A genetic linkage map for an apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) BC<Subscript>1</Subscript> population mapping <Emphasis Type="Italic">plum pox virus</Emphasis> resistance

Plum pox virus (sharka; PPV) can cause severe crop loss in economically important Prunus species such as peach, plum, apricot, and cherry. Of these species, certain apricot cultivars (‘Stark Early Orange’, ‘Goldrich’, ‘Harlayne’) display significant levels of resistance to the disease and are the genetic substrate for studies of several xlaboratories working cooperatively to genetically characterize and mark the resistance locus or loci for marker-assisted breeding. The goals of the work presented in this communication are the characterization of the genetics of PPV resistance in ‘Stark Early Orange’ and the development of co-dominant molecular markers for marker-assisted selection (MAS) in PPV resistance breeding. We present the first genetic linkage map for an apricot backcross population of ‘Stark Early Orange’ and the susceptible cultivar ‘Vestar’ that segregates for resistance to PPV. This map is comprised of 357 loci (330 amplified fragment length polymorphisms (AFLPs), 26 simple sequence repeats (SSRs), and 1 morphological marker for PPV resistance) assigned to eight linkage groups. Twenty-two of the mapped SSRs are shared in common with genetic reference map for Prunus (T × E; Joobeur et al. 1998) and anchor our apricot map to the general Prunus map. A PPV resistance locus was mapped in linkage group 1 and four AFLP markers segregating with the PPV resistance trait, identified through bulk segregant analysis, facilitated the development of SSRs in this region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lalli, D.A. and Salava, J. contributed equally to this work.     

Flanking the major Plum pox virus resistance locus in apricot with co-dominant markers (SSRs) derived from candidate resistance genes

Plum pox virus (PPV) is a potyvirus that causes sharka disease in infested stone fruit trees (Prunus species, peach, apricot, plum). In apricots, the resistance is controlled by a major quantitative trait locus that explains up to 70% of the phenotypic variance; it is localised in the upper part of linkage group 1. In this report, we transformed candidate genes that mapped in the region of the apricot resistance locus into polymerase chain reaction markers (SSCP and SSR) and tested for their co-localisation with the major PPV resistance locus in related and unrelated populations. Three populations of F1 and F2 individuals issued from crosses between the PPV-resistant cultivar ‘Stark Early Orange’ or ‘Goldrich’ and three susceptible parents were used in this study. Molecular-marker data were collected to determine the linkage relationship between the PPV resistance locus in apricots and markers that target candidate disease-resistance genes. In addition, SSR markers linked to resistance-gene candidates were mapped to positions flanking the PPV resistance locus in different apricot populations. Therefore, we demonstrate that this strategy helps to saturate the major genomic region controlling resistance to PPV in apricot with valuable co-dominant markers. O. Sicard and G. Marandel have contributed equally to this work.     

Rust mite resistance in apple assessed by quantitative trait loci analysis

The aim of this study was to assess the genetic basis of rust mite (Aculus schlechtendali) resistance in apple (Malus × domestica). A. schlechtendali infestation of apple trees has increased as a consequence of reduced side effects of modern fungicides on rust mites. An analysis of quantitative trait loci (QTLs) was carried out using linkage map data available for F₁ progeny plants of the cultivars ‘Fiesta’ × ‘Discovery’. Apple trees representing 160 different genotypes were surveyed for rust mite infestation, each at three different sites in two consecutive years. The distribution of rust mites on the individual apple genotypes was aggregated and significantly affected by apple genotype and site. We identified two QTLs for A. schlechtendali resistance on linkage group 7 of ‘Fiesta’. The AFLP marker E35M42-0146 (20.2 cM) and the RAPD marker AE10-400 (45.8 cM) were closest positioned to the QTLs and explained between 11.0% and 16.6% of the phenotypic variability. Additionally, putative QTLs on the ‘Discovery’ chromosomes 4, 5 and 8 were detected. The SSR marker Hi03a10 identified to be associated to one of the QTLs (AFLP marker E35M42-0146) was traced back in the ‘Fiesta’ pedigree to the apple cultivar ‘Wagener’. This marker may facilitate the breeding of resistant apple cultivars by marker assisted selection. Furthermore, the genetic background of rust mite resistance in existing cultivars can be evaluated by testing them for the identified SSR marker. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Linkage maps of grapevine displaying the chromosomal locations of 420 microsatellite markers and 82 markers for <Emphasis Type="Italic">R</Emphasis>-gene candidates

Genetic maps functionally oriented towards disease resistance have been constructed in grapevine by analysing with a simultaneous maximum-likelihood estimation of linkage 502 markers including microsatellites and resistance gene analogs (RGAs). Mapping material consisted of two pseudo-testcrosses, ‘Chardonnay’ × ‘Bianca’ and ‘Cabernet Sauvignon’ × ‘20/3’ where the seed parents were Vitis vinifera genotypes and the male parents were Vitis hybrids carrying resistance to mildew diseases. Individual maps included 320–364 markers each. The simultaneous use of two mapping crosses made with two pairs of distantly related parents allowed mapping as much as 91% of the markers tested. The integrated map included 420 Simple Sequence Repeat (SSR) markers that identified 536 SSR loci and 82 RGA markers that identified 173 RGA loci. This map consisted of 19 linkage groups (LGs) corresponding to the grape haploid chromosome number, had a total length of 1,676 cM and a mean distance between adjacent loci of 3.6 cM. Single-locus SSR markers were randomly distributed over the map (CD = 1.12). RGA markers were found in 18 of the 19 LGs but most of them (83%) were clustered on seven LGs, namely groups 3, 7, 9, 12, 13, 18 and 19. Several RGA clusters mapped to chromosomal regions where phenotypic traits of resistance to fungal diseases such as downy mildew and powdery mildew, bacterial diseases such as Pierce’s disease, and pests such as dagger and root-knot nematode, were previously mapped in different segregating populations. The high number of RGA markers integrated into this new map will help find markers linked to genetic determinants of different pest and disease resistances in grape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Identification and mapping of a locus conferring plum pox virus resistance in two apricot-improved linkage maps

Sharka disease, caused by the plum pox virus (PPV), is one of the major limiting factors for stone fruit crops in Europe and America. In particular, apricot is severely affected suffering significant fruit losses. Thus, PPV resistance is a trait of great interest for the apricot breeding programs currently in progress. In this work, two apricot maps, earlier constructed with the F₁ ‘Goldrich × Currot’ (G×C) and the F₂ ‘Lito × Lito’-98 (L×L-98) populations, have been improved including 43 and 37 new simple sequence repeat (SSR) loci, respectively, to facilitate PPV resistance trait mapping. Screening of PPV resistance on the segregating populations classified seedling phenotypes into resistant or susceptible. A non-parametric mapping method, based on the Kruskal–Wallis (KW) rank sum test, was initially used to score marker–trait association, and results were confirmed by interval mapping. Contrary to the putative digenic model inferred from the phenotypic segregations, all significant markers for the KW statistic (P < 0.005) mapped in a unique region of ~21.0 and ~20.3 cM located on the upper part of the G1 linkage group in ‘G×C’ and ‘L×L-98’ maps, respectively. According to the data, PPV resistance is suggested to be controlled by at least one major dominant locus. The association between three SSRs distributed within this region and the PPV resistance was tested in two additional populations (‘Goldrich × Canino’ and ‘Lito × Lito’-00) and breeding program parents. The marker ssrPaCITA5 showed the highest KW value (P < 0.005) in all cases, pointing out its usefulness in marker-assisted selection. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pyramided QTL underlying tolerance to Phytophthora root rot in mega-environments from soybean cultivars ‘Conrad’ and ‘Hefeng 25’

Phytophthora root rot (PRR) of soybean (Glycine max (L.) Merr.) is the second most important cause of yield loss by disease in North America, surpassed only by soybean cyst nematode (Wrather et al. in Can J Plant Pathol 23:115–121, 2001). Tolerance can provide economically useful disease control, conditioning partial resistance of soybean to PRR. The aims of this study were to identify new quantitative trait loci (QTL) underlying tolerance to PRR, and to evaluate the effects of pyramided or stacked loci on the level of tolerance. A North American cultivar ‘Conrad’ (tolerant to PRR) was crossed with a northeastern China cultivar ‘Hefeng 25’ (tolerant to PRR). Through single-seed descent, 140 F_2:5 and F_2:6 recombinant inbred lines were advanced. A total of 164 simple sequence repeat (SSR) markers were used to construct a genetic linkage map. The percentage of seedling death was measured over 2 years (2007 and 2008) in the field at four naturally infested locations in Canada and China following additional soil infestation and in the greenhouse following inoculation with Phytophthora sojae isolate. A total of eight QTL underlying tolerance to PRR were identified, located in five linkage groups (F, D1b+w, A2, B1, and C2). The phenotypic variation contributed by the loci ranged from 4.24 to 27.98%. QPRR-1 (anchored in the interval of SSR markers Satt325 and Satt343 of LG F), QPRR-2 (anchored in the interval of Satt005 and Satt600 of LG D1b+w), and QPRR-3 (anchored in the interval of Satt579 and Sat_089 of LG D1b+w) derived their beneficial allele from ‘Conrad’. They were located at chromosomal locations known to underlie PRR tolerance in diverse germplasm. Five QTL that derived beneficial alleles from ‘Hefeng 25’ were identified. The QTL (QPRR-1 to QPRR-7) that were detected across at least three environments were selected for loci stacking and to analyze the relationship between number of tolerance loci and disease loss percentage. The accumulation of tolerance loci was positively correlated with decreases in disease loss percentage. The pyramid of loci underlying tolerance to PRR provided germplasm useful for crop improvement by marker-assisted selection and may provide durable cultivar tolerance against the PRR disease.     

QTL analysis of resistance to sharka disease in the apricot (Prunus armeniaca L.) ‘Polonais’ × ‘Stark Early Orange’ F1 progeny

Different hypotheses on the genetic control of the resistance to the plum pox virus (PPV) have been reported in apricot, but there was a lack of agreement about the number of loci involved. In recent years, apricot genetic maps have been constructed from progenies derived from ‘Stark Early Orange’ or ‘Goldrich’, two main sources of resistance, three of these including the mapping of the PPV resistance loci. As the location of the locus was not precisely established, we mapped the PPV resistance loci using interval mapping (IM), composite interval mapping (CIM), and the Kruskal–Wallis non-parametric test in the F₁ progeny derived from a cross between the susceptible cv. ‘Polonais’ and ’Stark Early Orange’. Four genomic regions were identified as being involved in PPV resistance. One of these mapped to the upper region of linkage group 1 of ‘Stark Early Orange’, and accounted for 56% of the phenotypic variation. Its location was similar to the one previously identified in ‘Goldrich’ and Prunus davidiana. In addition, a gene strongly associated to these major quantitative trait loci (QTL) was found to be related to PPV infection. Two putative QTLs were detected on linkage groups 3 of ‘Polonais’ and 5 of both ‘Polonais’ and ‘Stark Early Orange’ with both parametric and non-parametric methods at logarithm of odds (LOD) scores slightly above the detection threshold. The last QTL was only detected in the early stage of the infection. PPV resistance is, thus, controlled by a major dominant factor located on linkage group 1. The hypothesis of recessive factors with lower effect is discussed.     

Microsatellite diversity in tetraploid <Emphasis Type="Italic">Gossypium</Emphasis> germplasm: assembling a highly informative genotyping set of cotton SSRs

A series of 320 mapped simple sequence repeats (SSRs) have been used to screen the allelic diversity of tetraploid Gossypium species. Fourty-seven genotypes were analyzed representing (i) the wide spectrum of diversity of the cultivated pool and of the primitive landraces of species G. hirsutum (‘marie-galante’, ‘punctatum’, ‘richmondi’, ‘morrilli’, ‘palmeri’, and ‘latifolium’, and ‘yucatanense’), and (ii) species G. barbadense, G. darwinii and G. tomentosum. The polymorphism of 201 SSR loci revealed 1128 allelic variants ranging from 3 to 17 per locus. Neighbor-joining (NJ) method based on genetic dissimilarities produced groupings consistent with the assignments of accessions both at species and at race level. Our data confirmed the proximity of the Galapagos endemic species G. darwinii to species G. barbadense. Within species G. hirsutum, and as compared to the other 6 races, race yucatanense appeared as the most distant from cultivated genotypes. Race yucatanense also exhibited the highest number of unique alleles. The important informative heterogeneity of the 201 SSR loci was exploited to select the most polymorphic ones that were assembled into three series of genome-wide (i.e. each homoeologous AD chromosome pair being equally represented) and mutliplexable (× 3) SSRs. Using one of these ‘genotyping set’, consisting of 39 SSRs (one 3-plex for each of the 13 AD chromosomes pairs) or 45 loci, we were able to assess the relationships between accessions and the topology in the genetic diversity sampled. Such genotyping set of highly informative SSR markers assembled in PCR-multiplex, while increasing genotyping throughput, will be applicable for molecular genetic diversity studies of large germplasm collections. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

QTL analysis for aphid resistance and growth traits in apple

The rosy apple aphid (Dysaphis plantaginea), the leaf-curling aphid (Dysaphis cf. devecta) and the green apple aphid (Aphis pomi) are widespread pest insects that reduce growth of leaves, fruits and shoots in apple (Malus × domestica). Aphid control in apple orchards is generally achieved by insecticides, but alternative management options like growing resistant cultivars are needed for a more sustainable integrated pest management (IPM). A linkage map available for a segregating F₁-cross of the apple cultivars ‘Fiesta’ and ‘Discovery’ was used to investigate the genetic basis of resistance to aphids. Aphid infestation and plant growth characteristics were repeatedly assessed for the same 160 apple genotypes in three different environments and 2 consecutive years. We identified amplified fragment length polymorphism (AFLP) markers linked to quantitative trait loci (QTLs) for resistance to D. plantaginea (‘Fiesta’ linkage group 17, locus 57.7, marker E33M35–0269; heritability: 28.3%), and to D. cf. devecta (‘Fiesta’ linkage group 7, locus 4.5, marker E32M39–0195; heritability: 50.2%). Interactions between aphid species, differences in climatic conditions and the spatial distribution of aphid infestation were identified as possible factors impeding the detection of QTLs. A pedigree analysis of simple sequence repeat (SSR) marker alleles closely associated with the QTL markers revealed the presence of the alleles in other apple cultivars with reported aphid resistance (‘Wagener’, ‘Cox’s Orange Pippin’), highlighting the genetic basis and also the potential for gene pyramiding of aphid resistance in apple. Finally, significant QTLs for shoot length and stem diameter were identified, while there was no relationship between aphid resistance and plant trait QTLs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and characterization of simple sequence repeats for flowering dogwood (Cornus florida L.)

Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences. The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and ‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and gene tagging of flowering dogwood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Linkage disequilibrium based association mapping of fiber quality traits in <Emphasis Type="Italic">G. hirsutum</Emphasis> L. variety germplasm

Cotton is the world’s leading cash crop, but it lags behind other major crops for marker-assisted breeding due to limited polymorphisms and a genetic bottleneck through historic domestication. This underlies a need for characterization, tagging, and utilization of existing natural polymorphisms in cotton germplasm collections. Here we report genetic diversity, population characteristics, the extent of linkage disequilibrium (LD), and association mapping of fiber quality traits using 202 microsatellite marker primer pairs in 335 G. hirsutum germplasm grown in two diverse environments, Uzbekistan and Mexico. At the significance threshold (r ² ≥ 0.1), a genome-wide average of LD extended up to genetic distance of 25 cM in assayed cotton variety accessions. Genome wide LD at r ² ≥ 0.2 was reduced to ~5–6 cM, providing evidence of the potential for association mapping of agronomically important traits in cotton. Results suggest linkage, selection, inbreeding, population stratification, and genetic drift as the potential LD-generating factors in cotton. In two environments, an average of ~20 SSR markers was associated with each main fiber quality traits using a unified mixed liner model (MLM) incorporating population structure and kinship. These MLM-derived significant associations were confirmed in general linear model and structured association test, accounting for population structure and permutation-based multiple testing. Several common markers, showing the significant associations in both Uzbekistan and Mexican environments, were determined. Between 7 and 43% of the MLM-derived significant associations were supported by a minimum Bayes factor at ‘moderate to strong’ and ‘strong to very strong’ evidence levels, suggesting their usefulness for marker-assisted breeding programs and overall effectiveness of association mapping using cotton germplasm resources. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (<Emphasis Type="Italic">Erysiphe necator</Emphasis>) and their use in marker-assisted breeding

A limited genetic mapping strategy based on simple sequence repeat (SSR) marker data was used with five grape populations segregating for powdery mildew (Erysiphe necator) resistance in an effort to develop genetic markers from multiple sources and enable the pyramiding of resistance loci. Three populations derived their resistance from Muscadinia rotundifolia ‘Magnolia’. The first population (06708) had 97 progeny and was screened with 137 SSR markers from seven chromosomes (4, 7, 9, 12, 13, 15, and 18) that have been reported to be associated with powdery or downy mildew resistance. A genetic map was constructed using the pseudo-testcross strategy and QTL analysis was carried out. Only markers from chromosome 13 and 18 were mapped in the second (04327) and third (06712) populations, which had 47 and 80 progeny, respectively. Significant QTLs for powdery mildew resistance with overlapping genomic regions were identified for different tissue types (leaf, stem, rachis, and berry) on chromosome 18, which distinguishes the resistance in ‘Magnolia’ from that present in other accessions of M. rotundifolia and controlled by the Run1 gene on chromosome 12. The ‘Magnolia’ resistance locus was termed as Run2.1. Powdery mildew resistance was also mapped in a fourth population (08391), which had 255 progeny and resistance from M. rotundifolia ‘Trayshed’. A locus accounting for 50% of the phenotypic variation mapped to chromosome 18 and was named Run2.2. This locus overlapped the region found in the ‘Magnolia’-based populations, but the allele sizes of the flanking markers were different. ‘Trayshed’ and ‘Magnolia’ shared at least one allele for 68% of the tested markers, but alleles of the other 32% of the markers were not shared indicating that the two M. rotundifolia selections were very different. The last population, 08306 with 42 progeny, derived its resistance from a selection Vitis romanetii C166-043. Genetic mapping discovered a major powdery mildew resistance locus termed Ren4 on chromosome 18, which explained 70% of the phenotypic variation in the same region of chromosome 18 found in the two M. rotundifolia resistant accessions. The mapping results indicate that powdery mildew resistance genes from different backgrounds reside on chromosome 18, and that genetic markers can be used as a powerful tool to pyramid these loci and other powdery mildew resistance loci into a single line.     

Genetic stability of cryopreserved shoot tips of <Emphasis Type="Italic">Rubus</Emphasis> germplasm

Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study examined the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for more than 12 yr, then rewarmed and regrown. We analyzed the genetic stability of Rubus grabowskii, two blackberry cultivars (“Hillemeyer” and ‘Silvan’), and one raspberry cultivar (“Mandarin”) as in vitro shoots and as field-grown plants. No morphological differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants appeared similar but were more vigorous than mother plants, with larger leaves, fruit, and seeds. Single sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for 7 mo before analysis. Ten SSR primers developed from “Marion” and “Meeker” microsatellite-enriched libraries amplified one to 15 alleles per locus, with an average of seven alleles and a total of 70 alleles in the four genotypes tested. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants regardless of subculture. Although no polymorphisms were detected in shoots analyzed immediately after recovery from cryopreservation, AFLP polymorphisms were detected in three of the four Rubus genotypes after they were subcultured for 7 mo. Field-grown plants from the polymorphic shoot tips of R. grabowskii and ‘Silvan’ displayed the same AFLP fingerprints as their corresponding mother plants. Only long-cultured in vitro shoot tips displayed polymorphisms in vitro, and they were no longer detected when the plants were grown ex vitro. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability.     

Development and characterization of a codominant marker linked to root-knot nematode resistance, and its application to peach rootstock breeding

 The presence of a codominant AFLP marker, EAA/MCAT10, correlates with the primary source of resistance to root-knot nematodes (Meloidogyne incognita and M. javanica) in rootstock cultivars of peach [Prunus persica (L.) Batsch]. Two allelic DNA fragments of this AFLP marker were cloned, sequenced and converted to sequence tagged sites (STS). Four nucleotide differences (i.e. one addition and three substitutions) were observed between the two clones. Furthermore, there was a diagnostic Sau3 AI cleavage site (GATC) in the large fragment that was absent from the small fragment (GTTC at this site). The applicability of this STS marker system to peach germplasm improvement was evaluated: genomic DNAs of cross parents (i.e. ‘Lovell’ and ‘Nemared’), four F₁ hybrids (K62-67, K62-68, P101-40 and P101-41) and two F₂ populations (from K62-68 and P101-41), as well as DNA from a test panel of 18 rootstock cultivars or selections, were PCR-amplified with the Mij3F/Mij1R primer pair and then digested with Sau3 AI. The banding patterns showed that the EAA/MCAT10 STS markers can clearly distinguish the three genotypes – homozygous resistant, heterozygous resistant and homozygous susceptible – in the ‘Lovell’בNemared’ cross. In addition, results from the rootstock survey were consistent with each rootstock’s phenotypic response to nematode infection, except for ‘Okinawa’, ‘Flordaguard’ and ‘Yunnan’ where root-knot resistance may have arisen independently. Therefore, the EAA/MCAT10 STS markers will be a useful tool to initiate marker assisted selection studies in peach rootstock breeding for root-knot nematode resistance. Received: 4 January 1999 / Accepted: 4 January 1999     

Development of wheat–<Emphasis Type="Italic">Lophopyrum elongatum</Emphasis> recombinant lines for enhanced sodium ‘exclusion’ during salinity stress

Lophopyrum elongatum (tall wheatgrass), a wild relative of wheat, can be used as a source of novel genes for improving salt tolerance of bread wheat. Sodium ‘exclusion’ is a major physiological mechanism for salt tolerance in a wheat–tall wheatgrass amphiploid, and a large proportion (~50%) for reduced Na⁺ accumulation in the flag leaf, as compared to wheat, was earlier shown to be contributed by genetic effects from substitution of chromosome 3E from tall wheatgrass for wheat chromosomes 3A and 3D. Homoeologous recombination between 3E and wheat chromosomes 3A and 3D was induced using the ph1b mutant, and putative recombinants were identified as having SSR markers specific for tall wheatgrass loci. As many as 14 recombinants with smaller segments of tall wheatgrass chromatin were identified and low-resolution breakpoint analysis was achieved using wheat SSR loci. Seven recombinants were identified to have leaf Na⁺ concentrations similar to those in 3E(3A) or 3E(3D) substitution lines, when grown in 200 mM NaCl in nutrient solution. Phenotypic analysis identified recombinants with introgressions at the distal end on the long arm of homoeologous group 3 chromosomes being responsible for Na⁺ ‘exclusion’. A total of 55 wheat SSR markers mapped to the long arm of homoeologous group 3 markers by genetic and deletion bin mapping were used for high resolution of wheat–tall wheatgrass chromosomal breakpoints in selected recombinants. Molecular marker analysis and genomic in situ hybridisation confirmed the 524-568 recombinant line as containing the smallest introgression of tall wheatgrass chromatin on the distal end of the long arm of wheat chromosome 3A and identified this line as suitable for developing wheat germplasm with Na⁺ ‘exclusion’.     

Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (<Emphasis Type="Italic">Vaccinium macrocarpon</Emphasis> Ait.)

The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar ‘HyRed’. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540–590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2–15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.     

Development of two loquat [Eriobotrya japonica (Thunb.) Lindl.] linkage maps based on AFLPs and SSR markers from different Rosaceae species

Loquat [Eriobotrya japonica (Thunb.) Lindl.] is a Rosaceae fruit species of growing interest as an alternative to the main fruit crops. However, only a few genetic studies have been carried out on this species. This paper reports the construction of the first genetic maps of two loquat cultivars based on AFLP and microsatellite markers from Malus, Eriobotrya, Pyrus and Prunus genera. An F₁ population consisting of 81 individuals, derived from the cross between ‘Algerie’ and ‘Zaozhong-6’ cultivars, was used to construct both maps. A total of 111 scorable simple sequence repeat (SSR) loci resulted from the testing of 440 SSR primer pairs in the analyzed progeny and the SSR transferability to Eriobotrya was found to be 74% from apple, 58% from pear and 49% from Prunus spp. In addition, 183 AFLP polymorphic bands were produced using 42 primer combinations. The ‘Algerie’ map was organized in 17 linkage groups covering a distance of 900 cM and comprising 177 loci (83 SSRs and 94 AFLPs) with an average marker distance of 5.1 cM. Self-incompatibility trait was mapped at the distal part of the LG17 linkage group, as previously reported in Malus and Pyrus. The ‘Zaozhong-6’ map covered 870 cM comprising 146 loci (64 SSRs and 82 AFLPs) with an average marker distance of 5.9 cM. The 44 SSRs and the 48 AFLPs share in common by both maps were essentially collinear and, moreover, the order of the 75% of apple and pear SSRs mapped in Eriobotrya was shown to be consistent across the Maloideae subfamily. As a whole, these maps represent a useful tool to facilitate loquat breeding and an interesting framework for map comparison in the Rosaceae.     

First report on the presence of fire blight resistance in linkage group 11 ofPyrus ussuriensis Maxim

Fire blight, caused by the gram-negative bacteriumErwinia amylovora (Burrill) Winslow et al., is a dangerous disease of pome fruits, including pear. A pear breeding program for fire blight resistance was initiated in 2003 at the Department of Pomology, Warsaw University of Life Sciences, Poland. Since several Asian species are considered to be potential sources of resistance to fire blight, the susceptiblePyrus communis ‘Doyenne du Comice’ was crossed with the resistantP. ussuriensis. The F₁ full-sib progeny composed of 155 seedlings was tested for susceptibility to fire blight by artificial shoot inoculation. A framework linkage map of both parents was constructed based on 48 AFLP and 32 SSR markers and covered a length of 595 cM and 680 cM in ‘Doyenne du Comice’ andP. ussuriensis, respectively. For the first time a putative QTL for fire blight resistance inP. ussuriensis linkage group 11 was identified. Another putative QTL in linkage group 4 of ‘Doyenne du Comice’ seems to indicate that sources of fire blight resistance can be identified also in the susceptible cultivars.