摘要
DNA条形码技术是利用DNA保守片段对物种进行快速准确鉴定的新兴技术。本研究根据GenBank中禾本科牧草matK和rbcL基因的核苷酸序列,设计4对通用引物,建立并优化了针对禾本科7个主要牧草属8种牧草11个样品[高丹草(Sorghum bicolor×S.sudanense)、玉米(Zea mays)、针茅(Stipa capillata)、"贝克"多年生黑麦草(Lolium perenne‘Plxie)、"凯帝莎"多年生黑麦草(L.perenne‘Caddieshack’)、甘肃羊茅(Festuca kansuensis)、"百琪"紫羊茅(F.rubra‘Bargena’)、"梦神"紫羊茅(F.rubra‘Maxima’)、"百胜"草地早熟禾(Poa pratensis‘Barvictor’)、"钻石"草地早熟禾(P.pratensis‘Diamond’)和芨芨草(Achnatherum splendens)]的目的片段的扩增条件。对扩增产物进行测序和分析,经分别比对,筛选出8种牧草的4个标记位点5’端和3’端保守序列。对各标记位点保守区内的核苷酸进行单核苷酸多态性(SNPs)的单倍型分析。结果表明,matK1、matK2、matK3分别有6个单倍型(H1^A、H1^B、H1^C、H1^D、H1^E和H1^F)、7个单倍型(H2^A、H2^B、H2^C、H2^D、H2^E、H2^F和H2^G)和3个单倍型(H3^A、H3^B和H3^C),rbcL基因有5个单倍型(H4^A、H4^B、H4^C、H4^D和H4^E)。根据matK(matK1、matK2、matK3)和rbcL基因筛选的4个标记位点为8种牧草建立了相对应的特异DNA识别码。本研究可为混合禾本科牧草饲料中的高粱属、玉蜀黍属、芨芨草属、针茅属、黑麦草属、羊茅属和早熟禾属的8种牧草准确识别提供分子水平上的科学依据。
DNA barcoding is an emerging technology,which is used as a tool to indentify species accurately and quickly using the conservative DNA segments.Our research has established and optimized target fragments' the amplification conditions for eleven samples(Sorghum bicolor×Sorghum sudanense,Zea mays,Stipa capillata,Lolium perenne ‘Plxie',Lolium perenne ‘Caddieshack',Festuca kansuensis,Festuca rubra ‘Bargena',F.rubra ‘Maxima',Poa pratensis ‘Barvictor',P.pratensis‘Diamond',Achnatherum splendens)from seven major genera(Sorghum,Zea,Stipa,Lolium,Festuca,Poa,Achnatherum)and eight species according to nucleotide sequence of grass matK and rbcL genes in GenBank,and designed four universal primers.5'end and 3'end conservative fragments in four marked fragments were selected by amplification products sequencing and analyzing.Single nucleotide polymorphism(SNP)haplotype analysis of conservative fragments nucleotide sequences of each maker loci showed there were 6haplotypes(H1^A,H1^ B,H1^C,H1^ D,H1^Eand H1^F),7haplotypes(H2^A,H2^ B,H2^C,H2^ D,H2^E,H2^ Fand H2^G)and 3(H3^A,H3^ Band H3^C)haplotypes among matK1,matK2,matK3 respectively,by the same token,rbcL gene has 5haplotypes(H4^A,H4^ B,H4^C,H4^ D and H4^E).According to the haplotypes about matK(matK1,matK2 and matK3)and rbcL,we have constructed the DNA barcoding for eight grass species.The research results provide a scientific basis for identification of mixed feed grasses including eight species of the seven generas.
出处
《草业科学》
CAS
CSCD
北大核心
2016年第9期1702-1710,共9页
Pratacultural Science
基金
甘肃省高等学校科研项目(2013A-64)
公益性行业(农业)科研专项(201503134)