Photomorphogenic mutants of tomato

Summary Two cloned cassava genes (pCAS6, pHNL) and a barley gene (pBLT63) which is highly conserved in higher plants, were evaluated as potential multilocus probes for RFLP analysis of phylogenetic relationships of species within the genus Manihot. For M. rubricaulis, M. chlorosticta and M. esculenta subsp. flabellifolia dendrograms based on 13 probe-restriction enzyme combinations show very little variation between accessions of the same species. A close relationship is demonstrated between the Mexican species M. chlorosticta and the M. esculenta subsp. flabellifolia accessions, which does not support the classification of flabellifolia as a true South American wild species.     

The European Prunus mapping project Progress in the almond linkage map

Summary Six European research groups are collaborating to develop genetic markers and linkage maps for use inPrunus breeding programmes. A basic map with 200 RFLPs and 50 more markers including isozymes and RAPDs will be constructed using two highly segregating populations: an interspecific peach × almond F₂ and a cherry F₂. Then, the parents of eleven almond, cherry, peach or plum breeding progenies segregating for target characters will be screened for polymorphisms at the marker loci, and a set of reduced maps, one per progeny, will be constructed with markers spaced 20–30 cM and covering the whole genome. Cosegregation analysis of markers and characters of interest will allow us to find linkages between markers and major genes or quantitative trait loci responsible for the expression of these traits. A map with 72 markers, 7 isozymes and 65 RFLPs, has been developed at the IRTA-Cabrils laboratory using an intraspecific almond progeny, ‘Ferragnes’ × ‘Mono’. Probes for the analysis of RFLPs were obtained from almond genomic and cDNA libraries. The level of polymorphism for RFLPs and the distribution of markers in the chromosomes of almond are discussed.     

RFLP analysis of cytoplasmic male-sterile lines of pigeonpea [Cajanus cajan (L.) Millsp.] developed by interspecific crosses

Total DNA from three putative cytoplasmic male sterile (CMS) progenies derived from crosses between the wild species Cajanus sericeus and the cultivated species Cajanus cajan, five C. cajan, one accession of C. sericeus and two genetic male sterile lines of pigeonpea were compared for their RFLP patterns using maize mitochondrial DNA (mtDNA) specific probes. Three putative cytoplasmic male sterile (CMS) progenies from the multiple cross genome transfer of pigeonpea lines (CMS 7–1, CMS 12–3, and CMS 33–1) showed hybridization patterns identical to that of C. sericeus when DNA was digested with EcoRI and HindIII and probed with maize mtDNA clones. The results suggested that these putative CMS progenies have the mitochondria of the female wild species parent. The hybridization patterns of the three male parental lines used in the development of the CMS progenies were similar in all the restriction enzyme-probe combinations except HindIII-atp6. The genetic male sterile lines, MS Prabhat and QMS 1 differed from each other in their hybridization pattern. The genomic DNA hybridization pattern of HindIII digested DNA from ICPL 87 differed from the other pigeonpea lines when probed with the maize mtDNA clones. The cluster analysis of the hybridization data suggested the occurrence of variation in the mitochondrial genome even among the cultivated species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic characterization of 218 elite CIMMYT maize inbred lines using RFLP markers

Characterization of genetic diversity among maize inbred lines can facilitate organization of germplasm and improve efficiency of breeding programs. A set of 218 phenotypically diverse inbred maize lines developed at CIMMYT for hybrid production was characterized using 32 RFLP markers to: (1) analyze the genetic diversity present; (2) define potential heterotic groups based on clusters formed with marker data; and (3) identify the most representative testers for each potential heterotic group. Lines were clustered using five different genetic distance measurements to find consensus non-hierarchical clusters. Dendrograms were produced to study hierarchical classification within smaller groups of lines. A very high average allelic diversity was seen in this germplasm. Lines did not cluster based on phenotype, environmental adaptation, grain color or type, maturity, or heterotic response (as determined based on hybrid performance with testers), but lines related by pedigree usually did cluster together. Previously defined testers from opposite heterotic groups were not genetically differentiated, and did not represent well their heterotic group. Discrete clusters were difficult to find; thus, potential heterotic groups will be difficult to suggest using RFLP markers alone. However, suggestions on how to use molecular markers and cross performance information to refine heterotic groups and select representative testers are presented.     

Recurrent phenotypic selection for forage yield in rye

Summary Cultivars of Cucurbita pepo and other Cucurbita species were characterized by RFLP analysis using different fragments of the ribosomal intergenic spacer (IGS) of Cucurbita pepo as hybridization probes. Several cultivars could be distinguished by a specific rDNA restriction pattern, whereas some cultivars showed an identical RFLP pattern suggesting a closer relationship. Other species of the genus Cucurbita exhibited strong cross-reaction with the C. pepo spacer probes, in contrast to DNA of Cucumis species which did not cross-hybridize.Abbreviations IGS intergenic spacer - ITS internal transcribed spacer - kbp kilo base pairs - rDNA ribosomal DNA - RFLP restriction fragment length polymorphism - rRNA ribosomal RNA     

A genomic library of Lycopersicon pennellii in L. esculentum: A tool for fine mapping of genes

The cultivated tomato contains only a small fraction of the genetic variation present in its wild relatives. In order to use the wild germplasm in tomato breeding and genetic studies we developed a new kind of genetic resource which is composed of 50 L. esculentum lines each containing a single introgression from the green fruited species L. pennellii (LA 716). Each of the introgression lines is nearly isogenic to the cultivated tomato; these lines provide complete coverage of the wild species genome. The lines contain on the average an introgression of 33 cM from a total genome size of 1200 cM. The size and identity of the introgressed segments was determined based on RFLP analysis of 350 markers. This resource can be viewed as a genomic library of the wild species in the cultivated background. It covers the entire genome with single independent inserts per line and therefore every phenotypic difference between the introgression lines can be associated with the unique introgressed segment. The development and potential application of this resource are discussed.     

Searching for DNA introgressed from wheat and for wheat-like grain proteins in a rice x wheat hybridization derivative

The DNA of a putative rice x wheat hybridization derivative (X Oryticum oryzoides) from China, the DNA of its parental rice cultivar and the DNA of a wheat line were digested with ten different restriction endonucleases, resolved by agarose electrophoresis, Southern blotted and hybridized using genomic wheat DNA as a probe. Phenol extracted, ethanol and cetyltrimethylammonium bromide precipitated DNA of the putative hybrid showed a restriction fragment length polymorphism (RFLP) different from that of the parental rice. When the DNA was further purified by Qiagen chromatography, the RFLP differences were not detected. Hence the apparent RFLP differences were probably due partial digestion of the less pure DNA preparations by the restriction endonucleases. No real introgressed fragments from wheat genome could be shown. The HpaII/MspI sites were more frequently digested with MspI than with HpaII in rice and hybridization derivative DNA, but the sites were evidently more frequently methylated in wheat DNA. Thus, in terms of methylation of the DNA, the hybridization derivative was much more like the rice parent than the wheat parent. The hybridization derivative showed a single endospermal protein (mass 19 kg mol^-1) not detected in the parental rice cultivar. This minor protein was soluble in buffered 50% isopropanol and precipitable with methanol. The results indicate that there are no or only short introgressed sequences from wheat in the rice/wheat derivative, a result which might be considered in breeding efforts with the hybrid derivative.     

RFLP在大豆种质资源及遗传连锁研究中的应用

本综合论述了ＲＦＬＰ在大豆种质资源及遗传连锁研究中的应用。自８０年代后期ＲＦＬＰＹ以大上，现已明确了栽培大豆ＯＧｌｙｃｉｎｅ ｍａｘ），野生在豆（Ｇ．ｓｏｊａ）和半野生大豆（Ｇ．ｇｒａｃｉｌｉｓ）ＲＦＬＰ的变异性；、大量探针已被制备和筛选出来。利用Ｇ．ｍａｘ×Ｇ．ｍａｘ和Ｇ．ｍａｘ×Ｇ．ｓｏｊａ两类组合已绘制出含约５５０个基因座３０００ｃＭ的ＲＦＬＰ遗传连锁图。一些质量性状基因座和数量性状基因座     

Comparison of conventional and molecular techniques to investigate the intestinal microflora of rainbow trout (Oncorhynchus mykiss)

The concept of the use of probiotic organisms or prebiotic compounds to modify the fish gut microflora is becoming a popular topic for investigation. A major flaw in many such studies is a failure to consider fully the nature of the established microflora, which is to be modified pre-, or probiotically. Since it is widely accepted that a large proportion of bacteria are non-culturable, the use of conventional bacteriological (culture) techniques alone to investigate fish intestinal microflora may be expected to bias results. We report a study designed to investigate the normal intestinal microflora of rainbow trout (Oncorhynchus mykiss) using both conventional bacteriological and molecular methods.Over an 18 month period, the intestinal microflora of a single population of laboratory-raised rainbow trout was investigated. Bacteria isolated using bacteriological techniques were identified using the BiOLOG system and 16S rRNA gene sequencing. Dominant bacteria consistently were Aeromonas sp. and Carnobacterium piscicola, demonstrating that the microflora is stable in fish kept in defined conditions. Restriction Fragment Length Polymorphism (RFLP) and 16S rRNA gene sequence analysis was used to investigate anaerobic and non-culturable bacteria. An obligate anaerobe, Clostridium gasigenes, was shown to be among the dominating intestinal microflora.     

Genotyping Cephalosporium gramineum and development of a marker for molecular diagnosis

This study investigated the genetic variation of 40 isolates of Cephalosporium gramineum, the causal agent of cephalosporium stripe disease of wheat, based on variations in internal transcribed spacers (ITS) and intergenic spacers (IGS) of rDNA. Of the isolates, 29 were from Japan and the rest from the USA and Europe. The ITS region was about 600 bp and almost identical among these isolates. In the IGS region (～5 kbp), restriction fragment length polymorphism analysis detected four genotypes among the 40 isolates. One representative isolate was selected from each of the four genotypes, and the IGS region was sequenced. Attempts to design a genotype‐specific marker based on the size of PCR products amplified with selected primers failed to differentiate among the four genotypes. Alternatively, a species‐specific primer set (CGIGS1 and CGIGS2) was developed that annealed within the conserved region, producing a DNA fragment of about 1·8 kbp. Tests of this primer set on a wide range of other fungi from 11 genera confirmed that it was specific to C. gramineum. This primer set could serve as an effective tool in the molecular diagnosis of C. gramineum and has the potential to assist in a better understanding of the host–pathogen interaction.