A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.

Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.     

Linkage analysis of er-1, a recessive Pisum sativum gene for resistance to powdery mildew fungus (Erysiphe pisi D.C.)

Linkage analysis was used to determine the genetic map location of er-1, a recessive gene conditioning resistance to powdery mildew, on the Pisum sativum genome. Genetic linkage was demonstrated between er-1 and linkage group 6 markers after analyzing the progeny of two crosses, an F₂ population and a set of recombinant inbred lines. The classes of genetic markers surrounding er-1 include RFLP, RAPD and allozyme markers as well as the morphological marker Gty. A RAPD marker tightly linked to er-1 was identified by bulked segregant analysis. After DNA sequence characterization, specific PCR primers were designed to convert this RAPD marker into a sequence characterized amplified region (SCAR).     

Efficient fine mapping of the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>) by HEGS (High Efficiency Genome Scanning)/AFLP in barley

The hulled or naked caryopsis character of barley (Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F₂ population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1–7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.Communicated by G. Wenzel     

Targeted mapping and linkage analysis of morphological isozyme,and RAPD markers in peach

Nine different F2 families of peach [Prunus persica (L.) Batsch] were analyzed for linkage relationships between 14 morphological and two isozyme loci. Linkage was detected between weeping (We) and white flower (W), 33 cM; double flower (Dl) and pillar (Br), 10 cM; and flesh color (Y) and malate dehydrogenase (Mdh1), 26 cM. A leaf variant phenotypically distinct from the previously reported wavy-leaf (Wa) mutant in peach was found in progeny of Davie II. The new willow-leaf character (designated Wa2) was closely linked (0.4 cM) to a new dwarf phenotype (designated Dw3). Two families derived from the pollen-fertile cultivar White Glory segregated for pollen sterility, but segregation did not follow a 31 ratio. Evidence is presented suggesting that White Glory possesses a pollen-sterility gene (designated Ps2) that is non-allelic to the previously reported pollen-sterility gene (Ps) in peach. Ps2 was linked to both weeping (We-Ps2, 15.5 cM) and white flower (Ps2-W, 25.3 cM). A genomic map of peach containing 83 RAPD, one isozyme, and four morphological markers was generated using an F2 family obtained by selfing an NC174RL x Pillar F1. A total of 83 RAPD markers were assigned to 15 linkage groups. Various RAPD markers were linked to morphological traits. Bulked segregant analysis was used to identify RAPD markers flanking the red-leaf (Gr) and Mdh1 loci in the NC174RL x Pillar and Marsun x White Glory F2 families, respectively. Three markers flanking Mdh1 and ten markers flanking Gr were identified. The combination of RAPD markers and bulked segregant analysis provides an efficient method of identifying markers flanking traits of interest. Markers linked to traits that can only be scored late in development are potentially useful for marker-aided selection in trees. Alternatives for obtaining additional map order information for repulsion-phase markers in large F2 populations are proposed.This work was supported in part by the McKnight Foundation, North Carolina Biotechnology Center, North Carolina State University Forest Biotechnology Research Consortium, and the North Carolina Agricultural Research Service, Raleigh, North Carolina     

Genetic mapping in pea. 1. RAPD-based genetic linkage map of Pisum sativum

 A genetic linkage map of Pisum sativum L. was constructed based primarily on RAPD markers that were carefully selected for their reproducibility and scored in a population of 139 recombinant inbred lines (RILs). The mapping population was derived from a cross between a protein-rich dry-seed cultivar ‘Térèse’ and an increased branching mutant (K586) obtained from the pea cultivar ‘Torsdag’. The map currently comprises nine linkage groups with two groups comprising only 6 markers (n=7 in pea) and covers 1139 cM. This RAPD-based map has been aligned with the map based on the (JI281×JI399) RILs population that currently includes 355 markers in seven linkage groups covering 1881 cM. The difference in map lengths is discussed. For this alignment 7 RFLPs, 23 RAPD markers, the morphological marker le and the PCR marker corresponding to the gene Uni were used as common markers and scored in both populations. Received: 13 March 1998 / Accepted: 29 April 1998     

Identification of a codominant amplified polymorphic DNA marker linked to the verticillium wilt resistance gene in tomato

Resistance to verticillium wilt, a vascular disease causing yield losses in many crops, is conferred in tomato by a single dominant allele, Ve. A population segregating for the Ve allele was generated using near-isogenic tomato lines. Analysis of the parental tomato DNA using the polymerase chain reaction and 400 random primers, each 10 deoxyribonucleotides in length, produced 1,880 amplified DNA fragments. Of the four polymorphisms observed between the resistant and susceptible parental genotypes, only one was linked to the Ve gene. No recombination was observed between this DNA marker and the Ve locus, indicating that the linkage is less than 3.5±2.7 cM. The marker detected both the susceptible and resistant alleles, producing amplified DNA fragments of approximately 1,300 and 1,350 bp, respectively. The sequence of the primer, determined from cloned amplified products, was 5 CTCACATGCA 3 instead of the expected 5 CTCACATGCC 3. The marker will be of value to tomato breeding programs because of the tight linkage, Codominant nature, and analytical procedure utilized.     

RAPD linkage map of the genomic region encompassing the root-knot nematode (Meloidogyne javanica) resistance locus in carrot

Inheritance studies have indicated that resistance to the root-knot nematode (Meloidogyne javanica) in carrot inbred line ’Brasilia-1252’ is controlled by the action of one or two (duplicated) dominant gene(s) located at a single genomic region (designated the Mj-1 locus). A systematic search for randomly amplified polymorphic DNA (RAPD) markers linked to Mj-1 was carried out using bulked segregant analysis (BSA). Altogether 1000 ten-mer primers were screened with 69.1% displaying scorable amplicons. A total of approximately 2400 RAPD bands were examined. Four reproducible markers (OP-C2₁₇₀₀, OP-Q6₅₀₀, OP-U12₇₀₀, and OP-AL15₅₀₀) were identified, in coupling-phase linkage, flanking the Mj-1 region. The genetic distances between RAPD markers and the Mj-1 locus, estimated using an F₂ progeny of 412 individuals from ’Brasilia 1252’×’B6274’, ranged from 0.8 to 5.7 cM . The two closest flanking markers (OP-Q6₅₀₀ and OP-AL15₅₀₀) encompassed a region of 2.7 cM . The frequency of these RAPD loci was evaluated in 121 accessions of a broad-based carrot germplasm collection. Only five entries (all resistant to M. javanica and genetically related to ’Brasilia 1252’) exhibited the simultaneous presence of all four markers. An advanced line derived from the same cross, susceptible to M. javanica but relatively resistant to another root-knot nematode species (M. incognita), did not share three of the closest markers. These results suggest that at least some genes controlling resistance to M. incognita and M. javanica in ’Brasilia 1252’ reside at distinct loci. The low number of markers suggests a reduced amount of genetic divergence between the parental lines at the region surrounding the target locus. Nevertheless, the low rate of recombination indicated these markers could be useful landmarks for positional cloning of the resistance gene(s). These RAPD markers could also be used to increase the Mj-1 frequency during recurrent selection cycles and in backcrossing programs to minimize ’linkage drag’ in elite lines employed for the development of resistant F₁ hybrids. Received: 22 June 1999 / Accepted: 6 July 1999     

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.     

RAPD-based genetic linkage map of blueberry derived from a cross between diploid species (Vaccinium darrowi and V. elliottii)

An initial genetic linkage map for blueberry has been constructed from over 70 random amplified polymorphic DNA (RAPD) markers that segregated 11 in a testcross population of 38 plants. The mapping population was derived from a cross between two diploid blueberry plants: the F₁ interspecific hybrid (Vaccinium darrowi Camp x V. elliottii Chapm.) and another V. darrowi plant. The map currently comprises 12 linkage groups (in agreement with the basic blueberry chromosome number) and covers a total genetic distance of over 950cM, with a range of 3–30cM between adjacent markers. The use of such a map for identifying molecular markers linked to genes controlling chilling requirement and cold hardiness is discussed.     

A detailed linkage map around an apple scab resistance gene demonstrates that two disease resistance classes both carry the V f gene

A detailed genetic map has been constructed in apple (Malus x domestica Borkh.) in the region of the v _f gene. This gene confers resistance to the apple scab fungus Venturia inaequalis (Cooke) Wint. Linkage data on four RAPD (random amplified polymorphic DNA) markers and the isoenzyme marker PGM-1, previously reported to be linked to the v _f gene, are integrated using two populations segregating for resistance to apple scab. Two new RAPD markers linked to v _f (identified by bulked segregant analysis) and a third marker previously reported as being present in several cultivars containing v _f are also placed on the map. The map around v _f now contains eight genetic markers spread over approximately 28 cM, with markers on both sides of the resistance gene. The study indicates that RAPD markers in the region of crab apple DNA introgressed with resistance are often transportable between apple clones carrying resistance from the same source. Analysis of co-segregation of the resistance classes 3A (weakly resistant) and 3B (weakly susceptible) with the linked set of genetic markers demonstrates that progeny of both classes carry the resistance gene.This work was supported in part by grants from the New Zealand Foundation for Research Science and Technology (FoRST) Programme 94-HRT-07-366 and ENZA New Zealand (International)     

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.     

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.     

Varietal identification and assessment of genetic relationships in Hippeastrum using RAPD markers

The National Botanical Research Institute (NBRI) in Lucknow, India, maintains germplasm of Hippeastrum, a beautiful summer blooming ornamental. Germplasm collections comprise NBRI hybrids developed through selective breeding, hybrids with unknown parentage, local species, and Dutch hybrids for research purposes. Considering the importance of protecting plant breeders’ rights for commercial exploitation of hybrids, a PCR-based technique (random amplified polymorphic DNA—RAPD) was used to correctly identify known and unknown hybrids and to determine cultivar relatedness. RAPD profiles were used very successfully to trace and confirm the parentage of all the hybrids tested and to determine clear molecular relationships among varieties.     

A chromosomal linkage map of Azotobacter vinelandii

Summary A chromosomal map of Azotobacter vinelandii strain UW was constructed. The map was based on measures of cotransfer of various markers mediated by plasmids R68.45 and pJB3JI, on results obtained from conjugal experiments with R-primes, and on recombinants obtained by chromosomal transfer mediated by RP4/Tn5-Mob.     

DNA polymorphisms in grain sorghum (Sorghum bicolor (L.) Moench)

Molecular markers [random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)] were used to determine the frequency of DNA polymorphism in grain sorghum (Sorghum bicolor (L.) Moench). Twenty-nine oligonucleotide primers were employed for RAPDs, generating a total of 262 DNA fragments, of which 145 were polymorphic in at least one pairwise comparison between 36 genotypes. Individual primers differed significantly in their ability to detect genetic polymorphism in the species. The overall frequency of polymorphisms was low with a mean frequency of 0.117 polymorphisms per RAPD band being obtained from all pairwise comparisons between genotypes, with maximum and minimum values of 0.212 and 0.039, respectively. Results from phenetic analysis of bandsharing data were consistent with current sub-specific groupings of the species, with clusters of Durra, Zerazera, Caud-Nig, Caud-Kaura and Caffrorum being discernible. The results also indicated that individuals of a similar taxonomic grouping but different geographic origin may be genetically less identical than previously considered. Similar frequencies of polymorphism to that obtained with RAPDs were obtained with RFLPs. Results from these experiments indicated that a high level of genetic uniformity exists within S. bicolor.     

Identification of AFLP fragments linked to hydroxysafflor yellow A in Flos Carthami and conversion to a SCAR marker for rapid selection

Hydroxysafflor yellow A (HSYA), an important active compound in treating focal cardiac and cerebral ischemia, is uniquely present in flower petals of Carthamus tinctorius. In this study, inheritance and molecular marker analyses for HSYA trait in safflower were carried out. HSYA contents in parents, cross hybridized F₁ and F₂ individuals were analyzed by high performance liquid chromatography. Results revealed that the presence/absence of HSYA was controlled by one major nuclear gene termed HSya. A total of 48 AFLP primer combinations were screened, and bulked segregant analysis was performed by preparing two pools of 10 present-HSYA and ten absent-HSYA plants selected from the 498 individuals of the F₂ segregating population. Four AFLP markers, AFLP-5, AFLP-7, AFLP-15 and AFLP-16, were identified to be closely associated with HSya. Of those, AFLP-16 was the closest to HSya, estimated at about 9.4 cM in genetic distance. The dominant AFLP-16 marker was converted into a simple sequence characterized amplified region marker based on the sequence information of the cloned flanking regions of the AFLP fragment and was designated as SCM16. Our result has direct application for marker-assisted selection of quality breeding in safflower.     

The plastome of Citrus. Physical map,variation among Citrus cultivars and species and comparison with related genera

Summary A physical plastome map was constructed for Citrus aurantium, and the plastomes of species and cultivars of Citrus and of two Citrus relatives were analysed by Southern blot-hybridisation of labelled total tobacco cpDNA to digests of total Citrus DNA. A resemblance was found between the plastomes of cultivars of C. limon (lemon), C. sinensis (orange), C. aurantium (sour orange), C. paradisii (grapefruit) and C. grandis (pomello). The plastomes of other Citrus types such as mandarin (C. reticulata) and citron (C. medico) differed from each other as well as from the plastomes of the aforementioned group. The plastomes of Poncirus trifoliata and Microcitrus sp. are distinct from each other as well as from the Citrus types.     

Genetic characterization of the Pto locus of tomato: semi-dominance and cosegregation of resistance to Pseadomonas syringae pathovar tomato and sensitivity to the insecticide Fenthion

The Pto locus governs resistance to bacterial speck disease in tomato caused by race 0 strains of Pseudomonas syringae pathovar tomato (Pst). Large populations segregating for the Pto locus were generated and genetically characterized. Analysis of the locus has revealed that Pto acts in a semi-dominant manner and cosegegrates with sensitivity to an organophosphorous insecticide, Fenthion, suggesting that Pto may be a complex locus responsible for both phenotypes. We have redefined its map position on chromosome five of the classical genetic map and assigned its position on the molecular map, thus facilitating the alignment of the two genetic maps of the short arm of chromosome five of tomato. Furthermore, we have screened random amplified polymorphic (RAPD) markers for their ability to differentiate near-isogenic lines that differ only with respect to Pto and have identified and mapped seven of these markers. Our results suggest that Pto may be located in a euchromatic region on chromosome five which will be advantageous for the cloning of this locus by one of several molecular strategies.     

The integration of mutant loci affecting maize endosperm development in a dense genetic map using an AFLP-based procedure

In this paper, 10 mutations conditioning the appearance of defective, miniature or collapsed endosperm, but with normal sporophyte development, were considered. Homozygous mutant kernels have reduced grain weight, kernel size, density and, in some of these, higher than normal seed protein content. The mutant loci were integrated into a high-resolution genetic map in order to associate them to specific genes. We have placed 1167 AFLP markers on a consensus map using IBM2 as a backbone and reaching an average of 1 marker every 1.9 cM. We have identified AFLP markers linked to all individual mutant alleles. BSA was adopted to screen the largest possible number of primer combinations on homozygous F₃ mutant and wild type plants. The ten mutant loci are linked to the closest AFLP or SSR markers with distances ranging from 0 to 17.9 cM. The genes we have defined by the existence of mendelian mutants can now be considered good candidates for testing the association to QT loci. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

Summary The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 11 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.