Fine Mapping Of The Etiolation Gene 2(et2) In Maize

The leaf color mutant is the most ideal plant material for studying the molecular mechanism of chlorophyll synthesis degradation and chloroplast development,and is also a special material for studying photosynthesis mechanism.In the preliminary work of this subject,a chloroplast mutant derived from the progeny of maize inbred line HZ124 B and Lian87 was found,and two mutation sites were used to control the yellowing phenotype by BSR-Seq.Based on this work,this experiment conducted a more in-depth study on the phenotypic characteristics,chlorophyll and lipid content,chloroplast structure,photosynthetic parameters and genetic characteristics of yellowing mutants,and the yellowing mutation on the second chromosome.The loci(etiolation 2,et2)were finely located.The main findings obtained are as follows:1.Phenotypic investigation and genetic analysis.In the F2 population obtained by crossing HZ124 B with Lian87,the ratio of wild type and mutant phenotypes satisfies Mendelian inheritance law of 15:1(wild type: mutant type = 1379:76,X2=2.4645<X20.05=3.84,P>0.05)Moreover,both HZ124 B and Lian87 have a yellowing mutant phenotype,and this test proves that the yellowing phenotype is controlled by two nuclear recessive genes.Mutant seeds were obtained by self-separation of heterozygous wild-type plants.After sowing,the phenotypes from budding to late growth stage were pale yellow,no female tassels,and died during tasseling.2.Observation of chloroplast structure and determination of related physiological and biochemical indexes.The wild-type and mutant materials were observed by transmission electron microscopy.Compared with the wild type,the structure and shape of the mutant chloroplasts changed significantly,and the thylakoids were disorderly distributed.The mutant and wild-type chlorophyll a,chlorophyll b and carotene were measured by spectrophotometer,and the results showed that the pigment content in the mutant was significantly lower than that in the wild type.The photosynthetic parameters and chlorophyll fluorescence parameters of wild type and mutant were determined,and the mutant related parameters were found to be lower than the wild type.Mutant lipid content determination results show that the mutant has lower glycolipid content than wild type,and the decrease of glycolipid content will affect the normal function of chloroplast,thus affecting the photosynthetic capacity of the plant.3.Fine positioning of et2.Developed and screened 9 pairs of SSR markers and Indel markers with polymorphism between parents in chromosome 22 M-44 M of the original localization interval;76 yellowing mutations isolated by self-recombination of heterozygous plants in F2 population The body was located in the range of 5.4 M between markers 2-4 and umc1024;and a total of 3,229 yellowing mutants were obtained using an isolated population of 50,000 seeds;the original polymorphism marker and new development of the target segment of et2 were utilized.The polymorphic marker was screened for recombinant exchange of individual plants,and finally et2 was localized in the range of approximately 186 kb between the markers Indel2-19 and Indel2-21.According to the corn public database information,the positioning interval contains 9 candidate genes.4.Identification of key candidate genes for et2.The candidate gene function annotation and RNA-Seq analysis results of the comprehensive localization interval,only two differentially expressed genes Zmet06 and Zmet07 in the target segment;further through q RT-PCR expression analysis and candidate gene sequencing analysis,Zmet07 was finally identified as a key candidate for et2 gene.