Linkage maps of the apple ( Malus × domestica Borkh.) cvs ‘Ralls Janet’ and ‘Delicious’ include newly developed EST markers

Two apple genetic linkage maps were constructed using amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), random amplified polymorphic DNAs (RAPDs), and expressed sequence tag (EST)-derived markers in combination with a pseudo-testcross mapping strategy in which the cultivars ‘Ralls Janet’ and ‘Delicious’ were used as the respective seed parents. Mitsubakaido (Malus sieboldii) was used as the pollen parent for each of the segregating F₁ populations. Expressed sequence tag data were obtained from the random sequencing of cDNA libraries constructed from in vitro cultured shoots and maturing fruits of cv ‘Fuji’, which is the offspring of a cross between ‘Ralls Janet’ and ‘Delicious’. In addition, a number of published gene sequences were used to develop markers for mapping. The ‘Ralls Janet’ map consisted of 346 markers (178 AFLPs, 95 RAPDs, 54 SSRs, 18 ESTs, and the S locus) in 17 linkage groups, with a total length of 1082 cM, while that of ‘Delicious’ comprised 300 markers (120 AFLPs, 81 RAPDs, 64 SSRs, 32 ESTs, and the S, Rf, and MdACS-1 loci) on 17 linkage groups spanning 1031 cM. These maps are amenable to comparisons with previously published maps of ‘Fiesta’ and ‘Discovery’ (Liebhard et al., Mol Breed 10:217–241, 2002; Liebhard et al., Theor Appl Genet 106:1497–1508, 2003a) because several of the SSRs (one to three markers per linkage group) were used in all of the maps. Distorted marker segregation was observed in three and two regions of the ‘Ralls Janet’ and ‘Delicious’ maps, respectively. These regions were localized in different parts of the genome from those in previously reported apple linkage maps. This marker distortion may be dependent on the combinations of cultivars used for map construction.     

A microsatellite linkage map for the cultivated strawberry (Fragaria?×?ananassa) suggests extensive regions of homozygosity in the genome that may have resulted from breeding and selection

The linkage maps of the cultivated strawberry, Fragaria × ananassa (2n = 8x = 56) that have been reported to date have been developed predominantly from AFLPs, along with supplementation with transferrable microsatellite (SSR) markers. For the investigation of the inheritance of morphological characters in the cultivated strawberry and for the development of tools for marker-assisted breeding and selection, it is desirable to populate maps of the genome with an abundance of transferrable molecular markers such as microsatellites (SSRs) and gene-specific markers. Exploiting the recent release of the genome sequence of the diploid F. vesca, and the publication of an extensive number of polymorphic SSR markers for the genus Fragaria, we have extended the linkage map of the ‘Redgauntlet’ × ‘Hapil’ (RG × H) mapping population to include a further 330 loci, generated from 160 primer pairs, to create a linkage map for F. × ananassa containing 549 loci, 490 of which are transferrable SSR or gene-specific markers. The map covers 2140.3 cM in the expected 28 linkage groups for an integrated map (where one group is composed of two separate male and female maps), which represents an estimated 91% of the cultivated strawberry genome. Despite the relative saturation of the linkage map on the majority of linkage groups, regions of apparent extensive homozygosity were identified in the genomes of ‘Redgauntlet’ and ‘Hapil’ which may be indicative of allele fixation during the breeding and selection of modern F. × ananassa cultivars. The genomes of the octoploid and diploid Fragaria are largely collinear, but through comparison of mapped markers on the RG × H linkage map to their positions on the genome sequence of F. vesca, a number of inversions were identified that may have occurred before the polyploidisation event that led to the evolution of the modern octoploid strawberry species.     

Assaying polymorphism at DNA level for genetic diversity diagnostics of the safflower (Carthamus tinctorius L.) world germplasm resources

Carthamus tinctorius (2n = 2x = 24), commonly known as safflower, is widely cultivated in agricultural production systems of Asia, Europe, Australia, and the Americas as a source of high quality vegetable and industrial oil. Twenty-two RAPD primers, 18 SSR primers, and 10 AFLP primer combinations were used to assess: (1) the genetic diversity of 85 accessions (originating from 24 countries) representing global germplasm variability of safflower and (2) the interrelationships among safflower ‘centers of similarity’ or ‘regional gene pools’ proposed earlier. The RAPD and SSR primers and AFLP primer combinations revealed 57.6, 68.0, and 71.2% polymorphism, respectively, among 111, 72, and 330 genetic loci amplified from the accessions. The sum of effective number of alleles (66.44), resolving power (59.16), and marker index (51.3) explicitly revealed the relative superiority of AFLP as a marker system in uncovering variation in safflower. Overall, AFLP markers could recognize ‘centers of similarity’ or ‘regional gene pools’. Analysis of molecular variance and Shannon’s information index provided corroborating evidences for the present and previous studies that concluded fragmentation of safflower gene pool into many gene pools. Divergent directional selection is likely to have played an important role in shaping the diversity. From the practical applications standpoint, the diversity of Iran–Afghanistan gene pool is very high, equivalent to the total diversity of the species. The Far East gene pool is the least diverse. The present comprehensive input, first of its own kind in safflower, will assist marker based improvement programmes in the crop.     

Genotypic effects on the frequency of homoeologous and homologous recombination in <Emphasis Type="Italic">Brassica napus</Emphasis> × <Emphasis Type="Italic">B. carinata</Emphasis> hybrids

We investigated the influence of genotype on homoeologous and homologous recombination frequency in eight different Brassica napus (AACⁿCⁿ) × B. carinata (BBC^cC^c) interspecific hybrids (genome composition CⁿC^cAB). Meiotic recombination events were assessed through microsatellite marker analysis of 67 unreduced microspore-derived progeny. Thirty-four microsatellite markers amplified 83 A-, B-, Cⁿ- and C^c-genome alleles at 64 loci, of which a subset of seven markers amplifying 26 alleles could be used to determine allele copy number. Hybrid genotypes varied significantly in loss of A- and B-genome alleles (P < 0.0001), which ranged from 6 to 22% between hybrid progeny sets. Allele copy number analysis revealed 19 A–C, 3 A–B and 10 B–C duplication/deletion events attributed to homoeologous recombination. Additionally, 55 deletions and 19 duplications without an accompanying dosage change in homoeologous alleles were detected. Hybrid progeny sets varied in observed frequencies of loss, gain and exchange of alleles across the A and B genomes as well as in the diploid C genome. Self-fertility in hybrid progeny decreased as the loss of B-genome loci (but not A-genome loci) increased. Hybrid genotypes with high levels of homologous and homoeologous exchange may be exploited for genetic introgressions between B. carinata and B. napus (canola), and those with low levels may be used to develop stable synthetic Brassica allopolyploids.     

Multiple field and glasshouse assessments increase the reliability of linkage mapping of the Vf source of scab resistance in apple

 Apple scab, caused by the fungus Venturia inaequalis (Cke.) Wint., is an important disease in commercial apple production. A mapping population of 155 individuals, derived from a cross between the apple varieties ‘Prima’ (resistant)בFiesta’ (susceptible), was scored for response to the disease in replicated field and glasshouse trials throughout Europe. Twenty data sets were selected and cluster analysis was used to form a consensus score for the population fitting a 1 : 1 segregation ratio of resistance:susceptibility. The progeny were scored with molecular markers. A detailed map covering 54 cM of the ‘Prima’ linkage group containing the Vf gene for scab resistance was constructed using 24 molecular markers linked to the resistance gene. One isoenzyme marker (Pgm-1), six RFLP markers and 17 RAPD markers formed a linkage group with the consensus measure of resistance to scab. Four marker bridges were established with the corresponding ‘Fiesta’ linkage group with additional markers (one isozyme, one RFLP, three RAPD and one AFLP). A low chi-square value indicated a good fit of the marker ordering, which was in close agreement with previously reported linkage positions for some of the markers and Vf. Differences were observed in the ability of different scoring methods to resolve susceptible and resistant classes. The results obtained for the consensus classification of resistance to scab for the population may suggest the presence of virulent inocula at some sites, which could overcome the Vf gene for resistance. The consequences of relying on individual scoring occasions for studying Vf scab resistance are discussed in the context of linkage analysis, conventional breeding selection, and marker-assisted selection. Received: 23 July 1997 / Accepted: 31 October 1997     

Assessment of fire blight tolerance in apple based on plant inoculations with Erwinia amylovora and DNA markers

Fire blight (Erwinia amylovora) causes serious damage to pome fruit orchards, and identification of germplasm with heritable disease resistance is therefore crucial. Two dominant SCAR (sequence characterised amplified region) marker alleles (AE10-375 and GE-8019), flanking a previously identified QTL (quantitative trait locus) for resistance to fire blight on ‘Fiesta’ linkage group 7 in apple cultivars related to ‘Cox’s Orange Pippin’, were screened on 205 apple cultivars. Both marker alleles were present in 22% of the cultivars, indicating presence of the QTL allele for tolerance, and both were lacking in 25%, indicating homozygosity for absence of the QTL tolerance allele. However, 33% had only the marker allele AE10-375, while 20% had only GE-8019, suggesting that some cultivars with the dominant alleles for both of the flanking markers can carry these on separate chromosomes and may lack the QTL allele for tolerance. In 2009 and 2010, terminal shoots of greenhouse-grown grafted trees of 21 cultivars (only 20 in 2010) were inoculated with Erwinia amylovora. ‘Idared’ (susceptible) and ‘Enterprise’ (tolerant) were included as controls. Disease severity for each cultivar was expressed as percentage of necrosis in relation to entire length of shoot, and the ranking of cultivars in 2009 and 2010 was compared with a Spearman rank correlation test, P < 0.01. A relationship between presence of both flanking marker alleles for tolerance and level of fire blight tolerance was confirmed with a Mann–Whitney U-test, P < 0.01 in 2009, and P < 0.05 in 2010. A PCO (principal coordinate) analysis based on band profiles obtained with 12 SSR (simple sequence repeat) loci produced three loose clusters, two of which contained known offspring of ‘Cox’s Orange Pippin’, and one with cultivars that were either unrelated or had an unknown origin. Cases where DNA markers did not predict level of fire blight damage as expected, were, however, as common among descendants of ‘Cox’s Orange Pippin’ as among apparently unrelated cultivars. Obviously the ‘Fiesta’ LG 7 QTL has some predictive value, both for known ‘Cox’ relatives and others, but more efficient markers would be desirable for marker-assisted selection.     

A cherry map from the inter-specific cross Prunus avium ‘Napoleon’ × P. nipponica based on microsatellite, gene-specific and isoenzyme markers

One hundred and sixty microsatellite (simple sequence repeat (SSR)) and six gene-specific markers revealing 174 loci were scored in 94 seedlings from the inter-specific cross of Prunus avium ‘Napoleon’ × Prunus nipponica accession F1292. The co-segregation data from these markers were used to construct a linkage map for cherry which spanned 680 cM over eight linkage groups with an average marker spacing of 3.9 cM per marker and just six gaps longer than 15 cM. Markers previously mapped in Prunus dulcis ‘Texas’ × Prunus persica ‘Earlygold’ allowed the cherry map to be anchored to the peach × almond map and showed the high level of synteny between the species. Eighty-four loci segregated in P. avium ‘Napoleon’ versus 159 in P. nipponica. The segregations of 32 isoenzyme loci in a subset of 47 seedlings from the progeny were scored, using polyacrylamide gel electrophoresis and/or isoelectric focusing separation followed by activity staining, and the co-segregation data were analysed along with those for 39 isoenzymes reported previously and for the 174 sequence-tagged site loci plus an additional two SSR loci. The second map incorporates 233 loci and spans 736 cM over eight linkage groups with an average marker spacing of 3.2 cM per marker and just two gaps greater than 15 cM. The microsatellite map will provide a useful tool for cherry breeding and marker-assisted selection and for synteny studies within Prunus; the gene-specific markers and isoenzymes will be useful for comparisons with maps of other rosaceous fruit crops. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A framework map from grapevine V3125 (Vitis vinifera ‘Schiava grossa’?×?‘Riesling’)?×?rootstock cultivar ‘B?rner’ (Vitis riparia?×?Vitis cinerea) to localize genetic determinants of phylloxera root resistance

Grapevine rootstock cultivar ‘B?rner’ is a hybrid of Vitis riparia and Vitis cinerea Arnold that shows high resistance to phylloxera (Daktulosphaira vitifoliae Fitch). To localize the determinants of phylloxera root resistance, the susceptible grapevine V3125 (Vitis vinifera ‘Schiava grossa’ × ‘Riesling’) was crossed to ‘B?rner’. Genetic framework maps were built from the progeny. 235 microsatellite markers were placed on the integrated parental map. They cover 1,155.98 cM on 19 linkage groups with an average marker distance of 4.8 cM. Phylloxera resistance was scored by counting nodosities after inoculation of the root system. Progeny plants were triplicated and experimentally infected in 2 years. A scan of the genetic maps indicated a quantitative trait locus on linkage group 13. This region was targeted by six microsatellite-type markers newly developed from the V. vinifera model genome sequence. Two of these appear closely linked to the trait, and can be useful for marker-assisted breeding.     

Species abundance distributions in neutral models with immigration or mutation and general lifetimes

We consider a general, neutral, dynamical model of biodiversity. Individuals have i.i.d. lifetime durations, which are not necessarily exponentially distributed, and each individual gives birth independently at constant rate λ. Thus, the population size is a homogeneous, binary Crump–Mode–Jagers process (which is not necessarily a Markov process). We assume that types are clonally inherited. We consider two classes of speciation models in this setting. In the immigration model, new individuals of an entirely new species singly enter the population at constant rate μ (e.g., from the mainland into the island). In the mutation model, each individual independently experiences point mutations in its germ line, at constant rate θ. We are interested in the species abundance distribution, i.e., in the numbers, denoted I _n (k) in the immigration model and A _n (k) in the mutation model, of species represented by k individuals, k = 1, 2, . . . , n, when there are n individuals in the total population. In the immigration model, we prove that the numbers (I _t (k); k ≥ 1) of species represented by k individuals at time t, are independent Poisson variables with parameters as in Fisher’s log-series. When conditioning on the total size of the population to equal n, this results in species abundance distributions given by Ewens’ sampling formula. In particular, I _n (k) converges as n → ∞ to a Poisson r.v. with mean γ/k, where γ : = μ/λ. In the mutation model, as n → ∞, we obtain the almost sure convergence of n ⁻¹ A _n (k) to a nonrandom explicit constant. In the case of a critical, linear birth–death process, this constant is given by Fisher’s log-series, namely n ⁻¹ A _n (k) converges to α ^k /k, where α : = λ/(λ + θ). In both models, the abundances of the most abundant species are briefly discussed.     

Development of an STS map of an interspecific progeny of Malus

Simple sequence repeat (SSR) markers developed from Malus, as well as Prunus, Pyrus and Sorbus, and some other sequence-tagged site (STS) loci were analysed in an interspecific F₁ apple progeny from the cross ‘Fiesta’ × ‘Totem’ that segregated for several agronomic characters. A linkage map was constructed using 259 STS loci (247 SSRs, four SCARs and eight known-function genes) and five genes for agronomic traits—scab resistance (Vf), mildew resistance (Pl-2), columnar growth habit (Co), red tissues (Rt) and green flesh background colour (Gfc). Ninety SSR loci and three genes (ETR1, Rt and Gfc) were mapped for the first time in apple. The transferability of markers from other Maloideae to Malus was found to be around 44%. The loci are spread across 17 linkage groups, corresponding to the basic chromosome number of Malus and cover 1,208 cM, approximately 85% of the estimated length of the apple genome. Interestingly, we have extended the top of LG15 with eight markers covering 25 cM. The average map density is 4.7 cM per marker; however, marker density varies greatly between linkage groups, from 2.5 in LG14 to 8.9 in LG7, with some areas of the genome still in need of further STS markers for saturation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Construction of an intra-specific sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.) genetic linkage map and synteny analysis with the <Emphasis Type="Italic">Prunus</Emphasis> reference map

Linkage maps of the sweet cherry cultivar ‘Emperor Francis’ (EF) and the wild forest cherry ‘New York 54’ (NY) were constructed using primarily simple sequence repeat (SSR) markers and gene-derived markers with known positions on the Prunus reference map. The success rate for identifying SSR markers that could be placed on either the EF or NY maps was only 26% due to two factors: a reduced transferability of other Prunus-species-derived markers and a low level of polymorphism in the mapping parents. To increase marker density, we developed four cleaved amplified polymorphic sequence markers (CAPS), 19 derived CAPS markers, and four insertion–deletion markers for cherry based on 101 Prunus expressed sequence tags. In addition, four gene-derived markers representing orthologs of a tomato vacuolar invertase and fruit size gene and two sour cherry sorbitol transporters were developed. To complete the linkage analysis, 61 amplified fragment length polymorphism and seven sequence-related amplified polymorphism markers were also used for map construction. This analysis resulted in the expected eight linkage groups for both parents. The EF and NY maps were 711.1 cM and 565.8 cM, respectively, with the average distance between markers of 4.94 cM and 6.22 cM. A total of 82 shared markers between the EF and NY maps and the Prunus reference map showed that the majority of the marker orders were the same with the Prunus reference map suggesting that the cherry genome is colinear with that of the other diploid Prunus species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

A consensus map of rye integrating mapping data from five mapping populations

A consensus map of rye (Secale cereale L.) was constructed using JoinMap 2.0 based on mapping data from five different mapping populations, including ‘UC90’ × ‘E-line’, ‘P87’ × ‘P105’, ‘I_0.1-line’ × ‘I_0.1-line’, ‘E-line’ × ‘R-line’, and ‘Ds2’ × ‘RxL10’. The integration of the five mapping populations resulted in a 779-cM map containing 501 markers with the number of markers per chromosome ranging from 57 on 1R to 86 on 4R. The linkage sizes ranged from 71.5 cM on 2R to 148.7 cM on 4R. A comparison of the individual maps to the consensus map revealed that the linear locus order was generally in good agreement between the various populations, but the 4R orientations were not consistent among the five individual maps. The 4R short arm and long arm assignments were switched between the two population maps involving the ‘E-line’ parent and the other three individual maps. Map comparisons also indicated that marker order variations exist among the five individual maps. However, the chromosome 5R showed very little marker order variation among the five maps. The consensus map not only integrated the linkage data from different maps, but also greatly increased the map resolution, thus, facilitating molecular breeding activities involving rye and triticale.     

Extent and structure of linkage disequilibrium in canola quality winter rapeseed (Brassica napus L.)

Linkage disequilibrium was investigated in canola quality winter rapeseed to analyze (1) the prospects for whole-genome association analyses and (2) the impact of the recent breeding history of rapeseed on linkage disequilibrium. A total of 845 mapped AFLP markers with allele frequencies ≥0.1 were used for the analysis of linkage disequilibrium in a population of 85 canola quality winter rapeseed genotypes. A low overall level of linkage disequilibrium was found with a mean r ² of only 0.027 over all 356,590 possible marker pairs. At a significance threshold of P = 2.8 × 10⁻⁷, which was derived by a Bonferroni correction from a global α-level of 0.1, only 0.78% of the marker pairs were in significant linkage disequilibrium. Among physically linked marker pairs, the level of linkage disequilibrium was about five times higher with more than 10% of marker pairs in significant linkage disequilibrium. Linkage disequilibrium decayed rapidly with distance between linked markers with high levels of linkage disequilibrium extending only for about 2 cM. Owing to the rapid decay of linkage disequilibrium with distance association analyses in canola quality rapeseed will have a significantly higher resolution than QTL analyses in segregating populations by interval mapping, but much larger number of markers will be necessary to cover the whole genome. A major impact of the recent breeding history of rapeseed on linkage disequilibrium could not be observed.     

A new SNP haplotype associated with blue disease resistance gene in cotton (Gossypium hirsutum L.)

Resistance to cotton blue disease (CBD) was evaluated in 364 F_2.3 families of three populations derived from resistant variety ‘Delta Opal’. The CBD resistance in ‘Delta Opal’ was controlled by one single dominant gene designated Cbd. Two simple sequence repeat (SSR) markers were identified as linked to Cbd by bulked segregant analysis. Cbd resides at the telomere region of chromosome 10. SSR marker DC20027 was 0.75 cM away from Cbd. DC20027 marker fragments amplified from 3 diploid species and 13 cotton varieties whose CBD resistance was known were cloned and sequenced. One single nucleotide polymorphism (SNP) was identified at the 136th position by sequence alignment analysis. Screening SNP markers previously mapped on chromosome 10 identified an additional 3 SNP markers that were associated with Cbd. A strong association between a haplotype based on four SNP markers and Cbd was developed. This demonstrates one of the first examples in cotton where SNP markers were used to effectively tag a trait enabling marker-assisted selection for high levels of CBD resistance in breeding programs.     

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     

Molecular mapping of pea powdery mildew resistance gene <Emphasis Type="Italic">er2</Emphasis> to pea linkage group III

Powdery mildew caused by Erysiphe pisi D.C. is one of the most serious diseases that inflict heavy losses to pea crop world-wide. Identification of resistance sources and their incorporation into susceptible cultivars remains the most effective method of controlling the disease. The present study investigated the resistance phenotype, inheritance, and genomic location of gene(s) controlling resistance to powdery mildew in pea genotype ‘JI2480’. The powdery mildew resistance in ‘JI2480’ appeared to be a spatial phenomenon showing expression only in leaf tissues. By segregation analysis of an F₂ progeny of cross ‘Lincoln/JI2480’, the leaf resistance of ‘JI2480’ was shown to be controlled by a single recessive gene, presumed to be er2. Through linkage analysis of 111 resistant F₂ progeny plants with simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) markers adopted from the published linkage maps, the er2 gene was localized on pea linkage group III (LGIII). The assignment of er2 to LGIII, a position different from that reported for er1, has resolved the long standing controversy in the literature regarding the existence and genomic location of er2 gene. A RAPD marker OPX-17_1400, exhibiting cis phase linkage (2.6 cM) to er2 was successfully converted to a sequence characterized amplified region (SCAR) marker, ScX17_1400. The SCAR marker ScX17_1400 will ensure speedy and precise introgression of er2 into susceptible cultivars by permitting selection of er2 heterozygotes amongst BC _n F₁s without progeny tests and resistance screening.     

Interspecific somatic hybrids between <Emphasis Type="Italic">Cyclamen persicum</Emphasis> and <Emphasis Type="Italic">C. coum</Emphasis>, two sexually incompatible species

By applying polyethylene glycol (PEG)-mediated protoplast fusion, the first somatic hybrids were obtained between Cyclamen persicum (2n = 2x = 48) and C. coum (2n = 2x = 30)—two species that cannot be combined by cross breeding. Heterofusion was detected by double fluorescent staining with fluorescein diacetate and scopoletin. The highest heterofusion frequencies (of about 5%) resulted from a protocol using a protoplast density of 1 × 10⁶/mL and 40% PEG. The DNA content of C. coum was estimated for the first time by propidium iodide staining to be 14.7 pg/2C and was 4.6 times higher than that of C. persicum. Among 200 in vitro plantlets regenerated from fusion experiments, most resembled the C. coum parent, whereas only 5 plants showed typical C. persicum phenotypes and 46 had a deviating morphology. By flow cytometry, six putative somatic hybrids were identified. A species-specific DNA marker was developed based on the sequence of the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. The hybrid status of only one plant could be verified by the species-specific DNA marker as well as sequencing of the amplification product. RAPD markers turned out to be less informative and applicable for hybrid identification, as no clear additivity of the parental marker bands was observed. Chromosome counting in root tips of four hybrids revealed the presence of the 30 C. coum chromosomes and 2–41 additional ones indicating elimination of C. persicum chromosomes.     

Shoot organogenesis in leaf explants of <Emphasis Type="Italic">Hydrangea macrophylla</Emphasis> ‘Hyd1’ and assessing genetic stability of regenerants using ISSR markers

For the first time, an in vitro regeneration protocol of Hydrangea macrophylla ‘Hyd1’ was developed. Effects of different plant growth regulators (PGRs) on shoot regeneration were investigated jointly with selecting optimal basal media and cefotaxime concentrations. The highest frequency of shoot organogenesis (100%) and mean number of shoots per explant (2.7) were found on Gamborg B5 basal medium supplemented with 2.25 mg/l 6-benzyladenine (BA), 0.1 mg/l Indole-3-butyric acid (IBA), 100 mg/l cefotaxime and 30 g/l sucrose solidified by 7 g/l agar. Regenerated shoots were rooted by culturing on perlite plus half strength liquid B5 basal medium with 0.5 mg/l NAA. Rooted plantlets were transplanted to the greenhouse with 100% survival rate. Genetic stability of 32 plantlets (one mother plant and 31 regenerants) was assessed by 44 ISSR markers. Out of 44 ISSR markers, ten markers produced clear, reproducible bands with a mean of 5.9 bands per marker. The in vitro regeneration protocol is potentially useful for the genetic transformation of Hydrangea macrophylla ‘Hyd1’.     

Molecular mapping of leaf rust resistance gene <Emphasis Type="Italic">Rph14</Emphasis> in <Emphasis Type="Italic">Hordeum vulgare</Emphasis>

An incompletely dominant gene conferring resistance to Puccinia hordei, Rph14, identified previously in an accession of Hordeum vulgare, confers resistance to all known pathotypes of P. hordei in Australia. Knowledge of the chromosomal location of Rph14 and the identification of DNA markers closely linked to it will facilitate combining it with other important leaf rust resistance genes to achieve long lasting resistance. The inheritance of Rph14 was confirmed using 146 and 106 F₃ lines derived from the crosses ‘Baudin’/‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks using DArT markers located Rph14 to the short arm of chromosome 2H. DArT marker bPb-1664 was identified as having the closest genetic association with Rph14. PCR based marker analysis identified a single SSR marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cm in the ‘Baudin’/‘PI 584760’and ‘Ricardo’/‘PI 584760’ populations, respectively.