Phenotypic Identification And Genetic Analysis Of Tomato Seedling Yellowing Mutant Lmyl

Tomato(Solanum lycopersicum L.)is one of the model crops in the research of plant growth and development.It is also a significant vegetable crop widely planted in the south and north of China.Tomato leaf color is an vital agronomic trait.The leaf color mutant can be used as an important genetic material for the study of plant photosynthetic characteristics,chloroplast synthesis and degradation,and can also be used as a marker trait for the rapid identification of variety purity and the screening of hybrid progeny.It has a great application prospect in the breeding of new tomato varieties.In this research,the leaf seedling yellowing mutant lmyl and its wild type “404” were used as materials to study its horticulture traits,photosynthetic characteristics,genetic rules and gene identification,so as to provide a research basis for exploring the molecular mechanism of leaf color regulation.It will be a reference for the research on the structure of photosynthetic system,functional genomics and tomato genetics and breeding.The results are as follows:(1)It was found that the mutant lmyl showed yellow-cotyledon and yellow-green heart leaves at the seedling stage,while the heart leaves gradually turned green at the later stage,which was consistent with the phenotype of the control wild-type traits.(2)Studies on photosynthetic physiological characteristics indicated that the contents of chlorophyll a and chlorophyll b in the leaves of the yellow-green mutant lmyl were significantly lower than those of the wild type at the early growth stage,and the differentiation of grana lamellae and matrix lamellae in the chloroplasts of the mutant was incomplete,and the stacking was irregular and unclear.At the later growth stage,the mutant leaves turned green,and the chlorophyll content and chloroplast structure returned to normal.Consistent with this,the net photosynthetic rate of the mutants was significantly reduced in the early growth stage,while there was no difference between the mutants and the wild type in the late growth stage.(3)The mutant lmyl was crossed with the wild-type "404" to obtain F1 generation,and further self-hybridization was conducted to obtain F2 generation.The number of yellowing to green mutants and the number of normal plants with leaf color corresponded to the separation ratio of 1:3,which proved that the yellowing to green mutation in tomato leaves was controlled by a single recessive gene.(4)Mixed-population separation analysis combined with high-throughput sequencing technology(BSA-Seq)was used to map the key regulatory genes of tomato yellow-green mutation lmyl.The analysis results made clear that the difference sites between SNP and In Del were mainly concentrated on chromosome 3 of tomato,and only a few sites were different on other chromosomes.The region of chromosome 3 exceeding the threshold was identified as the primary candidate region.Two candidate genes,solyc03g025700 and solyc03g026080 located on chromosome 3 were screened using bioinformatics analysis.The solyc03g025700 gene encodes a Pentatricopeptide repeat-containing protein(PPR protein);while solyc03g026080 encodes a Rhomboid domain-containing protein 2.(5)CRISPR/Cas9 knockout vector of solyc03g025700 gene encoding PPR protein was constructed and the transgenic tomato lines were generated.Preliminary results demonstrated that single knockout of solyc03g025700 did not result in mutant phenotype in the transgenic plants,and the function of the candidate genes still needs to be further verified.