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.     

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci

Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC₁ progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70–80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.     

The inheritance of restriction fragment length polymorphisms in the flax rust Melampsora lini

Summary Random cDNA sequences synthesized from poly A⁺ RNA extracted from germinated urediospores of the flax rust fungus, Melampsora lini, were used as probes to detect restriction fragment length polymorphisms (RFLPs) in three races of M. lini originating from cultivated flax, Linum usitatissimum, and one race originating from Australian native flax, L. marginale. Fourteen out of 22 probes tested detected RFLPs in the three races from cultivated flax while 19 of the probes detected polymorphisms between these three races and the race from L. marginale. The segregation of seven RFLPs was determined in a family of 19 F₂ progeny derived from a cross between two of the rust races. With six of these the inheritance was consistent, in each case, with the segregation of alleles at a single locus. Inheritance of the seventh was unusual and an explanation involving two loci with null alleles at each was proposed. No linkage was detected between any of the RFLP loci and nine unlinked loci specifying avirulence.     

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.     

DNA restriction fragment length polymorphisms and heterozygosity in the human genome

Summary A list is presented of published reports of DNA polymorphisms found in the human genome by restriction enzyme analysis. While the list indicates the large number of restriction fragment length polymorphisms (RFLPs) detected to date, the information collated is insufficient to permit an estimate of heterozygosity for the genome as a whole. Data from our laboratory are therefore also presented on RFLPs detected using a random sample of cloned DNA segments. Such an analysis has permitted a first unbiassed estimate of heterozygosity for the human genome. Since this figure is an order of magnitude higher than previous estimates derived from protein data, the majority of polymorphic variation present in the human genome must, by implication, occur in noncoding sequences. In addition it was confirmed that enzymes containing the dinucleotide CpG in their recognition sequences detect more polymorphic variation than those that do not contain a CpG. Also presented are the clinical applications of DNA polymorphisms in the diagnosis of human genetic disease.Supported by the Deutsche Forschungsgemeinschaft     

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Summary Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.     

Development of genetic markers in celery based on restriction fragment length polymorphisms

Summary Linkage relationships are reported for 34 markers in celery (Apium graveolens L. var dulce) including 21 RFLP, 11 isozyme, and 2 morphological traits. The mapping was carried out in a cross between celery and an annual accession from Thailand, A143, and based on F₂ segregation of 136 plants. A total of 318 centiMorgans (cM) are covered by the markers distributed in 8 linkage groups. Probes for the identification of RFLPs were isolated from a celery cDNA library and were also obtained from heterologous sources. EcoRV, EcoRI, and HindIII were the most useful restriction enzymes in uncovering polymorphism. In our cross, 18% of the cDNA probes were found to be polymorphic for at least one of the enzymes used. Six of the markers showed significant deviations from expected F₂ ratios.     

Inheritance of restriction fragment length polymorphisms and random amplified polymorphic DNAs in coastal Douglas-fir

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29 RAPD loci was demonstrated based on single-locus segregation in a sample of F₂ progeny. One RFLP locus, PtIFG2025, showed segregation distortion. Probe pPtIFG2025 is a loblolly pine cDNA probe encoding for rbcS. The 16 RFLP loci and 23 allozyme loci were also assayed in a sample of 16 Douglas-fir seed-orchard clones. Allelism was determined at 11 of the 16 RFLP loci. RFLPs were able to detect slightly more variation (4.0 alleles per locus) than allozymes (3.1 alleles per locus). The inheritance of an additional 80 RAPD loci was determined based on haploid segregation analysis of megagametophytes from parent tree 013-1. Once 200–300 markers are identified and placed on a genetic map, quantitative trait loci affecting bud phenology will be mapped.     

Cytoplasmic DNAs and nuclear rDNA restriction fragment length polymorphisms in commercial witloof chicories

Restriction fragment length polymorphisms of cytoplasmic DNAs and nuclear rDNA were analyzed in several Cichorium intybus genotypes, comprising four white inbred lines, eight red witloof experimental lines, and a number of F₁ hybrids derived from two white parents. Chloroplast and mitochondrial restriction patterns led to the distinction between two different cytoplasms, called I and II. Southern hybridization using a nuclear rDNA probe revealed that all the lines possessed two types of rDNA repeat units. The shortest unit was 10 kb and was common to all lines. The largest rDNA repeat unit was 10.5 kb in lines I and 10.4 kb in lines II. In addition, a sequence heterogeneity between the 10.5 and 10.4-kb rDNA repeat units was revealed by Sac I digestion. A 10-kb rDNA unit was successively cloned, mapped, and used as a probe to check the genetic purity of F₁ hybrid seeds between line I and II white parents. We found a 30% average percentage of impurities, originating both from selfing and full-sib crossing, in different open-pollinated hybrid samples.     

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     

Mitochondrial genome size variation and restriction fragment length polymorphisms in threePhaseolus species

Summary Restriction patterns of mitochondrial DNA (mtDNA) from threePhaseolus species were examined to estimate their relative genome sizes and to determine the level of interspecific variability and relatedness. Three restriction endonucleases that produced relatively simple profiles were identified and used to determine the genome size of the three species. Taking into account fragment stoichiometries, the average estimates across enzymes were 456, 324, and 400 kb, respectively, forP. vulgaris, P. coccineus, andP. acutifolius. Restriction fragment length polymorphisms (RFLPs) differentiated the species when the mtDNAs were digested with seven endonucleases and hybridized with five cosmid clones covering ca. 200 kb of mtDNA sequences. Proportions of shared restriction fragments between every two species were computed as F-values and demonstrated thatP. vulgaris andP. coccineus are more related to each other than either is toP. acutifolius, and that the latter has a similar degree of relationship to the other two species.     

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.     

QTL analysis of yield and seed number in Citrus

Amount, regularity and low seed content of the crop are important properties of scion citrus cultivars. The genetic control of these traits was studied in a progeny derived from the cross Citrus volkameriana×Poncirus trifoliata using molecular marker analysis. Since the traits were not normally distributed, the Kruskal-Wallis non-parametric test was used for quantitative trait loci (QTLs) detection. Most of the QTLs detected correspond to the trait ”number of fruits per tree”, in agreement with its known physiological complexity. Related traits (fruit number, fruit size and seed number) are controlled by QTLs some of which are located in the same genomic regions, suggesting that undesired associations could be broken to some degree by recombination. QTL analysis over years revealed important effects of genotype-by-environment interaction on QTL detection. This result agrees with the differences found for the trait means among years, which was found to be related, among other causes, to the alternate bearing of some genotypes and the amount of rain before harvest. Received: 1 October 1999 / Accepted: 2 December 1999     

High rate of restriction fragment length polymorphisms between two population of the nematode Pristionchus pacificus (Diplogastridae)

Pristionchus pacificus (Diplogastridae, Nematoda) has recently been described as a ‘satelite’ organism for a functinal comparative approach, becuase genetic, molecular and cell-biological tools can be used in way similar to the genetic model organism Caenorhabditis elegans. This tudy describes the analysis of two previously isolated strains of P. pacificus for the occurrence of restriction fragment length polymorphisms (RFLPs). In all, 14 of 17 randomly chosen cDNA clones give polymorphisms after hybridization to EcoRI digested genomic DNA of the populations from California and Washington. This polymorphism is much higher than polymorphism found among different strains in C. elegans. Therefore this study compares most of the nucleotide sequence of the Ppa-let-60/ras gene between the two strains. No base-pair substitutions were found between these two sequences within the coding regions. However, within the untranslated region, four base-pair substitutions in introns and the deletion of three base-pairs in the 5′ sequence and in intron 4 have been observed. Since the two strains interbreed, RFLPs can be used as molecular markers for future chromosomal walking and gene cloning.     

Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms

Phylogenetic relationships among 30 species of the genusPinus were studied using restriction site polymorphism in the large subunit of nuclear rDNA. Of the 58 restriction sites scored, 48 were phylogenetically informative, and the 30 species reduced to ten taxa when species with identical restriction site patterns were combined. These ten taxa corresponded to the currently recognized subsections of the genus, with the sole exception ofP. leiophylla, which was identical in its pattern of restriction sites to all three species included from subsect.Oocarpae despite its being in a different section of subg.Pinus (Pinea instead ofPinus). A measure of the proportion of phylogenetic information contained within the data set (Homoplasy Excess Ratio, or HER) revealed that the character states were significantly non-randomly distributed among the ten taxa (HER = 0.71, p < 0.01). Branchand-bound searches using either Wagner or Dollo parsimony as the optimization criterion were carried out using PAUP in order to estimate phylogenetic relationships among the ten taxa. Three taxa (Picea pungens, Tsuga canadensis, andLarix decidua) were used independently as outgroups for purposes of rooting the trees. Despite the extreme differences in the assumptions underlying the Wagner and Dollo parsimony, the two gave surprisingly similar estimates of phylogeny, with both analyses supporting the monophyly of the two major subgeneraPinus andStrobus and differing in topology only in the placement of subsect.Ponderosae within subg.Pinus. The likelihood for the Wagner tree was only slightly higher than that computed for the Dollo tree.     

Association analysis of lipid levels and apolipoprotein restriction fragment length polymorphisms

Summary We have analyzed the correlation between restriction site variants (RFLPs; restriction fragment length polymorphisms) of the apolipoprotein AI, AII, B, CI and CII genes and serum lipid levels in a sample of male Norwegians. We find no significant association between any of the RFLPs and lipid levels.     

The use of restriction fragment length polymorphisms in paternity analysis.

This paper examines the utility of restriction fragment length polymorphisms (RFLPs) for paternity analysis. While, on the average, 99% of falsely accused males can be excluded with the standard battery of blood group antigens, red cell enzymes, serum proteins, and HLA antigens, there are still mother-child pairs for whom the exclusion probability is not high. It has been suggested that additional resolution would be available with RFLPs. We have examined the strategic aspects of using RFLPs for paternity analysis, comparing the efficacy and cost of a multimarker haplotypic set with those of a comparable set of unlinked RFLPs, using published frequencies for the beta-globin complex, the serum albumin region, and the growth hormone region. There are four major findings. (1) Greater resolution is obtained with a carefully chosen set of tightly linked RFLPs producing chromosomal haplotypes than with a comparable set (same allele frequencies) of unlinked markers, but only if it is possible to establish linkage phase unambiguously. (2) Assay of linked sets is cheaper than is the assay of unlinked markers, but the cost advantage is optimized with sets of no more than two or three linked markers. (3) Also, with more than two or three tightly linked markers, the haplotypic frequencies are too poorly estimated to provide a reliable measure of the probability of paternity for unexcluded males, given the sample sizes likely to be available in the near future. (4) Optimal resolution, minimal cost, and acceptable accuracy are obtained with several independent sets of no more than two or three tightly linked RFLP markers each. With current technology, RFLP analysis is more expensive for the same level of genetic resolution than is the standard battery, but gradual replacement of the latter can be anticipated as economies of scale reduce the cost of the DNA technology.     

Restriction fragment length polymorphisms at the growth hormone gene in pigs

Pigs from four Danish and two Swedish populations were examined for restriction fragment length polymorphism (RFLP) at the growth hormone (GH) gene. Polymorphism was detected with the restriction enzymes DraI and TaqI. A comparison of the Danish populations showed significant differences among their allelic frequencies.