Development Of E~e-Chromosome Specific AFLP And STS Molecular Marker For L. Elongatum In Chinese Spring Wheat Background

The amplified fragment length polymorphism(AFLP) is one kind of PCR based molecular markers for detecting of genomic restriction fragment length variations.It has many advantages,such as rapid,sensitive,reliable,repeatable,higher level of polymorphism and can be used without prior knowledge about the genome strucrare.For these reasons,it is widely used in different plant species.Diploid species of Lophopyrum elongatum is one of the valuable relatives for wheat quality and yielding improvement.For better utilization of the genes or superior characters deposited in L.elongatum species,a lot of wheat-L,elongatum disomic substitution and additional lines have been obtained through distance cross between wheat and L.elongatum.However,the markers for detection and tracking of alien genetic material of L.elongatum were very limited and thus impeded us better use of this plant resources.In this study,twenty eight Ee-Chromosome specific AFLP molecular markers were acquired and five of them were successfully converted into the sequence tagged site(STS) molecular markers.The results present here would provide new ways for rapid detection of alien genetic material transferred through L. elongatum into common wheat.The main results were as follows:1.Screen of E^e chromosome specific AFLP markers by analyzing of L. elongatum-wheat var.CS disomic substitution and additional linesFive AFLP primer combinations were used to screen common wheat var.Chinese Spring(CS)—L.elongatum disomic substitution and additional lines for development of the E^e chromosome specific molecular markers of L.elongaturn.It was showed that 28 AFLP markers,which were specific for 1E^e to 7E^e chromosome,respectively,were obtained.The chromosomes specific AFLP markers distributed in 1E^e,2E^e,3E^e,4E^e,5E^e, 6E^e and 7E^e chromosomes were 7,8,5,1,4,1,and 2,respectively. 2.Sequencing and sequence alignment of E^e chromosome specific AFLP fragmentThe full DNA sequences of these AFLP markers were obtained by sequencing of three different positive clones.After sequence alignments in NCBI database,it was indicted that in the 28 AFLP sequences,only 4 sequences find the similar sequence.The two sequences of M-CCAG/E-AAT 463bp and M-CCAG/E-AAC 367bp were the repeat sequences of genes,while M-CTAG/E-ATT 284bp and 205bp were the partial sequences of genes, respectively.3.E^e chromosome specific marker conversions:AFLP into STSFive AFLP markers of E-ATC/M-CGTA341bp,E-ATT/M-CTAG265bp,E-AAT/ M-CCAG139bp,E-ATT/M-CTAG205bp and E-AAT/M-CCAG 143bp were successfully converted into chromosome specific STS markers STS318,STS24I,STall6,STS182 and STS₁₂₀,respectively.Among them,STS₃₁₈ and STS₂₄₁ only exist in CS 1E^e substitution and addition lines,while STS₁₁₆ and STS₁₈₂ only presented in CS 2E^e substitution and addition lines,and STS₁₂₀ only exist in CS5E^e substitution and addition lines,they were thought to L.elongatum 1E^e,2E^e and 5E^e chromosome specific STS markers,respectively.4.STS marker polymorphisms on five L.elongatum accessionsThe STS marker polymorphisms were performed on five L.elongatum accessions.It was indicated that the STS polymorphism exhibited as non-band or the same length band as in the STS fragments.Among them,STS₃₁₈ can amplified the same size products in all accessions,while STS₂₄₁,STS₁₈₂ and STS₁₂₀ can respectively obtained the same size fragments in 3 different accessions,and STS₁₁₆ can amplified the same size fragments in only one accession.The results suggested that these STS marker fragments were present in some of the five L.elongatum accessions.As far as the five STS markers were concerned, they can amplify the target DNA products in at least one and at most four of the five L.elongatum accession.For example,in L.elongatum accession PI595139,there was only one marker obtained amplification,in accessions PI206624 and PI57416,there were three markers acquired target fragment,and in the remaining two accessions,there were four markers obtained expected fragment.5.Specificity analysis of STS markers in ditterent common wheatIn previously study,it was showed that the eight wheats,Chuan nong16,Chuan mai42,Mian nong 4,Mian yang26,Chuan mail07,Chuan mai28,Chuan yul2 and Chuan yul6, were no L.elongatum pedigree.In order to detecting the specificity of these STS markers in other common wheat,the above wheat eight cultivars were used.The results showed that none of the five STS markers could be detected in common wheat,suggesting that they can be used for detecting the alien genetic materials of L.elongatum in the CS-L. elongatum substitution and additional lines as well as in other wheat background.