Gene Mapping And Candidate Gene Prediction Of Purple Leaf Sheath In Maize (Zea Mays L.)

Pigment deposition, distribution and concentration within different maize varieties play an important role in functional genomics research and genetic breeding. Purple leaf sheath (PSH) is one of the common traits related with pigment in maize. PSH is an important morphological marker in maize breeding and it can be more intuitive to do the selection and genetic research of special traits associated with it. Some studies also prove that pigment from maize purple plant can delay aging. In addition, with the depth research on PSH, physiological mechanism of pigment synthesis would be further improved. Therefore, the mapping of maize purple leaf sheath gene (PSH) has very important theoretical significance and practical value.To carry out genetic analysis and gene mapping of PSH, a F2 population and a recombinant inbred line (RIL) population consisting of 208 inbred lines are constructed separately by crossing of maize inbred lines DH1M and T877. The main results are as follows:1. The separation ratio of purple and green leaf sheath appearing in the F2 population conforms to Mendelian segregation ratios 3:1. In addition, the separation ratio of purple and green leaf sheath pedigree in the RIL populations is consistent with 1:1. These show that the purple leaf sheath is controlled by dominant nuclear gene (PSH).2. It is turned out that PSH gene is preliminarily mapped on the chromosome 10 of maize by WinQTLCart in RIL population. Simultaneously by using gene chip to perform SNP genotyping in parents and 190 F2 plants, PSH gene is initially mapped on the chromosome 10. The closest linked marker is SNP marker SYN17666, which is transformed into CAPS to verify. Then PSH is quickly mapped by QTL-seq. Firstly, whole genome sequencing of two parents pools and recessive pool consisting of 30 green leaf sheath pedigrees are conducted using high-throughput sequencing. Then sequencing results are processed through filtration, alignment, SNP-index calculation and mapping. The results show that SNP-index value is greater than 0.9 in two regions (128M-132M and 134M-139M) on chromosome 10. Therefore, it is further inferred gene that controls purple leaf sheath locates in the range of 128-139Mb on chromosome 10. At the same time, PSH is located between markers IDP8334 and IDP7541 within about 2Mb via genotyping of 98 F2 plants and 76 RIL pedigree based on markers published on MaizeGDB. Research shows the result of QTL-seq is consistent with that of traditional gene mapping methods. Both experiments have an effect on mutual authentication. On the basis, positioning interval is further reduced to about 304Kb from marker Inde102A to Inde101C by developing new indel markers using resequencing information.3. There are 10 genes within the above range of 304 Kb through gene annotations, among which gene 5 (GRMZM5G822829) predictively encodes bHLH transcription factor, an important regulatory factor in the anthocyanin biosynthetic pathway. Homologous genes of gene 5 in rice and Arabidopsis also encode putative bHLH-type regulatory factors. Furthermore, sequence analysis reveals there are four sequence differences at gene 5 between parents. Thus, GRMZM5G822829 is regarded as the most likely candidate gene of purple leaf sheath gene. Then, the GRMZM5G822829 gene expression analysis carried out by Real-Time PCR indicates expression of the gene exists very significant differences between purple and green leaf sheath parents, which further proves GRMZM5G822829 gene is a candidate gene of purple leaf sheath gene PSH. These lay the foundation for cloning and functional verification of the target gene.