Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

Objective  Cellulose synthase (CesA) plays a key role in the biosynthesis pathway of plant cellulose,which controls the quality and yield of wood fiber. This work aims at cloning the homologous genes cellulose-related from Larix kaempferi, and further analyzing the nucleotide diversity and linkage disequilibrium, which may provide important genetic foundation associated with LkCesA gene and gene-assisted breeding of new germplasms with desirable wood fiber traits in L.kaempferi.Method  According to the expressed sequence tags (ESTs) of cellulose synthase from L.kaempferi transcriptome database, a cDNA fragment encoding CesA was isolated from L. kaempferi by gene-specific PCR amplification. The genomic sequences of LkCesA in 40 individuals were cloned and sequenced, then the single nucleotide polymorphisms (SNPs) diversity and linkage disequilibrium of LkCesA were analyzed using DnaSP5.0 software.Result  The CesA fragment was 1 209 bp in length with a partial open reading frame (ORF, 1 053 bp) which would be capable to encode a predicted protein of 350 AA. The sequence indicated that the deduced amino acids shared 95.4% identity with PtCesA2 from Pinus taeda. A total of 83 SNPs was detected, and the frequency and diversity of SNPs (πT) were 1/21 bp and 0.006 05, respectively. There were 69 SNPs belonging to transition type and 24 belonging to transversion type, including 19 common SNPs and 64 rare SNPs. In total, 54 SNPs were detected in the coding regions of LkCesA, among which 20 were synonymous mutation and 34 were missense mutation. The results of linkage disequilibrium analysis showed that the LD declined rapidly with the nucleotide length.Conclusion  The results of this study indicate that LkCesA is a member of CesA gene family. The linkage disequilibrium declined rapidly within the gene regions of LkCesA suggests that the gene could be used as the candidate gene for LD mapping, which contribute to the directional cultivation and wood quality improvement in Japanese larch. In addition, many common SNPs were detected in LkCesA which could be used in further LD mapping.