Study Of Purple Inheritance,Response To Light And Temperature And New Germplasm Creatation In Brassica Campestris.ssp.Chinensis Makino

The pakchoi?Brassica campestris ssp.chinensis Makino.?is one of the mainvegetables widely cultivated in southern China,and plays an important role in the annual supply of vegetables.It is well known anthocyanin is important to be human health,so it is necessary to breed new purple pakchoi germplasm with high anthocyanin content,analyze the inhertance of purple trait,and study the anthocyanin synthesis mechanism induced by low light stress and temperature.In this paper,we analyzed inheritance of purple trait,effects of low light stress on the physiology and biochemistry,response to temperature regulation of leaf anthocyanin synthesis by RNA-seq in purple pakchoi.The main results are as follows:1.The new inbred line Q14 was bred from the collected pakchoi germplasm,by hybridization and backcross,which is extremely purple in autumn and winter.The male sterile line C10-213 was bred from male sterile line C07-213,and its maintainer line is B10-4.As for male sterile line C10-213,its stamen is completely degenerated,but the nectary grows normally.Characteristics of C10-213 was analyzed and assessed by morphology,physiology and cytology for breeding hybrid.2.To study the genetic characteristics of purple leaf and its application in breeding,we obtained six generations?P₁,P₂,F_l,F₂,CB_l and CB₂?by crossing,self-crossing and back-crossing.The results showed that leaf colors of F₁ hybrid were purple,but the purple was lighter than its purple parent.The purple character is incomplete dominance inheritance.The relative anthocyanin content of each generation was determined by spectrophotometry at 540 nm.The inheritance of anthocyanin content in purple pakchoi was analyzed by the segregation analysis of quantitative traits in multiple generations.The results showed that relative anthocyanin content of purple pakchoi hybrid offspring was between two parents,and its genetic model was E-0 model,it was two additive-dominance-epistasis major genes plus additive-dominance-epitasis polygene model;F₂ and BC₁?F₁×purple?heritability of two major genes were 59.17%and 74.12%,polygene heritability of F₂is 27.58%,the environmental variance out of phenotypic variance was 19.48%.So it was suggested artificial selection to purple leaves trait of purple pakchoi could be carried out in the separation of the early generation,at suitable temperature and light in fall,and it is more efficient in BC₁.3.Light is an important environment factor to regulate the plant growth and the anthocyanin biosynthesis and accumulation,so we studied the effects of low light on the physiological responses of purple pakchoi plant leaves by shading with black shading in a phytotron.The responses in terms of photosynthetic properties,carbohydrate metabolism,antioxidant enzyme activity,anthocyanin biosynthetic enzyme activity,and the relative chlorophyll and anthocyanin content of leaves were measured.The results showed that chlorophyll b,intracellular CO₂ content,stomatal conductance and antioxidant activities of guaiacol peroxidase,catalase and superoxide dismutase transiently increased in the shade treatments at 5 d.The malondialdehyde content also increased under low light stress,which damages plant cells.With the extension of shading time?at 15 d?,the relative chlorophyll a,anthocyanin and soluble protein contents,net photosynthetic rate,transpiration rate,stomata conductance,antioxidant enzyme activities,and activities of four anthocyanin biosynthetic enzymes decreased significantly.Thus,at the early stage of low light treatment,the chlorophyll b content increased to improve photosynthesis.When the low light treatment was extended,antioxidant enzyme activity and the activity of anthocyanin biosynthesis enzymes were inhibited,causing the purple pakchoi seedlings to fade from purple to green.This study provides valuable information for further deciphering genetic mechanisms and improving agronomic traits in purple pakchoi under optimal light requirements.4.We setted three temperature treatments:low temperature was 5??LT?,normal temperature was 20??NT?and the high temperature was 35??HT?,and determined the anthocyanin content of three treatments at 0 d,2 d,4 d,6 d,8 d,10 d,and 12 d.The results showed that low temperature increase of anthocyanin contents in leaves,the purple was dark,while the high temperature reduced the anthocyanin content in leaves,the leaves turned green gradually.Therefore,according to phenotypic changes,samples of 10 d were treated for RNA-seq.After de nove assembly,114,043 unigenes were obtained.There were 8710 differentially expressed genes?DEGs?in LT vs NT contrast group,4,172 genes were up-regulated and 4,538genes were down-regulated,while in HT vs NT contrast group,3,916 genes were up-regulated and 2,096 genes down-regulated expression.A part of DEGs were significantly enriched on KEGG pathways,such as flavonoid biosynthesis pathway,anthocyanin biosynthesis pathway,including transcription factor MYB44 and MYB113and the stracture genes?PAL,C4H,FLS,CHS,F3H,F3M,DFR,U78D1 and UGT75C1?in anthocyanin biosynthesis pathway.These genes regulated the anthocyanin synthesis and accumulation by the different expression at different temperature treatments in purple pakchoi.It is an initial explaination of the mechanism of temperature regulation of purple leaf anthocyanin synthesis at the level of mRNA.