Exploring The Formation Mechanism Of Striped Leaves Of Clivia Miniata Var.variegata Based On High-throughput Sequencing

Clivia miniata is one of the "Top Ten New Year Festival Flowers" with both medicinal and ornamental values.Clivia miniata var.variegata is a mutant of C.miniata,and its typical plant has yellow-green striped leaves.The striped phenotype of C.miniata var.variegata is maternally inherited,and the formation mechanism of striped leaves is yet to be studied.In this study,we used high-throughput chloroplast genome sequencing,transcriptome high-throughput sequencing,PCR,first generation sequencing and bioinformatics analysis to reveal the formation mechanism of stripe phenotype.The main results are as follows:(1)The full-length sequences of chloroplast genome(cp DNA)for C.miniata and C.miniata var.variegata(158,114 bp or 157,689 bp)were obtained by high-throughput sequencing.The online tool Ge Seq was used to annotate the genes of cp DNA in C.miniata and C.miniata var.variegata and a total of 135 functional genes were obtained.Simple sequence repeat(SSR),codon preference,and phylogenetic analysis were performed based on the full length of cp DNA in C.miniata and C.miniata var.variegata.It was found that there are 61 SSR sites in C.miniata cp DNA.Forthe 88 protein-coding genes of C.miniata cp DNA,a total of 26,419 codons were detected.The codons of C.miniata cp DNA prefer end with A or U(T),and Leu has the highest frequency of codon usage.C.miniata was in the same branch as the Amaryllidaceae,showing the closest relationship with Amaryllidaceae,supporting that C.miniata belongs to the Amaryllidaceae.The phylogenetic analysis results based on chloroplast ycf2 are mostly the same as those based on full-length cp DNA,which supported that the ycf2 can replace the full-length cp DNA for plant phylogenetic analysis.The main results of C.miniata var.variegata are the same as the above.However,the number of codons and SSR sites in the C.miniata var.variegata was different from that of C.miniata.A total of26,273 codons and 62 SSR sites of cp DNA in yellow stripes of C.miniata var.variegata leaf were detected.(2)High-throughput transcriptome sequencing,PCR and first generation sequencing showed that compared with wild type,the coding region of chloroplast rpo C2 gene in yellow stripe region of C.miniata var.variegata was 425 bp missing,and the gene in green stripe region was normal,and the deletion could be transmitted to offspring through maternal parent.Ten differential hotspots(including rpo C2)in C.miniata cp DNA were found through differential hotspots analysis.Because rpo C2 encodes the β" subunit of chloroplast E.coli-like RNA polymerase(PEP),it is speculated that the 425 bp deletion may lead to abnormal chloroplast gene transcription.The transcripts of rpo C2 were detected by high-throughput transcriptome sequencing(RNA-seq).The deletion of rpo C2 transcript in theyellow stripe region of C.miniata var.variegata was consistent with that of the gene.The expression of rpo C2 gene in the yellow green stripe region of C.miniata var.variegata was not significantly different.The deletion of 425 bp resulted in the transcription from 4,125 nt to3,700 nt.Finally,the translated protein was shortened from 1,384 amino acids to 299 amino acids.Software analysis showed that the deletion resulted in changes in the secondary structure and three-dimensional structure of rpo C2 protein.High-throughput transcriptome sequencing(RNA-seq)analysis revealed 28 chloroplast differential expression genes(cp DEGs)between yellow stripes and green stripes of C.miniata var.variegata.The 28 cp DEGs were all down-regulated in the yellow stripes and included:(1)Photosystem I related genes psa A,psa B and psa C;(2)Photosystem II related genes psb A,psb B,psb C,psb J,psb N,psb T,psb M and psb Z;(3)Cytochrome b/f complex genes pet A,pet B,pet D,Pet N and pet G;(4)Encoding ATPase genes atp A,atp B,atp I,atp H and atp F;(5)Membrane protein gene cem A;(6)Coding ribosomal protein small subunit gene rps14;(7)Coding NADH oxidoreductase gene ndh K;(8)Transfer RNA gene trn L-UAA;(9)Ribulose diphosphate carboxylase large subunit gene rbc L;(10)Ribosomal RNA genes rrn16 and rrn23.Promoter analysis found that the forgoing cp DEGs of photosystem I and II,cytochrome b/f complex and ribosome synthesis can only be transcribed by PEP;pet A,atp A,atp F,atp H,atp I,cem A,ndh K,16 Sr DNA and 23 Sr DNA can be transcribed by both PEP and NEP;atp B can only be transcribed by NEP.It suggested that most cp DEGs were caused by rpo C2 deletion mutation directly but a few(atp B)are caused by the mutation indirectly.In summary,the expression product of rpo C2 was the β" subunit of PEP.It was speculated that the 425 bp deletion of the chloroplast gene rpo C2 of C.miniata led to the significant down-regulation of chloroplast photosynthesis-related genes(including most of the photosystem I and II genes psa A,psa B,psa C,psb A,psb B,psb C,psb J,psb N,psb T,psb M and psb Z)and then abnormal chloroplast and yellow stripes of leaves.This study excavated the genetic basis of the striped leaf phenotype of C.miniata var.variegata and established a model of the genetic mechanism of the special phenotype of C.miniata var.variegata,which layed the foundation for finally revealing the molecular mechanism of striped leaf formation and the breeding of ornamental plants.