Characterisation of wheat-rye recombinants with RFLP and PCR probes

Summary The introgression of genetic material from alien species into wheat has become an important tool in modern wheat breeding. Ideally, only the trait of interest and no flanking material should be transferred. Random recombination between the genetic material is therefore of paramount importance. In a model system, we examined 17 recombinants putatively between chromosome 1D of wheat and 1R of rye with 60 random RFLP and three PCR markers. The recombinants had been generated by removing the normal effect of the Ph1 gene in the wheat background. Amongst the nine short-arm recombinants, three breakpoints were identified but no differentiation could be made between the five proximal recombinants. For the eight long-arm recombinants analysed only two breakpoints were identified with 36 markers. However, only a single RFLP marker was able to differentiate between the recombinants. Indeed the long-arm results are consistent with the possibility that only the rye telomeric region had been transferred. These results indicate either a strong clustering of the RFLP markers near the centromere or else imply that recombination induced between wheat and rye in the absence of the normal effect of the Ph1 gene occurs at only restricted sites. The results allow new primary recombinants to be selected for intercrossing to generate secondary recombinants which are expected to have a smaller interstitial rye segment than that present in DR-A1.     

用PCR直接筛选和鉴定虎纹捕鸟蛛毒素Ⅰ的重组阳性克隆

以合成的两段插入序列为上、下游引物用PCR法直接筛选插入有虎纹捕鸟蛛毒素Ⅰ（HWTX－Ⅰ）cDNA的重组阳性克隆。并用PCR法快速鉴定重组体中插入片段的正、反连接方向,扩增用引物是以位于克隆位点上游的一段载体序列上游引物,以插入序列为下游引物。对100个单克隆进行了上述两次PCR筛选鉴定,选取2个有靶片段插入并且为正向连接的重组子进行测序,其结果证实了插入片段及其方向的正确性。     

The applicability of consensus PCR primers across species and genera: the use of wheat Em sequences to develop markers for orthologues in rye

Two wheat consensus primer sets, directed to ”early-methionine-labelled” (Em) gene sequences, were tested for their ability to amplify beyond their original source. A range of widely diverse templates, including other Triticeae species and sample monocot and dicot species, was assayed. Primer set EMC5/EMC3, amplifying the entire coding region with its intron and part of the 3’ untranslated region, targets Triticeae and sorghum Em sequences. The other set, EMC5/EMCO31, directed to the coding region and its intron, amplifies templates from all the grass species. Both primer sets fail to amplify Em sequences from more distant monocots and the dicots. Using set EMC5/EMC3, we isolated and sequenced ten members of the rye Em gene family from five different rye sources. Significant DNA sequence variation between wheat and rye sequences in the non-coding regions was found, and this was used to develop seven sequence-specific primers. Twelve primer combinations were analysed, 7 of which were Em-R1-specific, amplifying a product in at least one of the tested rye or rye-carrying genotypes but not in wheat. Four sets exhibited clear amplification length polymorphisms which allowed discrimination between and within the rye sources. The primers also discriminated between wheat-rye recombinants with proximal 1RL rye chromatin and those carrying distal 1RL rye chromatin. These results show that wheat consensus primer sets can be used to isolate orthologous sequences, especially from species that are used for alien gene transfer in wheat. Subsequently, species-specific assays can be designed that are useful tools for this application. Received: 7 December 1998 / Accepted: 21 July 1999