The Research On CpDNA, MtDNA And ITS Sequence And The Possibility To Identify Larix Species

In this paper, partial chloroplast DNA(cpDNA),mitochondrial DNA(mtDNA) and nuclear internal transcribed spacer sequence(ITS) was studied for the limitation to identify larix species.One single nucleotide polymorphism(SNP) was obtained in rbcL gene that can divide the larix species into two groups.Combining with mitochondrial marker f-13 and B-11,we can identify larix species exactly and quickly.1. The total DNA were extracted from seeds of larix species by improved CTAB method.Adding pheno/chloroform/isopentanol(25:24:1) to extraction buffer and extracting for three times,we can get rid of the lipid and protein in seeds,and the quality of DNA is good.2. CpDNA rbcL and matK, nuclear ITS1 were amplified using PCR primers. rbcL, matK and ITS1 were digested by six restriction endonucleaseas respectively,including Haeâ…¢,EcoRâ…¤,Mspâ… ,Xspâ… ,Taqâ… ,Sphâ… .An effective SNP was obtained,a restriction fragment of 601bp was observed in L. kaempferi,L.occidentalis and L.laricina while two fragments of 120bp and 481bp were observed in the others. This polymorphism is due to a single nucleotide substitution in the restriction site:TTGA in L. kaempferi,L.occidentalis and L.laricina,not recognized by Taqâ… and TCGA recognized by Taqâ… .This SNP can divide the larix species into two groups.3.The mutation rate of ITS1 and ITS2 was high considered by the other researchers.These sequences were amplified using primers,of which the lenth were 250bp.They were analysised by optimized SSCP method,the result showed that there weren't mutation gene.The result was also validated by sequences process.4. In order to indentify the seeds ,mitochondrial markers f-13 and B-11 were amplified in all larix specials,which were converted SCAR(sequence characterized amplified region) by RAPD(random amplified polymorphic DNA) technique.The specific sequence f-13 were present in L. kaempferi,L.Laricina,L.gmelinii and L.olegensis while absent in the others,the specific fragment B-11 were present in L.kaempferi,L.occidentalis and L.gmelinii while absent in the other specials.RbcL-taqâ… ,combined with mtDNA f-13 and B-11,can be used to identify larix specials fastly and exactly.