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

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

A consensus linkage map of barley

A consensus linkage map of the barley genome was constructed. The map is based on six doubled haploid and one F₂ population. The mapping data for three of the doubled haploid populations was obtained via the GrainGenes database. To allow merger of the maps, only RFLP markers that produce a single scorable band were included. Although this reduced the available markers by about half, the resultant map contains a total of 587 markers including 87 of known function. As expected, gene order was highly conserved between maps and all but two discrepancies were found in closely linked markers and are likely to result from the small population sizes used for some maps. The consensus map allows the rapid localisation of markers between published maps and should facilitate the selection of markers for high-density mapping in defined regions.     

Development of simple sequence repeat markers and construction of a high-density linkage map of Capsicum annuum

To facilitate marker-assisted breeding and genetic analyses of pepper (Capsicum annuum), we developed non-redundant 2- or 3-base simple sequence repeat (SSR) markers from enriched C. annuum genomic libraries and from C. annuum cDNA sequences in public databases. The SSR-enriched libraries were constructed using combinations of three restriction enzymes (AluI, HaeIII, and RsaI) and two biotinylated oligonucleotides [b(GA)₁₅ and b(CA)₁₅]. Ultimately, we obtained 1,736 genomic SSR markers and 1,344 cDNA-derived SSR markers from 6,528 clones and 13,003 sequences, respectively. We mapped 597 markers, including 265 of the newly developed SSR markers, onto a linkage map by using doubled-haploid (DH) lines derived from an intraspecific cross of two pure lines of C. annuum (K9-11 × MZC-180). The map, designated as the KL-DH map, consisted of 12 linkage groups. The map covered a genetic distance of 2,028 cM, and the average distance between markers was less than 4 cM. The frame structure of the KL-DH map was compared with the published standard conserved ortholog set II (COSII) map, which was derived from an interspecific F₂ population (C. frutescens × C. annuum), by using tomato (Solanum lycopersicum) chromosomal sequences to bridge the two maps. The intraspecific KL-DH map constructed in this study and the interspecific COSII map were similar in map length and marker distribution, suggesting that the KL-DH map covers nearly the whole genome of C. annuum.     

One hundred and one new simple sequence repeat-based markers for the canine genome

One hundred and one new dinucleotide repeat polymorphisms specific for the canine genome have been identified and characterized. Screening of both primary libraries and marker selected libraries enriched for simple sequence repeats led to the isolation of large numbers of genomic clones that contained (CA)_n repeats. Over 200 of these clones were sequenced, and PCR primers that bracket the repeat were developed for those that contained ten or more continuous (CA)_n units. This effort led to the production of 101 polymorphic markers, which were assigned to one of four categories depending on their degree of polymorphism. Fiftyfour markers were found to be highly or very highly polymorphic as they had four or more alleles when tested on a panel of unrelated dogs. This group of markers will be useful for following inheritance of traits in crosses between dogs.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers indicated in Table 1.     

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.     

The development of simple sequence repeat markers for Magnaporthe grisea and their integration into an established genetic linkage map

Although microsatellite or simple sequence repeat (SSR) markers have several advantages, few have been developed in fungi. The goal of this study was to identify and characterize SSR-containing loci in the filamentous ascomycete Magnaporthe grisea, the causal agent of rice blast disease, and to add these markers to an integrated genetic map of this species [Theor. Appl. Genet. 95 (1997) 20]. We have constructed and screened a microsatellite-enriched small-insert genomic library as well as exploited both publicly available and one proprietary databases for identification of M. grisea SSR containing sequences. Twenty-four out of 49 primer pairs designed to amplify SSR, produced unambiguous polymorphic products in our test population of six isolates. The number of alleles at each locus ranged from two to six when assayed on 3% agarose gels. Twenty-three of the primer pairs amplified polymorphic products between Guy11 and 2539, the parents of a cross from which a genetic map for M. grisea has been established. Genetic analysis showed that all the markers segregated in the expected 1:1 ratio and map positions were determined for all 23 loci.     

A molecular linkage map of rye

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

A genetic linkage map of an apple rootstock progeny anchored to the Malus genome sequence

An apple rootstock progeny raised from the cross between the very dwarfing ??M.27?? and the more vigorous ??M.116?? (??M.M.106???×???M.27??) was used for the construction of a linkage map comprising a total of 324 loci: 252 previously mapped SSRs, 71 newly characterised or previously unmapped SSR loci (including 36 amplified by 33 out of the 35 novel markers reported here), and the self-incompatibility locus. The map spanned the 17 linkage groups (LG) expected for apple covering a genetic distance of 1,229.5?cM, an estimated 91% of the Malus genome. Linkage groups were well populated and, although marker density ranged from 2.3 to 6.2?cM/SSR, just 15 gaps of more than 15?cM were observed. Moreover, only 17.5% of markers displayed segregation distortion and, unsurprisingly in a semi-compatible backcross, distortion was particularly pronounced surrounding the self-incompatibility locus (S) at the bottom of LG17. DNA sequences of 273 SSR markers and the S locus, representing a total of 314 loci in this investigation, were used to anchor to the ??Golden Delicious?? genome sequence. More than 260 of these loci were located on the expected pseudo-chromosome on the ??Golden Delicious?? genome or on its homeologous pseudo-chromosome. In total, 282.4?Mbp of sequence from 142 genome sequence scaffolds of the Malus genome were anchored to the ??M.27???×???M.116?? map, providing an interface between the marker data and the underlying genome sequence. This will be exploited for the identification of genes responsible for traits of agronomic importance such as dwarfing and water use efficiency.     

Efficient construction of high-density linkage map and its application to QTL analysis in barley

Using a High Efficiency Genome Scanning (HEGS) system and recombinant inbred (RI) lines derived from the cross of Russia 6 and H.E.S. 4, a high-density genetic map was constructed in barley. The resulting 1,595.7-cM map encompassed 1,172 loci distributed on the seven linkage groups comprising 1,134 AFLP, 34 SSR, three STS and vrs1 (kernel row type) loci. Including PCR reactions, gel electrophoresis and data processing, 6 months of work by a single person was sufficient for the whole mapping procedure under a reasonable cost. To make an appraisal of the resolution of genetic analysis for the 95 RI lines based on the constructed linkage map, we measured three agronomic traits: plant height, spike exsertion length and 1,000-kernel weight, and the analyzed quantitative trait loci (QTLs) associated with these traits. The results were compared on the number of detected QTLs and their effects between a high-density map and a skeleton map constructed by selected AFLP and anchor markers. The composite interval mapping on the high-density map detected more QTLs than the other analyses. Closely linked markers with QTLs on the high-density map could be powerful tools for marker-assisted selection in barley breeding programs and further genetic analyses including an advanced backcross analysis or a map-based cloning of QTL. Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by J.S. Heslop-Harrison     

QTL analysis of Fusarium head blight resistance using a high-density linkage map in barley

Fusarium head blight (FHB) resistance was evaluated in a set of recombinant inbred (RI) lines from a cross between Russia 6 (resistant) and H.E.S. 4 (susceptible), which had one of the widest differences of FHB resistance reactions among ca. 5,000 barley germplasm accessions in Okayama University. Field-grown spikes were sampled and inoculated by the ‘cut-spike test’. Resistance reactions on the parents and RI lines were scored by eleven grades, from resistant (0) to susceptible (10). Quantitative trait loci (QTL) analysis detected three QTL: two located on the long arm of chromosome 2H, and another on the short arm of chromosome 5H. A QTL located on chromosome 2H was coincident with the vrs1 locus, which governs inflorescence row type. The other QTL on chromosome 2H was positioned in the vicinity of cleistogamy locus (cly1 or Cly2) that determines inflorescence opening/closing. Resistant gene analog (RGA) and expressed sequence tag (EST) markers with homology for disease resistance genes were integrated into the high-density linkage map. Most of these markers were not localized near the identified resistance QTL, except for one RGA marker (FXLRRfor_XLRRrev170) localized in the vicinity of the cly1/Cly2 locus. Five AFLP markers localized in the vicinity of the identified QTL were sequenced to convert them into sequence tagged site (STS) markers. Genotyping of each RI line using two AFLP–STS markers and the vrs1 locus indicated that the RI lines with three Russia 6 QTL alleles exhibited the same level of high FHB resistance reactions as Russia 6. In contrast, RI lines with three susceptible alleles showed reactions close to H.E.S. 4. Therefore, the markers closely linked to the QTL can be efficiently used for the selection of resistance.     

A sequence-tagged linkage map of Brassica rapa

A detailed genetic linkage map of Brassica rapa has been constructed containing 545 sequence-tagged loci covering 1287 cM, with an average mapping interval of 2.4 cM. The loci were identified using a combination of 520 RFLP and 25 PCR-based markers. RFLP probes were derived from 359 B. rapa EST clones and amplification products of 11 B. rapa and 26 Arabidopsis. Including 21 SSR markers provided anchors to previously published linkage maps for B. rapa and B. napus and is followed as the referenced mapping of R1-R10. The sequence-tagged markers allowed interpretation of the pattern of chromosome duplications within the B. rapa genome and comparison with Arabidopsis. A total of 62 EST markers showing a single RFLP band were mapped through 10 linkage groups, indicating that these can be valuable anchoring markers for chromosome-based genome sequencing of B. rapa. Other RFLP probes gave rise to 2-5 loci, inferring that B. rapa genome duplication is a general phenomenon through 10 chromosomes. The map includes five loci of FLC paralogues, which represent the previously reported BrFLC-1, -2, -3, and -5 and additionally identified BrFLC3 paralogues derived from local segmental duplication on R3.     

A microsatellite linkage map of Drosophila mojavensis

Background

Drosophila mojavensishas been a model system for genetic studies of ecological adaptation and speciation. However, despite its use for over half a century, no linkage map has been produced for this species or its close relatives.

Results

We have developed and mapped 90 microsatellites in D. mojavensis, and we present a detailed recombinational linkage map of 34 of these microsatellites. A slight excess of repetitive sequence was observed on the X-chromosome relative to the autosomes, and the linkage groups have a greater recombinational length than the homologous D. melanogaster chromosome arms. We also confirmed the conservation of Muller's elements in 23 sequences between D. melanogaster and D. mojavensis.

Conclusions

The microsatellite primer sequences and localizations are presented here and made available to the public. This map will facilitate future quantitative trait locus mapping studies of phenotypes involved in adaptation or reproductive isolation using this species.     

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.     

A genetic linkage map of durum wheat

 A genetic linkage map of tetraploid wheat [Triticum turgidum (L.) Thell.] was constructed using segregation data from a population of 65 recombinant inbred lines (RILs) derived from a cross between the durum wheat cultivar Messapia and accession MG4343 of T. turgidum (L.) Thell. ssp dicoccoides (Korn.) Thell. A total of 259 loci were analysed, including 244 restriction fragment length polymorphisms (RFLPs), one PCR (polymerase chain reaction) marker (a sequence coding for a LMW (low-molecular-weight) glutenin subunit gene located at the Glu-B3 locus), seven biochemical (six seed-storage protein loci and one isozyme locus) and seven morphological markers. A total of 213 loci were mapped at a LOD≥3 on all 14 chromosomes of the A and B genomes. The total length of the map is 1352 cM and the average distance between adjacent markers is 6.3 cM. Forty six loci could not be mapped at a LOD≥3. A fraction (18.6%) of the markers deviated significantly from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on chromosomes 1B, 3AL, 4AL, 6AL and 7AL. The durum wheat map was compared with the published maps of bread wheat using several common RFLP markers and general features are discussed. The markers detected the known structural rearrangements involving chromosomes 4A, 5A and 7B as well as the translocation between 2B-6B, but not the deletion on 2BS. This map provides a useful tool for analysing and breeding economically important quantitative traits and for marker-assisted selection, as well as for studies of genome organisation in small grain cereal species. Received: 5 January 1998 / Accepted: 31 March 1998     

A genetic linkage map of chromosome 17

We have developed a genetic linkage map of 19 markers (including nine genes) on human chromosome 17, providing 13 reference points along virtually the entire length of this chromosome. The map covers an estimated 149 cM in length (sex-averaged), with a total length of 214 cM in females and 95 cM in males. This sex difference appears to be significant along virtually the entire length of the map. This map will be useful both for providing reference points for fine structure genetic and physical mapping and for genetic linkage studies of diseases, including von Recklinghausen neurofibromatosis and Charcot-Marie-Tooth disease.     

Genetic mapping of ascochyta blight resistance in chickpea (Cicer arietinum L.) using a simple sequence repeat linkage map.

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Lab., is one of the most devastating diseases of chickpea (Cicer arietinum L.) worldwide. Research was conducted to map genetic factors for resistance to ascochyta blight using a linkage map constructed with 144 simple sequence repeat markers and 1 morphological marker (fc, flower colour). Stem cutting was used to vegetatively propagate 186 F2 plants derived from a cross between Cicer arietinum L. 'ICCV96029' and 'CDC Frontier'. A total of 556 cutting-derived plants were evaluated for their reaction to ascochyta blight under controlled conditions. Disease reaction of the F1 and F2 plants demonstrated that the resistance was dominantly inherited. A Fain's test based on the means and variances of the ascochyta blight reaction of the F3 families showed that a few genes were segregating in the population. Composite interval mapping identified 3 genomic regions that were associated with the reaction to ascochyta blight. One quantitative trait locus (QTL) on each of LG3, LG4, and LG6 accounted for 13%, 29%, and 12%, respectively, of the total estimated phenotypic variation for the reaction to ascochyta blight. Together, these loci controlled 56% of the total estimated phenotypic variation. The QTL on LG4 and LG6 were in common with the previously reported QTL for ascochyta blight resistance, whereas the QTL on LG3 was unique to the current population.     

Integration of simple sequence repeat (SSR) markers into a molecular linkage map of common bean (Phaseolus vulgaris L.)

Microsatellite or simple sequence repeat (SSR) markers have been successfully used for genomic mapping, DNA fingerprinting, and marker-assisted selection in many plant species. Here we report the first successful assignment of 15 SSR markers to the Phaseolus vulgaris molecular linkage map. A total of 37 SSR primer pairs were developed and tested for amplification and product-length polymorphism with BAT93 and Jalo EEP558, the parental lines of an F7 recombinant inbred (RI) population previously used for the construction of a common bean molecular linkage map. Sixteen of the SSRs polymorphic to the parental lines were analyzed for segregation and 15 of them were assigned to seven different linkage groups, indicating a widespread distribution throughout the bean genome. Map positions for genes coding for DNAJ-like protein, pathogenesis-related protein 3, plastid-located glutamine synthetase, endochitinase, sn-glycerol-3 phosphate acyltransferase, NADP-dependent malic enzyme, and protein kinase were determined for the first time. Addition of three SSR loci to linkage group B4 brought two separated smaller linkage groups together to form a larger linkage group. Analysis of allele segregation in the F7 RI population revealed that all 16 SSRs segregated in the expected 1:1 ratio. These SSR markers were stable and easy to assay by polymerase chain reaction (PCR). They should be useful markers for genetic mapping, genotype identification, and marker-assisted selection of common beans.     

A microsatellite genetic linkage map for Xiphophorus

Interspecies hybrids between distinct species of the genus Xiphophorus are often used in varied research investigations to identify genomic regions associated with the inheritance of complex traits. There are 24 described Xiphophorus species and a greater number of pedigreed strains; thus, the number of potential interspecies hybrid cross combinations is quite large. Previously, select Xiphophorus experimental crosses have been shown to exhibit differing characteristics between parental species and among the hybrid fishes derived from crossing them, such as widely differing susceptibilities to chemical or physical agents. For instance, genomic regions harboring tumor suppressor and oncogenes have been identified via linkage association of these loci with a small set of established genetic markers. The power of this experimental strategy is related to the number of genetic markers available in the Xiphophorus interspecies cross of interest. Thus, we have undertaken the task of expanding the suite of easily scored markers by characterization of Xiphophorus microsatellite sequences. Using a cross between Xiphophorus maculatus and X. andersi, we report a linkage map predominantly composed of microsatellite markers. All 24 acrocentric chromosome sets of Xiphophorus are represented in the assembled linkage map with an average intergenomic distance of 7.5 cM. Since both male and female F1 hybrids were used to produce backcross progeny, these recombination rates were compared between "male" and "female" maps. Although several genomic regions exhibit differences in map length, male- and female-derived maps are similar. Thus Xiphophorus, in contrast to zebrafish, Danio rerio, and several other vertebrate species, does not show sex-specific differences in recombination. The microsatellite markers we report can be easily adapted to any Xiphophorus interspecies and some intraspecies crosses, and thus provide a means to directly compare results derived from independent experiments.     

Simple sequence repeat-based assessment of genetic relationships among Prunus rootstocks

Ten SSR loci, previously developed for Prunus, were analyzed to examine genetic relationships among 23 rootstock candidates for sweet and sour cherries, of the species P. avium, P. cerasus, P. mahaleb, and P. angustifolia. Five genotypes of P. laurocerasus, not used as rootstock, were included in the molecular analysis. The number of alleles per locus ranged from 8 to 12, with a mean of 9, while the number of microsatellite genotypes varied from 8 to 17, indicating that the SSRs were highly informative. The degree of heterozygosity (0.61) was high. Clustering analysis resulted in two main clusters. The first cluster was divided into two subclusters; the first subcluster consisted of P. avium and P. cerasus, and the second subcluster consisted of P. laurocerasus. The second cluster was divided into two subclusters. The first subcluster consisted of P. mahaleb genotypes and the second consisted of P. angustifolia genotypes. The reference rootstocks also clustered with their associated botanical species. Unweighted pair-group method with arithmetic mean analysis demonstrated that P. laurocerasus genotypes had less genetic variation and that P. avium genotypes were more closely related to P. cerasus. The SSR-based phylogeny was generally consistent with Prunus taxonomy information, suggesting the applicability of SSR analysis for genotyping and phylogenetic studies in the genus Prunus.     

Expressed sequence tag-simple sequence repeat-based molecular variance in two Salicornia (Amaranthaceae) populations

Salicornia spp is one of the most salt-tolerant vascular plants and is native to salt marshes and estuaries. We developed expressed sequence tag derived-simple sequence repeat (EST-SSR) markers for estimating genetic diversity and marker-assisted Salicornia breeding. Six polymorphic EST-SSRs of 40 detected 27 alleles, ranging from three to five alleles per locus. The average number of alleles per locus was 4.33 and 4.17, and the major allele frequency at locus DY529765 was high, being 0.859 and 0.857 in S. bigelovii and S. europea, respectively. Gene diversity, heterozygosity and polymorphism information content were highest at locus DY529950 and similar in these two species. Gene diversity increased with increase in the number of alleles that had a low major allele frequency at a locus. Six polymorphic loci effectively discriminated 46 taxa into three clusters via different analyses. Significant deviation of F(ST) from zero in three suggested populations for six loci indicated population differentiation and limited gene flow among them. A reduced median network established that taxon SB65 is primitive. SMART (simple modular architecture research tool) analysis of peptide sequences of six EST-SSRs showed that loci DY529765, DY529950 and EC906203 contained transmembrane, TLC, AgrB and NTR domains and might be involved in salinity stress tolerance. These EST-SSRs are a valuable resource for marker development and may be useful in marker-assisted Salicornia breeding.