Development Of Pan-grass CNS-AFLP Markers And Their Usability Assay In Cereal Crops

Using the bioinformatic software of GEvo(Genome Evolution Analysis),129 gene sequences from maize,wheat,sorghum,rice and pearl millet respectively were conducted to identify their CNS(Conserved noncoding sequences) containing pattern.Of the 129 genes, 111 genes are from maize,12 genes are from wheat,3 genes are from sorghum,one gene(tb1) is from pearl millet and 2 genes are from rice.There are 79 genes have a high CNS synteny, of which 37 genes are enzyme coding genes and their percentage of the CNS synteny on average is 50.64%;27 genes are structural protein coding genes and their percentage of the CNS synteny on average is 46.50%;15 genes are other types,the percentage of the CNS synteny on average is 70.11%.The high synteny of CNS in orthologs of cereal crops is in favor of the comparative study of cereal crops,it also eatablished the theoretic base for the development of a pan-grass markers system.Based on their CNS density and degree of synteny,27 gene sequences including PHYB, pgp1,tb1 and lg2 were chosen as templates to design primers based on CNS.A total of 124 pairs of primers,including CNS—CNS and CNS—Exon combinations,were designed.For more CNS—AFLP primers designing,we download a database including 16571 pairs of homologous sequences from CoGe,identified their CNS from the homologous sequences using GEvo develop,selected sequences which have a high synteny,and used sorghum genes as templates designed 86 pairs of CNS—CNS primers.Ten important cereal crops,which represent different subfamilies of the grass species, were chosen to test the feasibility and the amplified fragment length polymorphism of the designed primers.The result shows that 74 pairs of the 210 primer pairs designed can be used as pan-grass primers,and most others can be used in some of cereal crops.The amplified fragments and their enzyme digested products were also polymorphic among different samples.Then,we use the Setaria materials to test the primers which screened out from all of the primers.The result shows that the primers not only can be used as pan-grass primers,but also can be used in the same category of different species,and have a better result,therefore,we can use the CNS primers analyze the evolution between species.The molecular markers of CNS—AFLP not only can analyze the differences of the selection pressure in evolution between different positions of homogeneity gene,but also can analyze the conservation between different genes in evolution,so CNS—AFLP is a powerful tool for studying biological evolution.The characteristic of CNS—AFLP is that primer pairs were designed according to existed genes analyzed in known species and they can also be transferably used in other species.CNS—AFLP is suitable for genetic diversity and evolution study of those species that lack SSR and other specific markers,and the method can also help the comparative study of cereal crops.This research indicated that CNS—AFLP is a new kind of molecular marker,which is suitable for genetic diversity and evolution study of those species that lack SSR and other specific markers,and the method can also help the comparative study of cereal crops.