Identification Of Three Type Leaf Phenotypic Related Mutants In Chinese Cabbage

Leaves are the most important photosynthetic organs of plants and the production organs of some vegetables.The leaves provide energy for plant growth and development through photosynthesis,respiration and nutrient storage.The typical characteristics of leaves mainly involve three aspects:shape,size and color.The mutants of the above three characteristics are mainly used in leaf functional genomics.Chinese cabbage is an important vegetable crop.With the release of genome sequence information of Chinese cabbage in 2011 and the subsequent two large-scale improvements,the research focus of Chinese cabbage genome has transformed from structural genomics to functional genomics.In this study,DH line?FT‘of Chinese cabbage was used as the material,and two leaf mutants with stable inheritance were obtained by 0.16%EMS mutagenesis Isolated microspore culture of‘FT‘,which were crinkled leaf mutant?lcm?and samller leaf mutant?grm1?.Five leaf shape related mutants were obtained from the germinated seeds of?FT‘by 0.8%EMS mutagenesis,including smaller leaf mutant?grm2?and leaf anthocyanin accumulation mutants?lam1,lam2,lam3,lam4?.The mutant genes were precisely located using BSR-Seq and MutMap methods,combined with whole genome resequencing,the candidate genes were predicted,and the functions of some candidate genes were verified by using parallel mutations.1.Fine mapping of a leaf flattening gene Brlcm through BSR-Seq in Chinese cabbage?Brassica rapa L.ssp.pekinensis?we obtained a stably inherited leaf crinkled mutant?lcm?,derived from the Chinese cabbage doubled haploid?DH??FT‘line using EMS mutagenesis combined with isolated microspore culture.The crinkled phenotype was controlled by a single recessive nuclear gene,Brlcm,preliminarily mapped to chromosome A01 by bulked segregant analysis RNA-seq,and further between markers SSRS-1 and Indel D-20 using 1,575 recessive homozygous individuals in F₂ population by a map-based cloning method.The target region physical distance was 126.69 kb,containing 23 genes;the marker SSRMG-4 co-segregated with the crinkled trait.Further,we found SSRMG-4 to be located on Bra A01g007510.3C,a homolog of AHA2,which encodes H⁺-ATPase2,an essential enzyme in plant growth and development.Sequence analysis indicated a C to T transition in exon 7 of Bra A01g007510.3C,resulting in a Thr?ACT?to Ile?ATT?amino acid change.Genotyping revealed that the leaf crinkled phenotype fully co-segregated with this SNP within the recombinants.q RT-PCR demonstrated that Bra A01g007510.3C expression in lcm mutant leaves was dramatically higher than that in wild-type?FT‘.Thus,Bra A01g007510.3C is a strong candidate gene for Brlcm.2.Cloning of smaller leaf gene?Brgrm1?Brgrm2?through BSR-Seq in Chinese Cabbage?Brassica rapa L.ssp.pekinensis?We obtained two stably inherited smaller leaf mutants?grm1 and grm2?by EMS mutagenesis of?FT‘?a DH line in Chinese cabbage?isolated microspores and germinated seeds,respectively.These two mutants had an identical phenotype that exhibited growth retardation with smaller leaf.Genetic analysis suggested that both mutations were controlled by single recessive nuclear genes,Brgrm1 and Brgrm2.An allelism test indicated that both mutations were allelic.Brgrm1 was mapped on chromosome A09 through BSR-Seq.A total of 3022 F₂ recessive individuals were used to map Brgrm1,which was finally mapped in a target region between indel marker XD-11 and SSR marker ZMD-63 with a physical distance of approximately 172.85 kb including 20 genes by map-based cloning.A comparison analysis of whole genome resequencing between grm1 and?FT‘plants indicated that only one indel occurred in the target region.Bra A09g024830.3C was the candidate gene?Br DDB1A?and encoded damaged DNA binding protein 1A?DDB1A?.A base?A?deletion occurred in the17th exon of grm1,which led to an amino acid coding termination.Genotyping revealed that the growth retardation phenotype was co-segregated with this indel in recombinants of the closest linkage markers.Cloning and sequencing of Bra A09g024830.3C in grm2 revealed a base substitution?G-A?in the first intron,resulting in the first intron having a 263-bp retention in CDs of Bra A09g024830.3C and leading to a termination codon?TAA?.We named Bra A09g024830.3C as Br DDB1A,and its expression in both mutants was significantly lower than that in?FT‘.The different sequence variations in the same Br DDB1A gene resulted in similar phenotypes,which confirmed that Br DDB1A is associated with growth and development in Chinese cabbage.3.Cloning of leaf anthocyanin accumulation gene?Brlam1,Brlam2,Brlam3,Brlam4?through MutMap in Chinese cabbage?Brassica rapa L.ssp.pekinensis?A class of anthocyanin accumulation mutant materials in leaves were obtained by EMS treated?FT‘germination seeds of Chinese cabbage,and four anthocyanin accumulation mutant materials with stable inheritance?lam1,lam2,lam3,lam4?were selected.Genetic analysis showed that the four mutants?lam1,lam2,lam3,lam4?were all regulated by single recessive nuclear gene,and named as Brlam1,Brlam2,Brlam3 and Brlam4,respectively.Allelic test results showed that the mutation sites of four?lam1,lam2,lam3,lam4?mutants were allelic.The lam1 mutant was selected for mapping the candidate gene,and Bra A10g030950.3C gene was predicted as the most likely candidate gene of lam1 mutant by MutMap and KASP validation methods.The results of cloning and sequencing showed that the 202^th base of the first exon in Bra A10g030950.3C gene from the lam1 mutant was replaced from G to A,resulting in the conversion of amino acid from glycine?Gly,GGG?to arginine?Arg,AGG?.Because the mutant genes of three mutants lam2,lam3 and lam4 allele with the mutant gene of lam1,we cloned and sequenced the Bra A10g030950.3C from three mutants lam2,lam3 and lam4,and the results showed that the 256^th base of the first exon of Bra A10g030950.3C in lam2 mutant was replaced from C to T,leading to the early termination of amino acid coding.The 695^th base in the first intron of Bra A10g030950.3C from the mutant lam3 was instread from G to A.The CDs sequence cloning results showed that 11 bp bases from the mutation site to the first intron termination were inserted into the second exon of Bra A10g030950.3C and resulting in the early termination of amino acid coding.The mutant lam4 had the same mutation site as lam1,and the 202^th base of the first exon changes from G to A,and the amino acid also changes from glycine?Gly,GGG?to arginine?Arg,AGG?.Bra A10g030950.3C encodes a FLS enzyme,which is a catalytic enzyme for the synthesis of flavonols from dihydroflavanols.