The Key Genes Mining Associated With Leaf Etiolation Of Hau Cytoplasmic Male Sterile Line In Chinese Cabbage(Brassica Rapa L.ssp.Pekinensis)

Chinese cabbage is the cruciferous vegetable with the largest cultivated area in China.It is popular among people because of its good taste,rich nutrition and low price.Cytoplasmic male sterility(CMS)is the main way of heterosis utilization in Chinese cabbage.Brassica juncea hau CMS is a new type of cytoplasmic male sterility,and its sterility is stable and thorough.However,the newly developed cytoplasmic male sterility sources are not coordinated during the genera(species)hybridization due to nuclear exchange caused by nuclear substitution,and the CMS phenotypes are presented with some undesirable traits,such as abnormal development of nectary glands,etiolated leaves and so on.In this study,interspecific hybridization and backcrossing of mustard CMS with Chinese cabbage were used to obtain hau cytoplasmic male sterile line 1409 A and its maintainer line 1409 B of Chinese cabbage,but there were different degrees of yellowing leaf in 1409 A plants.However,the hybrids between normal mustard(maintainer line)and Chinese cabbage have normal leaf color,the same as the hybrids between mustard hau CMS and mustard vegetables.This was a n entry point to utilize 1409 A and 1409 B as experimental materials by reverse genetics.This study systematically explained the molecular mechanism of leaf etiolation of hau CMS line in Chinese cabbage by the following methods: the identification of the leaf phenotype and genetic verification of the two lines,ultrastructural observation of chloroplasts,the quantification of main types of pigment and the determination photosynthesis index,transcriptome sequencing,chloroplast genome sequencing,RNA editing sites and functional analysis of key genes,etc..This will provide a theoretical basis for the biological problems of incompatible nucleoplasm interaction that produce undesirable traits,and will practically accelerate the utilization of mustard hau CMS in cabbage vegetables.The primary findings are as follows:1.Phenotypic observation and genetic validation of 1409 A and 1409BThe leaf etiolation of the hau CMS line in Chinese cabbage started from the cotyledon stage.During the whole growth period,the leaves had different degrees of etiolation,and this was more obvious at the seedling stage;The results of genetic verification of 1409 A and 1409 B were as following: the leaves of hybrid progeny of mustard hau CMS × 1409 B were normal green so that 1409 B was needed to as the recurrent parent to observe the leaf phenotype of the plants of the later backcross generation;the hybrid offspring of the mustard cultivar × 1409 B was without etiolated leaf,this indicated that the leaf yellowing of 1409 A was caused by the transformation of mustard hau sterile cytoplasm;the leaves of hybrid progeny of 1409 A × Brassica juncea maintainer line were etiolated without separation of leaf yellowing traits,this means that it was impossible to the genetic population.2.Chloroplast ultrastructure of 1409 A and 1409BThe chloroplast ultrastructure of 1409 A and 1409 B leaves was observed by transmission electron microscopy.It was found that the chloroplasts of 1409 A had no mature starch granules and no sufficient supply of energy.;the lack of complete granule structure and thylakoid membrane as well as loose thylakoid structure were obvious;some of the granules were degraded and larger chloroplast cavities were formed during flowering stage.3.Determination of pigment content and photosynthetic index in 1409 A and 1409BQuantitative determination of main pigments in leaves of 1409 A and 1409 B revealed that the contents of chlorophyll,carotenoids,anthocyanins and isoflavones in 1409 A decreased by 57.0%,58.9%,64.6% and 61.3,respectively.However,the total flavonoid content of 1409 A increased by 13.3%.Abnormal development of chloroplasts and reduced chlorophyll content also led to a decrease of 34.3% in the net photosynthetic rate of 1409 A plants.4.Transcriptome sequencing in 1409 A and 1409BBased on the above results,the transcriptome of Chinese cabbage was sequenced to analyze the differences in the expression levels of 1409 A and 1409 B genes.485 DEGs were obtained,of which 189 up-regulated genes and 296 down-regulated genes.The DEGs were screened for genes that affect chloroplast development(GLK1,GLK2),genes that regulate chlorophyll and carotenoid biosynthesis(PORB,CYP707A4),23 transcription factors involved in anthocyanin and flavonoid biosynthesis,and genes that touched upon posttranscriptional regulation in RNA editing(PPR),etc.These key genes may be associated with leaf etiolation in 1409 A.5.Chloroplast genome sequencing in 1409 A and 1409BA high-salt-low-pH method was developed to extract chloroplast DNA with highquality from Chinese cabbage,and the chloroplast genome of Chinese cabbage was sequenced.The annotations of 94 protein-encoding genes in 1409 A were performed using DOGMA,in which including 8 rRNAs and 36 tRNAs;1409B has 92 gene encoding genes including 8 rRNAs,and 37 tRNAs.The complete chloroplast genome in Chinese cabbage was assembled finally.On this basis,comparative genomic analysis revealed that there were INDELs in the accD,ccsA,rpoC2,and trnL-UAA,of which rpoC2 had 12 INDELs,and accD and ccsA each had 6 INDELs.In addition,there were 758 SNPs in 71 genes including rpoC2,ndhF,matK,rpoB,and ccsA,of which 15 genes had more than 20 SNPs.6.RNA edting analysisMultiple genes encoding PPR proteins were found to be differentially expressed between 1409 A and 1409 B from the transcriptome sequencing data.Using the CUREchloroplast platform,six differential RNA editing sites were predicted in matK,psbE,rpoC2,rps14,and rps16 between 1409 A and 1409 B.In addition,38 common RNA editing sites were predicted in an additional 13 genes.So far,the differential editing sites in psbE,ndhG,and rps14 had been validated.More RNA editing sites and the relationship between these sites and leaf etiolation in Chinese cabbage require further identification.