Olive genetic diversity assessed using amplified fragment length polymorphisms

 Amplified fragment length polymorphism (AFLP) analysis was used to study the genetic variation within and among populations of genus Olea. A group of genotypes, all of them cultivated varieties of a single species, Olea europaea, was compared with wild olives and with a group of individuals belonging to different Olea species. Five primer combinations were used which produced about 290 polymorphic bands. The data obtained were elaborated with the Nei’s genetic similarity coefficient, applying different clustering methods and the Principal Coordinate Analysis. Cultivars, wild olives and North-West African species formed groups clustering together at a similarity level of 0.56, while the Olea species from East Africa and Asia grouped separately. Species from the Indian Ocean and Australia showed the highest diversity. We hypothesize that cultivars and wild plants are different forms of the same O. europaea species. The Olea from East Africa and Asia may be assigned to a different species, while the role of O. laperrini as well as that of O. maroccana as an intermediary form is confirmed. Received: 30 April 1998 / Accepted: 13 August 1998     

Distinct genetic groups of Giardia intestinalis distinguished by restriction fragment length polymorphisms.

The taxonomic status of the parasitic protozoal species Giardia intestinalis depends on the morphological similarity of all Giardia isolated from humans and the presumption that Giardia are host-specific. On the basis of electrophoretic data derived from examination of 26 enzyme loci in Australian isolates, it has been proposed that G. intestinalis is a species complex comprising three or four genetically distinct (but morphologically cryptic) species. These received the tentative designations of genetic groups I-IV (R. H. Andrews, M. Adams, P. F. L. Boreham, G. Mayrhofer & B. P. Meloni. International Journal for Parasitology 19, 183-190, 1989). In the present study, two unrelated DNA probes (one specific for a gene encoding a trophozoite surface protein, the other detecting a non-coding repetitive sequence within the G. intestinalis genome) were used in Southern hybridization analyses to examine 10 axenic isolates of G. intestinalis, established from diverse geographical regions in Australia, together with the Portland-1 isolate from the USA. Both probes identified every isolate unambiguously as belonging to one or other of two genetic clusters. Electrophoretic analysis of the same samples indicated that these clusters correspond to the previously defined genetic groups I and II. No heterogeneity was apparent within the seven group I isolates using either probe. However, when probed with the repetitive sequence, the four isolates belonging to group II exhibited small differences in banding patterns, suggesting that this group may be less homogeneous than group I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of restriction fragment length polymorphisms for genetic counseling: Population genetic considerations

Two-locus population genetic models are analyzed to evaluate the utility of restriction fragment length polymorphisms for purposes of genetic counseling. It is shown that the linkage disequilibrium between a neutral marker and a tightly linked overdominant mutant will increase rapidly as the mutant moves to its polymorphic equilibrium. The linkage disequilibrium decays for deleterious recessive mutants. Two measures involving the linkage disequilibrium are investigated to determine how much information the transmission of the neutral marker provides about the transmission of the selected gene. In certain kinds of matings, where the parental two-locus genotypes and linkage phases are known, it is possible to determine whether or not a progeny is homozygous for the selected gene on the basis of the fetal genotype at the marker locus. A quantity of primary interest is the fraction of matings between individuals heterozygous for the selected gene in which exact diagnosis can be made in this way. The expected proportion of such matings, taken over all two-locus matings involving heterozygotes at the selected locus, is calculated as a function of the gene frequencies at the two loci and the linkage disequilibrium between them. This expected value is maximized when the linkage disequilibrium is at its maximum in absolute value. Fewer than half of all matings are informative if the linkage disequilibrium is small in magnitude or if the gene frequencies at the two loci are quite different. Consideration is also given to various conditional measures of association that may be useful when the parental two-locus genotypes are unknown. The results suggest that the utility of tightly linked neutral marker genes in predicting the transmission of a selected gene is generally less when selection acts against a recessive gene than for overdominant selection.     

The suitability of restriction fragment length polymorphisms as genetic markers in maize

Summary Strain identification in Zea mays by restriction fragment length polymorphism should be feasible due to the high degree of polymorphism found at many loci. The polymorphism in maize is apparently higher than that currently known for any other organism. Five randomly selected maize inbred lines were examined by Southern filter hybridization with probes of cloned low copy sequences. Typically, several alleles could be distinguished among the inbred lines with any one probe and an appropriately selected restriction enzyme. Despite considerable polymorphism at the DNA level, 16 RFLP markers in three inbred lines of maize were examined for six to 11 generations and found be stable. Mapping of RFLP markers in maize can be accelerated by the use of B-A translocation stocks, which enable localization of a marker to chromosome arm in one generation. The use of recombinant inbred lines in further refinement of the map is discussed.     

Genetic diversity of oilseedBrassica napus germ plasm based on restriction fragment length polymorphisms

Oilseed rape (Brassica napus) is an important oilseed crop worldwide. Cultivars have been developed for many growing regions, however little is known about genetic diversity inB. napus germ plasm. The purpose of the research presented here was to study the genetic diversity and relationships ofB. napus accessions using restriction fragment length polymorphisms (RFLPs). Eighty threeB. napus accessions were screened using 43 genomic DNA clones which revealed 161 polymorphic fragments. Each accession was uniquely identified by the markers with the exception of the near-isogenic cvs Triton and Tower. The RFLP data were analyzed by cluster analysis of similarity coefficients and by principal component analysis. Overall, there were three major groups of cultivars. The first group included only spring accessions, the second mostly winter accessions and the third, rutabagas and oilseed rape accessions from China and Japan. These results indicate that withinB. napus, winter and spring cultivars represent genetically distinct groups. The grouping of accessions by cluster analysis was generally consistent with known pedigrees. This consistency included the grouping of lines derived both by backcrossing or self-pollination with their parents.     

Estimating genetic diversity of Arabidopsis thaliana ecotypes with amplified fragment length polymorphisms (AFLP)

The extensive natural variation of Arabidopsis thaliana ecotypes is being increasingly exploited as a source of variants of genes which control (agronomically) important traits. We have subjected 19 different Arabidopsis thaliana ecotypes to an analysis using the anplified fragment length polymorphism (AFLP) technique in order to estimate their genetic diversity. The genetic diversity was estimated applying the method of Nei and Li (1979) and a modified version of it and using 471 informative polymorphisms. The data obtained revealed that within this small set of ecotypes a group of three ecotypes and a further single ecotype exhibit considerable genetic diversity in comparison to the others. These ecotypes clustered at positions significantly separated from the bulk of the ecotypes in the generated similarity plots. The analysis demonstrated the usefulness of the AFLP method for determinating intraspecies genetic diversity as exemplified with Arabidopsis thaliana ecotypes. Results are discussed and compared with data obtained with other methods. Received: 18 June 1999 / Accepted: 28 July 1999     

Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms

Summary Genetic linkage maps were constructed for both maize and tomato, utilizing restriction fragment length polymorphisms (RFLPs) as the source of genetic markers. In order to detect these RFLPs, unique DNA sequence clones were prepared from either maize or tomato tissue and hybridized to Southern blots containing restriction enzyme-digested genomic DNA from different homozygous lines. A subsequent comparison of the RFLP inheritance patterns in F2 populations from tomato and maize permitted arrangement of the loci detected by these clones into genetic linkage groups for both species.     

Genetic diversity among elite Sorghum lines revealed by restriction fragment length polymorphisms and random amplified polymorphic DNAs

The genetic diversity of sorghum, as compared to corn, is less well characterized at the genetic and molecular levels despite its worldwide economic importance. The objectives of this study were to: (1) investigate genetic diversity for restriction fragment length polymorphism (RFLPs) and random amplified polymorphic DNAs (RAPDs) in elite sorghum lines, (2) compare similarities based on molecular markers with pedigree relationships, and (3) examine the potential of RFLPs and RAPDs for assigning sorghum lines to the A/B (sterile) and R (restorer) groups. Using four restriction enzymes, polymorphism was detected with 61% of the RFLP probes used, compared to 77% of the random primers. One hundred and sixteen (64%) probe-enzyme combinations yielded multiple-band profiles compared to 98% of the random primers. RFLP profiles generated 290 polymorphic bands compared to 177 polymorphic RAPDs. Pair-wise comparisons of polymorphic RFLPs and RAPDs were used to calculate Nei and Jaccard coefficients. These were employed to generate phenograms using UPGMA and neighborjoining clustering methods. Analysis of RFLP data with Jaccard's coefficient and neighbor-joining clustering produced the phenogram with the closest topology to the known pedigree.Contribution of the College of Agricultural Sciences, Texas Tech University, Journal No. T-4-365     

Construction of a genetic linkage map in man using restriction fragment length polymorphisms.

We describe a new basis for the construction of a genetic linkage map of the human genome. The basic principle of the mapping scheme is to develop, by recombinant DNA techniques, random single-copy DNA probes capable of detecting DNA sequence polymorphisms, when hybridized to restriction digests of an individual's DNA. Each of these probes will define a locus. Loci can be expanded or contracted to include more or less polymorphism by further application of recombinant DNA technology. Suitably polymorphic loci can be tested for linkage relationships in human pedigrees by established methods; and loci can be arranged into linkage groups to form a true genetic map of "DNA marker loci." Pedigrees in which inherited traits are known to be segregating can then be analyzed, making possible the mapping of the gene(s) responsible for the trait with respect to the DNA marker loci, without requiring direct access to a specified gene's DNA. For inherited diseases mapped in this way, linked DNA marker loci can be used predictively for genetic counseling.     

Bamboo germplasm screening with nuclear restriction fragment length polymorphisms

Summary Bamboo species are difficult to identify because flowering material is seldom available and taxonomy is of necessity based on vegetative characters. To evaluate the utility of restriction fragment length polymorphism (RFLP) analysis in bamboo systematics and germplasm screening, a library of random genomic probes from a Phyllostachys nigra PstI library was constructed. Probes from the library were used to screen bamboo germplasm consisting mostly of temperate bamboos of the genus Phyllostachys. RFLP variation was abundant, and species-specific patterns were readily obtained. Chloroplast DNA showed little variation among the bamboo accessions analyzed.     

Human restriction fragment length polymorphisms and cancer risk assessment

The polymorphic restriction fragments of the human Ha-ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the "rare" class (individual frequencies less than 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha-ras restriction fragment length polymorphism is useful in cancer risk assessment.     

Some combinatorial problems of DNA restriction fragment length polymorphisms

Recombinant DNA techniques provide a means of defining new polymorphisms at the DNA sequence level. Polymorphisms arise when individuals differ in the location and number of sites where restriction endonucleases can cleave their DNA. Each such site exhibits two possible states: one for the presence of a specific endonuclease recognition sequence, the other for its absence. The states of a system of adjacent sites can be revealed experimentally by cleaving a person's DNA into a set of fragments. For experimentally well-understood systems of sites, we consider problems of counting numbers of possible fragments, haplotypes, genotypes, and phenotypes, and the means of resolving phenotype-genotype ambiguities. The degree of polymorphism generated by such systems and the importance to gene mapping are discussed.     

The parental origin of X chromosomes in XX males determined using restriction fragment length polymorphisms.

The inheritance of several X-linked restriction fragment length polymorphisms ( RFLPs ) is examined in seven 46,XX males and their immediate relatives. The XX males are shown to have inherited a paternal and a maternal RFLP allele in each of the five (of seven) families in which these X-linked markers are informative. In the other two families, a maternal X-chromosomal contribution is demonstrated, but a paternal contribution cannot be determined. We conclude that most, if not all, XX males inherit one paternal and one maternal X chromosome. A segment of single-copy DNA specific to the short arm of the Y chromosome is found to be absent from the genomes of eight XX males. In one of these XX males, an Xp-Yp translocation had previously been inferred from chromosome-banding studies. Our findings argue against mosaicism involving a Y-containing cell line in the XX males examined here, but they do not exclude an X-Y (or Y-autosome) translocation during paternal meiosis. If such a translocation has occurred, the translocation product received by the XX males does not include the Yp-specific sequence tested here.     

Chloroplast DNA restriction fragment length polymorphisms (RFLPs) in Musa species

Summary Taxonomic and phylogenetic determinations within the genus Musa are established using a numerical, morphology-based scoring system. However, within this system, the classification and relationships of some types are disputed. The application of chloroplast DNA (cpDNA) restriction fragment length polymorphism (RFLP) analysis to Musa taxonomy provided valuable, supplemental information about the classification of, and relationships between, Musa species and subspecies. Whole-cell DNA was extracted from lyophilized Musa leaf-blade tissue and digested with various restriction enzymes, Southern blotted onto nylon membranes, and probed using radioactively labeled heterologous orchid cpDNA fragments. Phylogenies were inferred from cpDNA RFLP patterns using PAUP software. The relationships between most species examined were as expected; however, some species (M. beccarii and M. basjoo) did not conform to the conventional morphology-based phylogeny.     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary RFLPs were used to study genome evolution and phylogeny in Brassica and related genera. Thirtyeight accessions, including 10 accessions of B. rapa (syn. campestris), 9 cultivated types of B. oleracea, 13 nine-chromosome wild brassicas related to B. oleracea, and 6 other species in Brassica and allied genera, were examined with more then 30 random genomic DNA probes, which identified RFLPs mapping to nine different linkage groups of the B. rapa genome. Based on the RFLP data, phylogenetic trees were constructed using the PAUP microcomputer program. Within B. rapa, accessions of pak choi, narinosa, and Chinese cabbage from East Asia constituted a group distinct from turnip and wild European populations, consistent with the hypothesis that B. rapa had two centers of domestication. A wild B. rapa accession from India was positioned in the tree between European types and East Asian types, suggesting an evolutionary pathway from Europe to India, then to South China. Cultivated B. oleracea morphotypes showed monophyletic origin with wild B. oleracea or B. alboglabra as possible ancestors. Various kales constitute a highly diverse group, and represent the primitive morphotypes of cultivated B. oleracea from which cabbage, broccoli, cauliflower, etc. probably have evolved. Cauliflower was found to be closely related to broccoli, whereas cabbage was closely related to leafy kales. A great diversity existed among the 13 collections of nine-chromosome wild brassicas related to B. oleracea, representing various taxonomic states from subspecies to species. Results from these studies suggested that two basic evolutionary pathways exist for the diploid species examined. One pathway gave rise to B. fruticulosa, B. nigra, and Sinapis arvensis, with B. adpressa or a close relative as the initial ancestor. Another pathway gave rise to B. oleracea and B. rapa, with Diplotaxis erucoides or a close relative as the initial ancestor. Raphanus sativus and Eruca sativus represented intermediate types between the two lineages, and might have been derived from introgression or hybridization between species belonging to different lineages. Molecular evidence for an ascending order of chromosome numbers in the evolution of Brassica and allied genera was obtained on the basis of RFLP data and phylogenetic analysis.     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary Restriction fragment length polymorphisms (RFLPs) of nuclear DNAs have been used to explore the origin and evolution of the six cultivated Brassica species. Extensive RFLP variation was found at the species, subspecies and variety levels. Based on RFLP data from Brassica and related genera, a detailed phylogenetic tree was generated using the PAUP microcomputer program, which permits a quantitative analysis of the interrelationships among Brassica species. The results suggested that 1) B. nigra originated from one evolutionary pathway with Sinapis arvensis or a close relative as the likely progenitor, whereas B. campestris and B. oleracea came from another pathway with a possible common ancestor in wild B. oleracea or a closely related nine chromosome species; 2) the amphidiploid species B. napus and B. juncea have evolved through different combinations of the diploid morphotypes and thus polyphyletic origins may be a common mechanism for the natural occurrence of amphidiploids in Brassica; 3) the cytoplasm has played an important role in the nuclear genome evolution of amphidiploid species when the parental diploid species contain highly differentiated cytoplasms. A scheme for the origins of diploid and amphidiploid species is depicted based on evidence gathered from nuclear RFLP analysis, cpDNA RFLP analysis, cytogenetic studies and classical taxonomy.     

Restriction fragment length polymorphisms as genetic markers in soybean,Glycine max (L.) merrill

Summary Restriction Fragment Length Polymorphisms (RFLP) have been identified between widely distant cultivars (Minsoy and Noir 1 ) of soybean Glycine max (L.) Merrill. Using as probes randomly chosen clones of DNA, one in five probes revealed a polymorphism. More than half of these polymorphisms appear to result from rearrangements of the genomic DNA. Twenty seven markers were analyzed for linkage in F₂ plants. Eleven of these markers were contained in four linkage groups. Five cultivars were compared in a search for new alleles. When RFLP markers corresponding to low copy DNA were used to analyze three other cultivars — Sooty, Forrest and Mandarin (Ottawa) — few new alleles were found. Using these probes, five different markers could be used to differentiate the five cultivars. Complex probes, which correspond to repeated DNA, revealed different polymorphisms in different cultivars and a single such probe could be used to distinguish the five cultivars from each other.     

Genetic diversity for restriction fragment length polymorphisms and heterosis for two diallel sets of maize inbreds

Summary Changes that may have occurred over the past 50 years of hybrid breeding in maize (Zea maize L.) with respect to heterosis for yield and heterozygosity at the molecular level are of interest to both maize breeders and quantitative geneticists. The objectives of this study were twofold: The first, to compare two diallels produced from six older maize inbreds released in the 1950's and earlier and six newer inbreds released during the 1970's with respect to (a) genetic variation for restriction fragment length polymorphisms (RFLPs) and (b) the size of heterosis and epistatic effects, and the second, to evaluate the usefulness of RFLP-based genetic distance measures in predicting heterosis and performance of single-cross hybrids. Five generations (parents, F₁; F₂, and backcrosses) from the 15 crosses in each diallel were evaluated for grain yield and yield components in four Iowa environments. Genetic effects were estimated from generation means by ordinary diallel analyses and by the Eberhart-Gardner model. Newer lines showed significantly greater yield for inbred generations than did older lines but smaller heterosis estimates. In most cases, estimates of additive x additive epistatic effects for yield and yield components were significantly positive for both groups of lines. RFLP analyses of inbred lines included two restriction enzymes and 82 genomic DNA clones distributed over the maize genome. Eighty-one clones revealed polymorphisms with at least one enzyme. In each set, about three different RFLP variants were typically found per RFLP locus. Genetic distances between inbred lines were estimated from RFLP data as Rogers' distance (RD), which was subdivided into general (GRD) and specific (SRD) Rogers' distances within each diallel. The mean and range of RDs were similar for the older and newer lines, suggesting that the level of heterozygosity at the molecular level had not changed. GRD explained about 50% of the variation among RD values in both sets. Cluster analyses, based on modified Rogers' distances, revealed associations among lines that were generally consistent with expectations based on known pedigree and on previous research. Correlations of RD and SRD with f₁ performance, specific combining ability, and heterosis for yield and yield components, were generally positive, but too small to be of predictive value. In agreement with previous studies, our results suggest that RFLPs can be used to investigate relationships among maize inbreds, but that they are of limited usefulness for predicting the heterotic performance of single crosses between unrelated lines.Joint contribution from Cereal and Soybean Research Unit, USDA, Agricultural Research Service and Journal Paper no. J-13929 of the Iowa Agric and Home Economics Exp Stn, Ames, IA 50011. Projects no. 2818 and 2778A.E.M. is presently at the Iowa State University on leave from University of Hohenheim, D-7000 Stuttgart 70, Federal Republic of Germany     

Linkage of restriction fragment length polymorphisms and isozymes in Citrus

Summary Genetic linkage analysis was performed using two segregating populations of citrus. One population arose from an intergeneric backcross of Citrus grandis (L.) Osb. cv Thong Dee and Poncirus trifoliata (L.) Raf. cv Pomeroy, using the former as the recurrent (female) parent. The other population came from an interspecific backcross of C. reticulata Blanco cv Clementine and C. x paradisi Macf. cv Duncan, using the former as the recurrent (male) parent. A total of 11 isozyme and 58 restriction fragment length polymorphisms were found to segregate in a monogenic fashion in one or both populations. Linkage analysis revealed that 62 of the loci examined mapped to 11 linkage groups, while 7 loci segregated independently from all other markers. Gene order was highly conserved between the maps generated from the two divergent segregating populations. Possible applications of the use of such maps in tree fruit breeding are discussed.