Environmental contribution to needle variation among natural populations of Pinus tabuliformis

Variations in the phenotypic characteristics of conifer needles is a consequence of genetic evolution that has been widely used in geographic variation and ecological studies.Although many studies are based on an in situ sampling strategy and generally realize the contribution of environmental effects to variation in needle traits,it is still uncertain which needle traits are most influenced by genetic effects and which are most influenced by the environment.Using both a common garden experiment to eliminate environmental heterogeneity and an in situ sampling strategy,we compared 18 Pinus tabuliformis needle traits among 10 geographical populations.Using both sampling strategies,we found significant differences in needle traits among populations and among individuals within populations.Differences in the‘‘among-population’’variance component between the two sampling strategies revealed the environmental contribution among natural populations for each trait.The among-population variance in the following traits exceeded 8%:needle length,number of stomata within 2 mm(NS2),number of stomatal lines on the planar side,number of resin canals(RCN)and the resin canal area(RCA).For the stability of needle traits,NS2,RCN,RCA,ratio of the vascular bundle area to the RCA(VBA/RCA),and MA/RCA differed significantly in more than five provenance changes between the common garden populations and natural populations,which may be susceptible to environmental effects.Conversely,the crosssectional area,mesophyll area(MA),MA/(VBA+RCA),and MA/VBA were phenotypically stable.Geographic variation patterns and systematic relation of needle traits differed between the two sampling strategies,suggesting that in situ sampling results may reflect environmental effects and deviate statistical parameters for genetic study.Future studies of genetic evolution in the context of geographic variation should be based on appropriate sampling strategies and stable phenotypic traits.

Isolation,expression and single nucleotide polymorphisms(SNPs) analysis of LACCASE gene(LkLAC8) from Japanese larch(Larix kaempferi)

Nucleotide diversity (pi) and linkage disequilibrium (LD) analysis based on SNP marker could provide a sound basis for choosing an association analysis method. Japanese larch (Larix kaempferi) is an important timber coniferous tree species for pulping and papermaking, but its high lignin content has significantly restricted it application potential. In this study, the LACCASE gene, that plays an important regulatory role for lignin biosynthesis, was selected as research target. The full-length cDNA and genomic sequences of the encoding LkLAC8 gene were isolated from the LACCASE expressed sequence tags of the Japanese larch transcriptome database using the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA was determined to be 1940 bp, with an open reading frame (ORF, 1734 bp) that encoded a protein of 577 AA. This protein contains four highly specific Cu2+ binding sites and 11 glycosylation sites, thus belonging to the LACCASE family. The deduced protein sequence shared an 89% identity with the PtaLAC from Pinus taeda. A real-time PCR analysis showed that the LkLAC8 transcript was expressed predominantly in mature xylem, with moderate levels in the immature xylem, cambium and mature leaves, the lowest in the roots. Lastly, the genomic sequences of LkLAC8 in 40 individuals from six naturally distributed populations of Japanese larch were amplified, and a total of 201 SNPs (103 and 98 mutation types of transition and transversion, respectively) were detected; the frequency of the SNPs was 1/19 bp. Nucleotide diversity among the six populations ranged from 0.0034 to 0.0053, which suggested that there were no significant differences among the populations. The LD analysis showed that the LD level decayed rapidly within the increasing length of the LkLAC8 gene. These results implied that LD mapping and association analysis based on candidate gene may be feasible for the marker-assisted breeding of new germplasms with low lignin in Japanese larch.