Screening Of Universal DNA Barcodes For Common Forages And Establishment Of Database

The traditional classification methods are susceptible to restrictions by grass characteristic,developmental stage,and the appearance of individual species form similar in the study of forage identification.It lead to classical taxonomy method make based on the morphology exist some difficulties in identification of common forage grass.If forages were digested by animal gastrointestinal tract or artificial processing,identification will become extremely difficult.DNA Barcoding can avoid recognizing errors and provide a classification basis on the molecular level,become more accurate and more convenient.It technology can boost identification speed of grass,improve scientific research efficiency and to benefit of new species found.But most importantly is make sure classification subject development more faster and more deeply.DNA barcoding is an emerging technology based on specificity within a species and diversity between species to build biological identification database by using one or more standard DNA barcode,which is used as a tool to indentify species accurately and efficiently.In the study,we designed seven universal primers according to nucleotide sequence of common forages' matK,rbcL,trnH-psbA and ITS genes in GenBank to amplify 40 species in leguminosae forages and grass forages by MEGA 5.0 software and in conservative district of the base polym area on both sides.4 universal primers was designed in matK gene' four market loci(matK1?matK2?matK3 and matK4),3 universal primers was designed in rbcL,trnH-psbA and ITS genes.Due to PCR amplification efficiency lower,universal primers were designed in trnH-psbA and ITS genes unfit as forages DNA Barcoding candidate sequences.Our research has established and optimized target fragmen'the amplification conditions for 40 variety forages.5? end and 3? end conservative fragments in four marked fragments were selected by amplification products' purification,sequencing and analyzing.Single nucleotide polymorphism(Single nucleotide polymorphism,SNPs)haplotype analysis of conservative fragments nucleotide.Sequences of each maker loci showed there were 12,17 and 6 haplotypes among mat K1,mat K2 and matK3 respectively,by the same token,rbcL gene has 13 haplotypes.According to the haplotypes about matK(mat K1,matK2 and mat K3)and rbcL,we have constructed the DNA barcoding for 20 species forages.It proved that the combination of mat K and rbc L could be used as candidate DNA barcode for leguminosae and grass forages,but trnH-psbA and ITS cannot be used as DNA Barcoding candidate sequences due to amplification efficiency lower.The results can provide a molecular basis for accurate identification of various pasture species in mixed legumes and gramineous pasture.