A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

Molecular genetics of growth and development in Populus. I. Triploidy in hybrid poplars

Summary While constructing a genetic linkage map of a hybrid poplar genome (Populus trichocarpa x P. deltoides), we identified several restriction fragment length polymorphismus (RFLPs) for which the parental trees are heterozygous. Although 8 of the 11 F₁ hybrid offspring inherited, as expected, single RFLP alleles from each parent, 3 F₁ trees in the mapping pedigree inherited both maternal alleles along with a single paternal allele at some loci. Aneuploidy or polyploidy in these 3 F₁ trees due to partial or complete nondisj unction during female gametogenesis is the simplest explanation for this finding. Of the 3 f₁ offspring with supernumerary RFLP alleles 2 have triploid nuclear DNA contents as measured by fluorescence flow cytometry; the 3rd F₁ with supernumerary alleles has a sub-triploid nuclear DNA content and is probably aneuploid. Among the tri/aneuploid hybrids, leaf quantitative traits either are skewed toward those values characteristic of the P. trichocarpa female parent (adaxial stomate density, petiole length: blade length ratio; abaxial color) or show transgressive variation (epidermal cell size). Abaxial leaf color was used to screen a large population of P. trichocarpa x P. deltoides hybrids for further evidence of tri/aneuploidy. In each case where a white abaxial leaf surface was observed and the nuclear DNA content measured, the hybrid proved to be tri/aneuploid. All sexually mature female triploids examined were sterile, although the inflorescences completed their development in the absence of embryo formation. The (probably) aneuploid F₁ hybrid is a fertile female. Of 15 female P. trichocarpa parents used in crosses to P. deltoides, 10 produced one or more tri/aneuploid hybrid offspring. In an intraspecific cross using a P. trichocarpa female that had produced triploid hybrids with five different P. deltoides males, no tri/aneuploid offpsring were found.     

Molecular genetics of growth and development in Populus. II. Segregation distortion due to genetic load

Distortion of expected Mendelian segregation ratios, commonly observed in many plant taxa, has been detected in an experimental three-generation inbred pedigree of Populus founded by interspecific hybridization between P. trichocarpa and P. deltoides. An RFLP linkage map was constructed around a single locus showing severe skewing of segregation ratio against F₂ trees carrying the P. trichocarpa allele in homozygous form. Several hypotheses for the mechanism of segregation distortion at this locus were tested, including directional chromosome loss, segregation of a pollen lethal allele, conflicts between genetic factors that isolate the parental species, and inbreeding depression as a result of genetic load. Breeding experiments to produce inbred and outcrossed progenies were combined with PCR-based detection of RFLPs to follow the fate of the deficient allele throughout embryo and seedling development. A recessive lethal allele, lth, inherited from the P. trichocarpa parent, was found to be tightly linked to the RFLP marker locus POP1054 and to cause embryo and seedling mortality. Heterozygotes (lth/+) appear to be phenotypically normal as embryos, seedlings, and young trees.Abbreviations RFLP restriction fragment length polymorphism - PCR polymerase chain reaction - STS sequence-tagged site - SDS sodium dodecyl sulfate     

A genetic linkage map of Picea abies Karst., based on RAPD markers,as a tool in population genetics

Norway spruce (Picea abies Karst.) is a most important species among European forest trees for both economical and ecological reasons. However, this species has suffered from a lack of information on the genetic side due to the scarcity of linkage data. In this study we have used a population of 72 megagametophytes from a single tree in a natural Italian stand to produce a genetic linkage map by means of RAPD markers. Ninety-six random decamers used as primers yielded 185 polymorphic loci showing Mendelian inheritance. Analysis of the segregation by multipoint analysis allowed us to define 17 major linkage groups covering a total distance of 3584 cM, with an average spacing between markers of 22 cM. Possible uses of a genetic linkage map with respect to population ecology and genetics are discussed.     

Construction of a basic genetic map for alfalfa using RFLP, RAPD, isozyme and morphological markers

The genetic map for alfalfa presented here has eight linkage groups representing the haploid chromosome set of the Medicago species. The genetic map was constructed by ordering the linkage values of 89 RFLP, RAPD, isozyme and morphological markers collected from a segregating population of 138 individuals. The segregating population is self-mated progeny of an F1 hybrid plant deriving from a cross between the diploid (2n=2x=16) yellow-flowered Medicago sativa ssp. quasifalcata and the diploid (2n=2x=16) blue-flowered M. sativa ssp. coerulea. The inheritance of many traits displayed distorted segregation, indicating the presence of lethal loci in the heterozygotic parent plants. In spite of the lack of uniform segregation, linkage groups could be assigned and the order of the markers spanning > 659 centimorgans could be unambiguously determined. This value and the calculated haploid genome size for Medicago (1n=1x=1.0 x 10⁹ bp) gives a ratio of < 1500 kb per centimorgan.     

A linkage map with RFLP and isozyme markers for almond

Inheritance and linkage studies were conducted with seven isozyme genes and 120 RFLPs in the F₁ progeny of a cross between almond cultivars Ferragnes and Tuono. RFLPs were detected using 57 genomic and 43 cDNA almond clones. Eight of the cDNA probes corresponded to known genes (extensin, prunin (2), -tubulin, endopolygalacturonase, oleosin, actin depolymerizing factor and phosphoglyceromutase). Single-copy clones were found more frequently in the cDNA (65%) than in the genomic libraries (26%). Two maps were elaborated, one with the 93 loci heterozygous in Ferragnes and another with the 69 loci heterozygous in Tuono. Thirty-five loci were heterozygous in both parents and were used as bridges between both maps. Most of the segregations (91%) were of the 11 or 1111 types, and data were analyzed as if they derived from two backcross populations. Eight linkage groups covering 393 cM in Ferragnes and 394 in Tuono were found for each map. None of the loci examined in either map was found to be unlinked. Distorted segregation ratios were mainly concentrated in two linkage groups of the Ferragnes map.     

An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.     

Genetic linkage mapping in peach using morphological,RFLP and RAPD markers

We have constructed a genetic linkage map of peach [Prunus persica (L.) Batsch] consisting of RFLP, RAPD and morphological markers, based on 71 F₂ individuals derived from the self-fertilization of four F₁ individuals of a cross between New Jersey Pillar and KV 77119. This progeny, designated as the West Virginia (WV) family, segregates for genes controlling canopy shape, fruit flesh color, and flower petal color, size and number. The segregation of 65 markers, comprising 46 RFLP loci, 12 RAPD loci and seven morphological loci, was analyzed. Low-copy genomic and cDNA probes were used in the RFLP analysis. The current genetic map for the WV family contains 47 markers assigned to eight linkage groups covering 332 centi Morgans (cM) of the peach nuclear genome. The average distance between two adjacent markers is 8 cM. Linkage was detected between Pillar (Pi) and double flowers (Dl) RFLP markers linked to Pi and flesh color () loci were also found. Eighteen markers remain unassigned. The individuals analyzed for linkage were not a random sample of all F₂ trees, as an excess of pillar trees were chosen for analysis. Because of this, Pi and eight other markers that deviated significantly from the expected Mendelian ratios (e.g., 121 or 31) were not eliminated from the linkage analysis. Genomic clones that detect RFLPs in the WV family also detect significant levels of polymorphism among the 34 peach cultivars examined. Unique fingerprint patterns were created for all the cultivars using only six clones detecting nine RFLP fragments. This suggests that RFLP markers from the WV family have a high probability of being polymorphic in crosses generated with other peach cultivars, making them ideal for anchor loci. This possibility was examined by testing RFLP markers developed with the WV family in three other unrelated peach families. In each of these three peach families respectively 43%, 54% and 36% of RFLP loci detected in the WV family were also polymorphic. This finding supports the possibility that these RFLP markers may serve as anchor loci in many other peach crosses.     

A genetic linkage map of Nicotiana plumbaginifolia/ Nicotiana longiflora based on RFLP and RAPD markers

We have constructed a genetic linkage map for Nicotiana plumbaginifolia/Nicotiana longiflora (2n= 2x=20), based on the segregation of 69 RFLP and 102 RAPD loci in 99 F₂ plants from the cross N. plumbaginifolia×N. longiflora. The map consists of nine major linkage groups, each containing more than nine marker loci, and spans 1062 cM. Twenty of the RFLP markers were mapped previously to Nicotiana sylvestris (2n=2x=24) chromosomes using monosomic alien addition lines. Taxonomically, N. plumbaginifolia and N. sylvestris belong to the same section, namely the Alatae; however, cytogenetic evidence indicates that they are not closely related. Comparison of the distribution of markers common to both maps suggests that genome reorganization has occurred during the evolution of these two species. Evidence is also presented that genome reorganization may be accompanied by gain and loss of specific classes of DNA sequences in their genomes.     

A genetic map of melon (Cucumis melo L.) with RFLP, RAPD, isozyme, disease resistance and morphological markers

One hundred and ten markers were analysed for linkage in 218 F₂ plants derived from two divergent cultivars (Védrantais and Songwhan Charmi) of Cucumis melo (L.). Thirty-four RFLPs, 64 RAPDs, one isozyme, four disease resistance markers and one morphological marker were used to construct a genetic map spanning 14 linkage groups covering 1390 cM of the melon genome. RAPD and RFLP markers detected similar polymorphism levels. RFLPs were largely due to base substitutions rather than insertion/deletions. Twelve percent of markers showed distorted segregation. Phenotypic markers consisted of two resistance genes against Fusarium wilt (Fom-1 and Fom-2), one gene (nsv) controlling the resistance to melon necrotic spot virus, one gene (Vat) conferring resistance to Aphis gossypii, and a recessive gene for carpel numbers (3 vs 5 carpels: p).     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

A linkage map for sugi (Cryptomeria japonica) based on RFLP,RAPD, and isozyme loci

A linkage map for sugi was constructed on the basis of restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), and isozyme loci using a three-generation pedigree prepared for genetic analysis of heartwood color. A total of 128 RFLP (123 cDNA and 5 genomic probes), 33 RAPD, 2 isozyme, and 1 morphological (dwarf) loci segregated in 73 progeny. Of the 164 segregating loci, 145 loci were distributed in 20 linkage groups. Of these loci, 91 with confirmed map positions were assigned to 13 linkage groups, covering a total of 887.3 cM. A clustering of markers with distorted segregation was observed in 6 linkage groups. In the four clusters, distortions with a reduction in the number of homozygotes from one parent only were found.Abbreviations MAS marker-assisted selection - PAGE polyacrylamide gel electrophoresis - QTL quantitative traits of loci - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism This work was supported by a Grant-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan (Cooperative Research, no. 04304017)     

Molecular evidence for the hybrid origin of Paulownia Taiwaniana based on RAPD markers and RFLP of chloroplast DNA

Genomic DNA of Paulownia fortunei, P. kawakamii and P. taiwaniana were amplified with 10-base primers of arbitrary sequences using the polymerase chain reaction (PCR). A total of 351 DNA fragments were amplified from 23 primers and of these 265 fragments (75.5%) were polymorphic. Almost all of the PCR-amplified products of P. taiwaniana were shared by either P. fortunei or P. kawakamii, or both, and the number of polymorphic fragments shared by P. taiwaniana and P. fortunei was about equivalent to those shared by P. taiwaniana and P. kawakamii. Restriction fragments of chloroplast DNA (cpDNA) purified from Paulownia species and from reciprocal crosses between P. fortunei and P. kawakamii were analyzed. Restriction enzyme SalI-digested cpDNA showed an identical pattern in both P. kawakamii and P. taiwaniana. These results further support the hypothesis that P. taiwaniana is the natural hybrid between P. fortunei and P. kawakamii and that the maternal parent of P. taiwaniana is P. kawakamii.This work was supported in part by the National Science Council (NSC-80-0409-B-054-06), Republic of China     

Comparing the distribution of RAPD and RFLP markers in a high density linkage map of sugar beet.

The distribution of RAPD markers was compared with that of RFLP markers in a high density linkage map of sugar beet. The same mapping population of 161 F2 individuals was used to generate all the marker data. The total map comprises 160 RAPD and 248 RFLP markers covering 508 cM. Both the RAPD and the RFLP markers show a high degree of clustering over the nine linkage groups. The pattern is compatible with a strong distal localization of recombination in the sugar beet. It leads generally to one major cluster of markers in the centre of each linkage group. In regions of high marker density, dominant RAPD markers present in either linkage phase and codominant RFLP markers are subclustered relative to each other. This phenomenon is shown to be attributable to: (i) effects of the mapping procedure when dominant and codominant data are combined, (ii) effects of the mapping procedure when dominant data in both linkage phases are combined, and (iii) genuine differences in the way RAPD and RFLP markers are recruited.     

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

 An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM. Received: 26 March 1998 / Accepted: 30 April 1998     

A genetic linkage map of Theobroma cacao L.

A linkage map of the cocoa genome comprising 193 loci has been constructed. These loci consist of 5 isozymes, 101 cDNA/RFLPs, 4 loci from genes of known function, 55 genomic DNA/RFLPs and 28 RAPDs. A population of 100 individuals derived from a cross between two heterozygous genotypes was used. Segregation analyses were performed with the JoinMap program. Ten linkage groups, which putatively correspond to the ten gametic chromosomes of cocoa, were identified. The map covers a total length of 759 cM with a 3.9 cM average distance between 2 markers. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios.     

Molecular markers for plant breeding: comparisons of RFLP and RAPD genotyping costs

Three molecular marker protocols, chemiluminescent restriction fragment length polymorphisms (c-RFLPs), radioactivity-based restriction fragment length polymorphisms (r-RFLPs), and randomly amplified DNA polymorphisms (RAPDs) were compared in terms of cost and time efficiency. Estimates of cost of supplies and time requirements were obtained from simulations of maize (Zea mays L.) genotyping experiments utilizing protocols currently in use. The increase in total cost with increasing numbers of individuals genotyped and markers analyzed is higher for RAPDs than for RFLPs. RAPDs were generally found to be more cost and time efficient for studies involving small sample sizes, while RFLPs have the advantage for larger sample sizes. Because of the shorter exposure times involved, c-RFLPs require less time than r-RFLPs to obtain a given amount of information. Variations in the protocols, such as number of re-uses of Southern blots or cost of Taq DNA polymerase per reaction of amplification, also affect the relative merits of RAPDs and RFLPs. Two examples were analyzed where molecular markers are used: a germ plasm survey and quantitative trait loci (QTL) mapping in a segregating population. No protocol was found to be the most cost and time efficient over the entire range of sample sizes and number of marker loci studied.     

Construction of a genetic linkage map for roses using RAPD and AFLP markers

A segregating population of diploid rose hybrids (2n = 2x = 14) was used to construct the first linkage maps of the rose genome. A total of 305 RAPD and AFLP markers were analysed in a population of 60 F₁ plants based on a so-called ”double-pseudotestcross” design. Of these markers 278 could be located on the 14 linkage groups of the two maps, covering total map lengths of 326 and 370 cM, respectively. The average distances between markers in the maps for 93/1–117 and 93/1–119 is 2.4 and 2.6 cM, respectively. In addition to the molecular markers, genes controlling two phenotypic characters, petal number (double versus single flowers) and flower colour (pink versus white), were mapped on linkage groups 3 and 2, respectively. The markers closest to the gene for double flowers, Blfo, and to the gene for pink flower colour, Blfa, cosegregated without recombinants. The maps provide a tool for further genetic analyses of horticulturally important genes as, for example, resistance genes and a starting point for marker-assisted breeding in roses. Received: 22 September 1998 / Accepted: 12 March 1999     

A genetic linkage map of papaya based on randomly amplified polymorphic DNA markers

A genetic linkage map of papaya (Carica papaya L.) was constructed using randomly amplified polymorphic DNA (RAPD) markers and a F₂ population derived from a University of Hawaii UH breeding line 356 x Sunrise cross. A total of 596 10-mer primers were screened, and 96 polymorphisms were detected. At LOD 4.0, 62 of these markers mapped to 11 linkage groups comprising 999.3 cM. About 80% of the markers conformed to expected Mendelian segregation ratios. We have mapped the locus that determines sex to a 14-cM region flanked by RAPD markers. The results demonstrate the usefulness of RAPD markers for developing a basic genetic linkage map in papaya.Journal series No. 4146 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Molecular phylogeny of mangroves V. Analysis of genome relationships in mangrove species using RAPD and RFLP markers

 DNA from pooled leaf samples of 11 true major mangrove, three true minor mangrove, two mangrove associate, two mangrove parasite, three terrestrial and one cultivated species were isolated for the present study. In total, 198 random amplified polymorphic DNAs (RAPDs) and 180 restriction fragment length polymorphism (RFLP) loci were scored by using ten primers and 14 enzyme-probe combinations respectively. The polymorphism observed for these markers revealed a high degree of genetic diversity in mangroves at both inter-specific or inter-generic levels. A dendrogram, constructed after pooling both RAPD and RFLP data, using a similarity index was analysed for genome relationships among these species. The dendrogram showed clustering of all the major mangroves, except for Nypa fruticans (Arecaceae), into one group. All species under the tribe Rhizophorae formed a sub-cluster, to which Xylocarpus granatum was found to be the most closesly related species. The clustering pattern implied that Excoecaria agallocha and Acanthus ilicifolius should be considered as true minor mangroves. The present study also provided molecular data favouring the separation of Avicennia spp. from the Verbenaceae to create a monotypic family the Avicenniaceae. The separation of Viscum orientale into the Viscaceae was also favoured. Received: 5 March 1998 / Accepted: 1 April 1998