Genetic,Transcriptomic And Metabonomic Analysis Of Purple Heart Size Traits In Chinese Cabbage

Chinese cabbage[Brassica campestris L.ssp.pekinensis（Lour）Olsson],a traditional Chinese vegetable,has an important position in the vegetable production in China.In order to meet people’s demand for vegetable diversification,scientists began to explore and create new germplasm of purple Chinese cabbage.The purple characters of purple Chinese cabbage obtained from different sources and different ways are different.At present,there are many researches on purple characters,anthocyanin content and composition,and the conclusion is clear.But there are no related research reports on the purple heart size character of Chinese cabbage.Therefore,F₁and F₂populations were constructed by the purple heading inbred line‘14S838’and the common heading inbred line‘15S1080’,which were used for studing genetic analysis of the purple trait of purple heading Chinese cabbage and conducting transcriptome sequencing and flavonoids component detection on bigger purple heart leaf head and smaller purple heart leaf head.Which can not only enrich the knowledge of flavonoid metabolism in Brassica vegetables,but also provide theoretical basis for breeding excellent purple Chinese cabbage.The main results of this study are as follows:1.In F₁population,the inner leaf color is all purple,but the purple area is smaller than the parent‘14S838’.The F₂population shows that the separation proportion of purple heading individuals to non-purple heading individuals fitted to the ratio of 3︰1,which indicated that the purple trait was a quality trait and purple genes（Pr）were dominant genes compared with white genes（pr）.2.The three parameters of purple height ratio（PHR）,purple width ratio（PWR）and purple area ratio（PAR）were used to describe the purple size trait for measuring and analysing purple individuals in F2 population.The result shows that the board heritability values of the three parameters were 94.00%、87.72%and 83.33%,respectively,which suggested the purple size trait is mainly controlled by genetic factors and less affected by the environment.The frequency distribution shows that the PHR and the PWR conform to the normal distribution characteristics and the PAR shows a positive skewed distribution,indicating that the purple size trait is a quantitative trait.3.Three parameters of the purple size trait were comparised,which showed that there have a significant correlation between PHR,PWR and PAR,respectively.The PAR is accurate but complicated in practice application,while the PHR with high board-sense heritability is simple to measure by ruler can be used to estimate purple size of purple heading Chinese cabbage.4.The full length gDNA sequence of BrMYB2 gene of purple heading Chinese cabbage,common heading Chinese cabbage and orange heading Chinese cabbage was obtained by cloning.Sequence alignment results showed that the sequences of BrMYB2^pof purple heading Chinese cabbage were completely consistent with the sequences of BrMYB2^pof purple heading Chinese cabbage published in our laboratory.Compared with the BrMYB2^wof common heading Chinese cabbage published in the laboratory,the sequences of common heading Chinese cabbage and orange heading Chinese cabbage have 1 base deletion(A³¹⁷³),and the deletion site is on the intron,and the difference does not cause the coding region of BrMYB2^wgene sequence to change.5.According to the comparison of gDNA sequences,it was found that the non-purple heading Chinese cabbage has a large fragment insertion of the gene BrMYB2^wcompared with the purple heading Chinese cabbage.A new single dominant molecular marker was developed at the position of the insertion fragment,which can distinguish the heterozygous（Prpr）and homozygous genotypes（PrPr）of purple heading Chinese cabbage.6.The newly developed molecular marker was used to analyze the genetic effect of BrMYB2 genotype on purple heart size traits of F₂purple segregation population of purple heading Chinese cabbage.The results showed that maximum,minimum,median,upper and lower quartile of the three parameters of leaf-head with purple heart size traits of purple homozygote（PrPr）genotype,all of them are larger than the maximum,minimum,median,upper and lower quartile of the three parameters of leaf head with purple heterozygote（Prpr）genotype,which indicates that the purple heart size character of purple heading Chinese cabbage may be affected by the dose of the key gene BrMYB2 which controls anthocyanin synthesis.The largest purple heart leaf head is obtained in the homozygous state and the smallest purple heart leaf head is obtained in the heterozygous state.therefore,the gene of purple heart size may only be studied under the same background of BrMYB2genotype.7.RNA-seq analysis was carried out in the leaves about the lower part,middle part and upper part of the bigger purple heart leaf head and the smaller purple heart leaf head under the background of hybrid purple heading Chinese cabbage（Prpr）in F2 purple segregation population.Results found it was found that the early biosynthetic synthetic structural genes,late biosynthetic synthetic structural genes and transcription factors of flavonoid biosynthesis pathway were up-regulated mainly in the internal part of purple heart.Whereas,the genes differentially expressed in the middle part of purple heart of the bigger purple heart leaf head and the smaller purple heart leaf head are PAL,C4H,4CL which are phenylalanine biosynthesis pathway genes and transcription factors BrMYBL2.2 and LBD37.8.LC-MS/MS was used to carry out qualitative and quantitative analysis of flavonoid substances in the leaves about the lower part,middle part and upper part of the bigger purple heart leaf head and the smaller purple heart leaf head under the background of hybrid purple heading Chinese cabbage（Prpr）in F2 purple segregation population.A total of 102flavonoids components were detected,including 23 flavones,21 flavonols,18 flavanones,14flavone C-glycosides,12 anthocyanins,6 isoflavones,6 catechins and their derivatives,1flavonoid-lignan and 1 proanthocyanidin.The accumulation amounts of these flavonoids in the three purple heart parts of purple heading Chinese cabbage were different.Among them,Cyanidin O-acetylhexoside was the only anthocyanin not detected in the internal part of purple heart.