Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars

Summary We have applied the recently developed technique of random amplification of polymorphic DNA (RAPD) to the analysis of the relationships among ten cultivars of papaya (Carica papaya L.). Eleven ten-base synthetic oligonucleotides were chosen that gave multiple PCR amplification products using papaya DNA as template. These 11 primers amplified a total of 102 distinct fragments. Cultivars were scored for presence or absence of RAPD fragments and grouped by cluster analysis using simple matching coefficients of similarity. A dendrogram of the ten cultivars was constructed. Of the ten cultivars seven were of the Hawaiian type, and all of these grouped to one branch of the tree. Divisions within the Hawaiian, branch were mostly consistent with the known genetic background of these cultivars. Three non-Hawaiian, cultivars were also analyzed. The minimum similarity detected was 0.7 suggesting that the domesticated papaya germ plasm is quite narrow. Our results show that RAPD technology is a rapid, precise and sensitive technique for genomic analysis.Journal series No. 3692 of the Hawaii Institute of Tropical Agriculture and Human Resources     

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.     

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.     

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.     

A partial genetic linkage map of slash pine (Pinus elliottii Engelm. var. elliottii) based on random amplified polymorphic DNAs

A set of 420 random, 10-base, oligonucleotide primers was screened for random amplified polymorphic DNA (RAPD) fragments within a sample of eight megagametophyte DNAs of a single slash pine (Pinus elliottii Engelm. var. elliottii) tree. The apparently repeatable RAPD fragments were further characterized within a sample of 68 megagametophytes from the same tree. Fragments segregating in a 11, present-to-absent, ratio were classified and mapped using multi-point linkage analysis. The analysis revealed 13 linkage groups of at least three loci, ranging in size from 28 to 68 cM, and nine linked pairs of loci. The 22 groups and pairs included 73 RAPD markers and covered a genetic map distance of approximately 782 cM. Genome size estimates, based on linkage data, ranged from 2880 to 3360 cM. Using a 30-cM map scale and including the 24 unlinked markers and the ends of the 13 linkage groups and nine linked pairs, the set of RAPD markers accounts for approximately 2160 cM or 64–75% of the genome. This extent of genomic coverage should allow for the efficient mapping of genes responsible for a reaction to the causal agent of fusiform rust disease, Cronartium quercuum (Berk.) Miyabe ex Shirai f. sp. fusiforme.     

Evaluation of genetic relationship in Typhonium species through random amplified polymorphic DNA markers

Studies were undertaken to identify genetic relationships in three species of Typhonium and to evaluate the genetic variance within populations of Typhonium trilobatum, Typhonium roxburghii and Typhonium flagelliforme by using random amplified polymorphic DNA (RAPD) markers. A total of 193 distinct DNA fragments ranging from 0.2 to 3.2 kb, were amplified using 22 selected random decamer primers. The cluster analysis indicated that the three species of Typhonium formed two clusters: the first one consisted of T. trilobatum and T. roxburghii, the second one was represented by T. flagelliforme. A maximum similarity of 63 % was observed in T. trilobatum and T. roxburghii. T. flagelliforme shared up to 43 % similarity with T. trilobatum and T. roxburghii. The closest genetic distance was obtained within populations of different Typhonium species.     

Structure and organization of the B genome based on a linkage map in Brassica nigra

We constructed a genetic map on Brassica nigra based on a segregating population of 83 F₂ individuals. Three different types of molecular markers were used to build the map including isozymes, restriction fragment length polymorphisms (RFLP), and random amplified polymorphic DNA (RAPD). The final map contained 124 markers distributed in 11 linkage groups. The map covered a total distance of 677 cM with the markers distributed within a mean distance of 5.5cM. Of the sequences found in the B. nigra map, 40% were duplicated and organized into three different types of arrangements. They were either scattered throughout the genome, organized in tandem, or organized in blocks of duplicated loci conserved in more than 1 linkage group.     

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.     

Development of simple sequence repeat DNA markers and their integration into a barley linkage map

Simple sequence repeats (SSRs), or microsatellites, are a new class of PCR-based DNA markers for genetic mapping. The objectives of the present study were to develop SSR markers for barley and to integrate them into an existing barley linkage map. DNA sequences containing SSRs were isolated from a barley genomic library and from public databases. It is estimated that the barley genome contains one (GA)_n repeat every 330 kb and one (CA)_n repeat every 620 kb. A total of 45 SSRs were identified and mapped to seven barley chromosomes using doubled-haploid lines and/or wheat-barley addition-line assays. Segregation analysis for 39 of these SSRs identified 40 loci. These 40 markers were placed on a barley linkage map with respect to 160 restriction fragment length polymorphism (RFLP) and other markers. The results of this study demonstrate the value of SSRs as markers in genetic studies and breeding research in barley.     

The use of random amplified polymorphic DNA markers in wheat

Summary An evaluation was made of the use of random amplified polymorphic DNA (RAPD) as a genetic marker system in wheat. Reproducible amplification products were obtained from varietal, homozygous single chromosome recombinant line and wheat/alien addition line genomic DNA with selected primers and rigorously optimized reaction conditions. Factors influencing the RAPD patterns are DNA concentration, Mg²⁺ concentration, polymerase concentration and denaturing temperature. In wheat, the non-homoeologous, non-dose responsive and dominant behaviour of RAPD products devalues their use as genetic markers for the construction of linkage maps, and the high probability that the amplified fragments derive from repetitive DNA limits their use as a source of conventional RFLP probes. However, RAPD markers will most certainly find many applications in the analysis of genotypes where single chromosomes or chromosome segments are to be manipulated.     

A linkage map of rye

A linkage map of rye (Secale cereale L.) is presented which comprises 60 loci including RFLPs, RAPDs, isozyme, morphological and physiological markers. The genetics and linkage relationships of these markers were investigated in several inbred lines of rye. For the RFLP mapping a genomic library of PstI-digested DNA was constructed from which 50 size-selected clones were analysed. The portion of single-copy and multi-copy DNA and the frequency of polymorphic DNA was determined. The markers are unequally distributed over the seven chromosomes of rye. Many of them exhibit a distorted segregation. The main region of deviating segregation ratios could be localized near the self-incompatibility loci.     

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA (RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China. A captive population has been established in the Beijing Zoo since 1999. In order to determine the kinship of the offsprings in 2001, randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo. To amplify the genomic DNA of each individual, 53 arbitrary primers were selected. The results of amplifications showed that 14 primers had clear and distinct RAPD patterns. Totally, 226 amplified fragments were generated by RAPD in this study. Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male). This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds. __________ Translated from Journal of Beijing Normal University (Natural Science), 2005, 41(5): 507–509 [译自: 北京师范大学学报 (自然科学版), 2005, 41(5): 507–509]     

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)     

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.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Extending a RFLP-based genetic map of rye using random amplified polymorphic DNA (RAPD) and isozyme markers

RFLP-based genetic map of rye, developed previously using a cross of lines DS2×RXL10 (F₂ generation), was extended with 69 RAPD and 12 isozyme markers. The actual map contains 282 markers dispersed on all seven chromosomes and spans a distance of 1,140 cM. The efficiency of mapping RAPD markers was close to ten loci per 100-screened arbitrary primers. A strong selection of polymorphic, intensive and reproducible fragments was necessary to reveal individual marker loci that could be assigned to rye chromosomes. Newly mapped markers cover a substantial part of the rye genome and constitute a valuable tool suitable for map saturation, marker-aided selection and phenetic studies. A specific nomenclature for the RAPD loci mapped on individual rye chromosomes, which could be helpful in managing of accumulating data, is proposed. Received: 8 May 2000 / Accepted: 17 October 2000     

Development and application of microsatellite markers for genomic analysis of papaya

Papaya has a relatively small genome, displays high levels of phenotypic diversity, and is amenable to transformation, making it attractive as a fruit tree model system. The high level of phenotypic diversity seen among papaya cultivars in the field does not correlate with the low levels of genotypic polymorphism thus far elucidated. The highly mutable nature of microsatellites or simple sequence repeats (SSRs) make them potentially powerful markers for distinguishing deoxyribonucleic acid (DNA) polymorphisms between closely related genotypes. Genomic research for papaya has resulted in a significant quantity of sequence data. We mined 28.1 Mb of bacterial artificial chromosomes end sequences, 5.8 Mb of complementary DNA, and 1.6 Mb of random genomic sequences for SSRs. We generated 938 SSR markers and tested for polymorphism among seven varieties that had been used to produce five mapping populations. The level of polymorphism was highest for Kaek Dum × 2H94 with 210 markers, followed by UH928 × SunUp with 194, AU9 × SunUp with 189, UH918 × SunUp with 177, and Kapoho × SunUp displaying the lowest level with 97. Variation in levels of polymorphism, motif predominance, and motif length between the genomic and genic fractions indicated differential selection pressures acting on the microsatellites in these two fractions. The microsatellites developed in this study will greatly assist in the genetic and physical mapping of the papaya genome as well as enhance breeders’ ability to improve the crop. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A molecular linkage map of rye

A genetic map of six chromosomes of rye, (all of the rye chromosomes except for 2R), was constructed using 77 RFLP and 12 RAPD markers. The map was developed using an F₂ population of 54 plants from a cross between two inbred lines. A rye genomic library was constructed as a source of clones for RFLP mapping. Comparisons were made between the rye map and other rye and wheat maps by including additional probes previously mapped in those species. These comparisons allowed (1) chromosome arm orientation to the linkage groups to be given, (2) the corroboration of several evolutionary translocations between rye chromosomes and homoeologous chromosomes of wheat; (3) an increase in the number of available markers for target regions of rye that show colinearity with wheat. Inconsistencies in the location of markers between the wheat and rye maps were mostly detected by multi-copy probes.     

A genetic map of Maritime pine based on AFLP, RAPD and protein markers

TheAFLP (amplified fragment length polymorphism) technique was adapted to carry out genetic analysis in maritime pine, a species characterized by a large genome size (24 pg/C). A genetic linkage map was constructed for one F₁ individual based on 239 AFLP and 127 RAPD (randomly amplified polymorphic DNA) markers. Markers were scored on megagametophytes (1n) from 200 germinated F₂ seedlings. Polymorphism rate, labour time and cost of both AFLP and RAPD techniques were compared. The AFLP technique was found to be twice as fast and three-times less costly per marker than the RAPD technique. Thirteen linkage groups were identified with a LOD score ≥6 covering 1873 cM, which provided 93.4% of genome coverage. Proteins were extracted from needles (2n) of the F₂ progeny and revealed by 2-DE (two-dimensional electrophoresis). Thirty one segregating proteins were mapped using a QTL detection strategy based on the quantification of protein accumulation. Two framework maps of the same F₁ individual are now available. The first map (Plomion et al. 1996) uses RAPD markers and the second map, presented in this study, uses mostly AFLP markers. Although the total genetic length of both maps was almost identical, differences among homologous groups were observed. Received: 11 February 1999 / Accepted: 29 April 1999     

A genetic map of Prunus based on an interspecific cross between peach and almond

A genetic linkage map of Prunus has been constructed using an interspecific F₂ population generated from self-pollinating a single F₁ plant from a cross between a dwarf peach selection (54P455) and an almond cultivar Padre. Mendelian segregations were observed for 118 markers including 1 morphological (dw), 6 isozymes, 12 plum genomic, 14 almond genomic and 75 peach mesocarp specific cDNA markers. One hundred and seven markers were mapped to 9 different linkage groups covering about 800 cM map distance, and 11 markers remained unlinked. Three loci identified by three cDNA clones, PC8, PC5 and PC68.1, were tightly linked to the dw locus in linkage group 5. Segregation distortion was observed for approximately one-third of the markers, perhaps due to the interspecific nature and the reproductive (i.e. self-incompatibility) differences between peach and almond. This map will be used for adding other markers and genes controlling important traits, identifying the genomic locations and genetic characterizing of the economically important genes in the genus Prunus, as well as for markerassisted selection in breeding populations. Of particular interest are the genes controlling tree growth and form, and fruit ripening and mesocarp development in peach and almond.