed.The main research results are as follows:1.The genetic linkage map of B.oleracea containing 303 pairs of molecular markers,covering a total genome length of 767.2 cM,average genetic distance and physical distance of 2.53 cM and 2.07 Mb was constructed using RILs developed from different disease resistance types of high-generation homozygous R4-P1 and R2-P2 inbred lines.2.Using 181 markers evenly distributed on the 9 chromosomes of the constructed genetic map,a set of 160 cabbage CSSLs with the genetic background of cabbage inbred line R2-P2 were constructed.The coverage rate of the introduced donor R4-P1 chromosome segment to the receptor R2-P2 genome was 100%,and the recovery rate of the receptor parent genotype reached 97.21% on average.3.Through spraying inoculation of Xcc Race 3 at seedling stage,11 black rot resistance QTLs distributed on chromosomes 1,2,7 and 8 of cabbage were identified in RILs and CSSLs.Among them,the LOD values of the 3 major black rot resistance QTLs qBR-7-1,qBR-7-3 and qBR-8-2 were higher than 3.The qBR-1-2,qBR-7-1,qBR-8-1 are overlaps with qBR-1-4,qBR-7-3 and qBR-8-2,indicating that there may be disease-resistant genes related to black rot in these three overlapped regions.In particular,qBR-7-3 had the highest LOD scores and able to explain up to 16.72% phenotypic variation in the RILs and ABLs,indicating that qBR-7-3 is most likely a major QTL.4.According to the gene annotations in the major stable resistance region,a total of 9(5 NBS-LRR,3 STK and 1 Disease-resistance-related)candidate resistance genes were identified.Sequencing results of the 9 candidate gene showed that the sequence of NBS-LRR type gene Bo7g111290 was significantly different between the two parents,resulting in the difference of partial coded amino acids between the two parents.Meanwhile,the expression levels of 9 candidate genes between 0 and 48 hours after the inoculation of Xcc race 3 were different.Different from the other 8 candidate genes,only Bo7g111290 showed higher relative expression levels of R4-P1 than R2-P2 in each time period after inoculation.Transient expression indicated that Bo7g111290 may be involved in resistance to Xcc race 3,and the expression difference of Bo7g111290 in R4-P1 and R2-P2 may be one of the main reasons for the great difference in the resistance of the two parents.5.A total of 176 NBS-LRR genes including Bo7g111290 were identified in the B.oleracea genome,among which 88 genes were distributed in clusters,accounting for 51.5% of the total number of NBS-LRR genes while the Bo7g111290 is not in the cluster.The distribution of these gene clusters may be related to the gene evolution.The mean sequence length,CDS length and amino acid encoding of TNL genes in cabbage were all larger than those of CNL and NL genes.Meanwhile,the average exon number of TNL gene was also higher than that of CNL and NL types,indicating that the size of cabbage NBS-LRR gene was closely related to its structural domain and genotype.At the same time,p-loop,RNBS and kinase 2 motifs were detected in all NBS-LRR proteins,and the three motifs in 3 types of NBS-LRR genes were highly similar,indicating that the protein domain of cabbage NBS-LRR gene family was highly conserved.Genetic evolution analysis showed that the TNL subfamily expanded to a greater extent,indicating that the TNL subfamily may be older than the CNL and NL subfamily. |
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