Development and characterization of CATS markers for genetic linkage mapping in the house musk shrew, Suncus murinus.

To serve as an initial step in developing an ideal genetic marker map for the house musk shrew, Suncus murinus, 318 comparative anchor tagged sequence (CATS) primer pairs were assessed for polymorphism ascertainment and linkage mapping. Of the 112 (35.2%) CATS primer pairs that were successfully amplified by PCR in the shrew, 18 (16.1%) showed polymorphism between two mutant strains, BAN-kc, oeb and WZ. Linkage analysis of the polymorphic CATS markers and three visible mutant genes, kc, oeb and wz, genotyped in a 77 F2 mapping panel from a cross of the two mutant strains, assigned wz and five CATS markers into three linkage groups. Sequence analysis revealed that two (ADA and TXN) out of nine CATS amplified sequences had a total of six deletions of varying sizes and 17 single nucleotide polymorphisms (SNPs). BLAST search identified three CATS (ADA, CYP1A2, and TXN) products matching the genes from which they were originally designed, while the remaining six markers could not be identified. Together with the use of the detected SNPs as genetic markers, the five CATS markers linkage mapped in this species will serve as anchors in establishing the first framework map for locating loci affecting all heritable qualitative and quantitative traits in the musk shrew.     

Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites

Ten (TG)_n positive clones, isolated from an equine genomic library and sequenced, contained 12–19 uninterrupted TG repeats. Primers for polymerase chain reaction (PCR) were synthesized and nine of these (TG)_n loci (HTG7-15) were successfully amplified and utilized in this study together with five previously reported equine microsatellite loci (HTG2-6). The PCR products were analysed by polyacrylamide gel electrophoresis followed by automated laser fluorescence detection or autoradiography. All microsatellites showed polymorphism and stable Mendelian inheritance. Differences in microsatellite variability between horse breeds were detected. A linkage analysis comprising HTG2-15, one coat colour gene and 16 genetic blood markers enabled addition of HTG2 to linkage group U2 and a new linkage group (U6) was established comprising the loci HTG7 and HTG12. Close linkage was excluded within a set of eight microsatellites. The estimated probability of exclusion in four breeds for a parentage test based on these eight loci varied between 0.96 and 0.99.     

Conservation and synteny of SSR loci and QTLs for vegetative propagation in four Eucalyptus species

Conservation of microsatellite loci, heterozygous in Eucalyptus grandis, Eucalyptus urophylla, Eucalyptus tereticornis and Eucalyptus globulus, allowed us to propose homeologies among genetic linkage groups in these species, supported by at least three SSR loci in two different linkage groups. Marker-trait associations for sprouting and adventitious rooting ability were also compared in the four species. Putative quantitative trait loci (QTLs) influencing vegetative propagation traits were located on homeologous linkage groups. Our findings indicate high transferability of microsatellite markers between Eucalyptus species of the Symphyomyrtus subgenus and establish foundations for the use of synteny in the genetic analysis of this genus. Microsatellite markers should help integrate eucalypt genetic linkage maps from various sources. The availability of comparative linkage maps provides a basis of more-efficient use of genetic information for molecular breeding and evolutionary studies in Eucalyptus.     

Mapping of plumage colour and blood protein loci on the microsatellite linkage map of the Japanese quail

The objective of this work was to map classical markers (plumage colours and blood proteins) on the microsatellite linkage map of the Japanese quail (Coturnix japonica). The segregation data on two plumage colours and three blood proteins were obtained from 25 three-generation families (193 F2 birds). Linkage analysis was carried out for these five classical markers and 80 microsatellite markers. A total of 15 linkage groups that included the five classical loci and 69 of the 80 microsatellite markers were constructed. Using the BLAST homology search against the chicken genome sequence, three quail linkage groups, QL8, QL10 and QL13, were suggested to be homologous to chicken chromosomes GGA9, GGA20 and GGA24, respectively. Two plumage colour loci, black at hatch (Bh) and yellow (Y), and the three blood protein loci, transferrin (Tf), haemoglobin (Hb-1) and prealbumin-1 (Pa-1), were assigned to CJA01, QL10, QL8, CJA14 and QL13, respectively.     

A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci

A genetic map for the model legume Lotus japonicus has been developed. The F(2) mapping population was established from an interspecific cross between L. japonicus and L. filicaulis. A high level of DNA polymorphism between these parents was the source of markers for linkage analysis and the map is based on a framework of amplified fragment length polymorphism (AFLP) markers. Additional markers were generated by restriction fragment length polymorphism (RFLP) and sequence-specific PCR. A total of 524 AFLP markers, 3 RAPD markers, 39 gene-specific markers, 33 microsatellite markers, and six recessive symbiotic mutant loci were mapped. This genetic map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes as described in the accompanying article by Pedrosa et al. 2002(this issue). The length of the linkage map is 367 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in certain sections of the map and linkage group I could be assembled only by combining colormapping and cytogenetics (FISH). A fast method to position genetic loci employing three AFLP primer combinations yielding 89 markers was developed and evaluated by mapping three symbiotic loci, Ljsym1, Ljsym5, and Ljhar1-3.     

Constructing a genetic linkage map of <Emphasis Type="Italic">Vitis aestivalis</Emphasis>-derived “Norton” and its use in comparing Norton and Cynthiana

The first microsatellite-based linkage map for Vitis aestivalis was constructed using 183 progeny from the crosses of V. aestivalis-derived “Norton” and V. vinifera “Cabernet Sauvignon”. A total of 1157 simple sequence repeat (SSR) markers were tested, 859 were amplified via PCR and 413 were polymorphic for at least one parent. The map for Norton consisted of 376 markers and covered 1496.6 centimorgan (cM) on 19 chromosomes. The consensus map consisted of 411 markers on 19 linkage groups with a total distance of 1678.6 cM. Although isozyme and SSR marker analyses in 1993 and 2009 provided preliminary evidence that Norton and Cynthiana grapes are genetically identical, only five banding patterns and four microsatellite loci were reported. This study characterized the relationship between these two cultivars using 185 microsatellites spanning 19 linkage groups for a genome-wide analysis. Four accessions of Norton and three accessions of Cynthiana were used; capillary electrophoresis results revealed Norton and Cynthiana to be identical at all selected loci.     

Polymorphic microsatellite markers for the Mojave desert tortoise, Gopherus agassizii

We describe primers and polymerase chain reaction (PCR) conditions to amplify 14 tri- and tetranucleotide microsatellite loci for the Mojave desert tortoise (Gopherus agassizii). Across three populations (87 individuals) located in the Mojave Desert, USA, the markers yielded a range of four to 33 alleles and an average observed heterozygosity of 0.733 (range 0.433 to 0.933). We neither detected linkage disequilibrium between any pair of loci nor did we find a consistent pattern of deviation from Hardy-Weinberg equilibrium. These microsatellites are designed for PCR multiplexing, and provide higher throughput capacity to aid in conservation genetics studies for this threatened species.     

Mapping 28 erythrocyte antigen, plasma protein and enzyme polymorphisms using an efficient genomic scan of the porcine genome

One hundred and fifty-four microsatellite markers were selected for genomic scanning of the porcine genome and were grouped into amplification sets to reduce the cost and labour required. Thirty amplification sets had two markers (duplex), 20 sets had three markers (triplex) and five sets had four markers (quadruplex) while 14 markers were analysed separately. The selection criteria for microsatellites were: ease of scoring, level of polymorphism, genetic location and ability to be genotyped in a multiplexed polymerase chain reaction (PCR). The selected microsatellites were chosen to span the entire genome flanked by the porcine linkage map with intervals between adjacent markers of 15–20 cM where possible. The utility of this set of markers was demonstrated by linkage analyses with loci controlling blood plasma protein and red cell enzyme polymorphisms ( n = 13), erythrocyte antigens ( n = 15), the S blood group, coat colour and ryanodine receptor from 174 backcross Meishan-White Composite pigs. These loci displayed various forms of inheritance and most (24 loci) have been placed in linkage groups. Significant two-point linkages (lod > 3·0) were detected for each polymorphic marker. These results provide the first linkage assignments for phosphoglucomutase (PGM2) and erythrocyte antigen F (EAF) to SSC8; and serum amylase (AMY) and erythrocyte antigen I (EAI) to SSC18. All of the remaining polymorphic loci ( n = 24) mapped to previously identified regions confirming earlier results. Most of the markers used in this study should be useful in resource populations of various breed crosses as the number of alleles detected in a multibreed reference population was one of the selection criteria.     

Quantitative trait loci for spawning date and body weight in rainbow trout: testing for conserved effects across ancestrally duplicated chromosomes

We incorporated 69 microsatellite loci into an existing data set of 132 markers to test for quantitative trait loci (QTLs) affecting spawning date and body weight in a backcross between two outbred strains of rainbow trout (Oncorhynchus mykiss). Twenty-six linkage groups were identified and synteny of duplicated microsatellite markers was used to confirm 13 homeologous chromosome pairs. Gene-centromere data were used to localize the centromeres for 13 linkage groups whose orientations were previously unknown. We applied a combination of interval mapping and single marker analysis to the segregating maternal and paternal alleles at 201 microsatellite loci. Four spawning date QTLs with suggestive evidence for an additional two QTLs were detected in female trout spawning at 3 and 4 years of age. Similarly we detected three QTLs for body weight in females at 2 years of age plus four suggestive QTLs for this trait. We found marginal evidence that three pairs of ancestral homeologues contained detectable QTLs for the same trait. In one of the three pairs of homeologues, the duplicated QTL regions mapped to the same relative chromosomal location, while the exact localization of the QTL position in one of the other pairs was difficult to infer since it was based on data from a male-derived map. The existing data were unable to refute a hypothesis that duplicated functional genes will be maintained within the telomeric regions of salmonids due to preferential male-mediated crossing over in this region. Two of the four spawning date QTLs were detected on linkage groups with unknown homeologous relationships. QTLs with possible pleiotropic effects on both spawning date and body size were localized to two linkage groups.     

A genetic and cytogenetic map for the duck (Anas platyrhynchos)

A genetic linkage map for the duck (Anas platyrhynchos) was developed within a cross between two extreme Peking duck lines by linkage analysis of 155 polymorphic microsatellite markers, including 84 novel markers reported in this study. A total of 115 microsatellite markers were placed into 19 linkage groups. The sex-averaged map spans 1353.3 cM, with an average interval distance of 15.04 cM. The male map covers 1415 cM, whereas the female map covers only 1387.6 cM. All of the flanking sequences of the 155 polymorphic loci--44 monomorphic loci and a further 41 reported microsatellite loci for duck--were blasted against the chicken genomic sequence, and corresponding orthologs were found for 49. To integrate the genetic and cytogenetic map of the duck genome, 28 BAC clones were screened from a chicken BAC library using the specific PCR primers and localized to duck chromosomes by FISH, respectively. Of 28 BAC clones, 24 were detected definitely on duck chromosomes. Thus, 11 of 19 linkage groups were localized to 10 duck chromosomes. This genetic and cytogenetic map will be helpful for the mapping QTL in duck for breeding applications and for conducting genomic comparisons between chicken and duck.     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Polymorphic microsatellite DNA markers from the Oriental Fruit Fly Bactrocera dorsalis (Hendel)

Six polymorphic microsatellite loci are isolated from the Oriental fruit fly Bactrocera dorsalis (Hendel), an agricultural pest in Asia, including Taiwan. To assess their potential utility as high‐resolution genetic markers, polymerase chain reaction (PCR) primers, amplification conditions, and an automated fluorescence detection protocol were developed. In analyses of 71 individual flies from six different areas of Taiwan, allele numbers ranged from five to 25 were detected for each locus. The observed heterozygosity ranged between 0.268 and 0.737 among these loci. No linkage disequilibrium was found. These microsatellite markers have potential utility to population structure and gene flow studies of B. dorsalis (Hendel).     

A preliminary microsatellite genetic map of the ostrich (Struthio camelus)

Molecular genetic maps can provide information for the identification and localization of major genes associated with quantitative traits. However, there are currently no published genetic linkage maps for any ratites. Herein, a preliminary genetic map of ostrich was developed using a two-generation ostrich reference family by linkage analysis of 104 polymorphic microsatellite markers, including 40 novel markers reported in this study. A total of 35 microsatellite markers were placed into 13 linkage groups. Five linkage groups are composed of three or more loci, whereas the remaining eight groups each contained two markers. The sex-averaged map spans 365.4 cM. The marker interval of each linkage group ranges from 5.3 to 25.4 cM, and the average interval distance is 16.61 cM. The male map covers 342.7 cM, with an average intermarker distance of 15.58 cM, whereas the female map is 456.7 cM, with the average intermarker spacing of 20.76 cM. In order to screen the orthologous loci between ostrich and chicken, all of the flanking sequences of the 104 polymorphic loci, nine monomorphic loci and a further 12 reported microsatellite loci for ostrich were screened against the chicken genomic sequence using the BLAST algorithm (Altschul et al., 1990), and corresponding orthologs were found for 13 sequences. The microsatellite loci and genetic map developed in this study will be useful for QTL mapping, population genetics and phylogenetic studies in the ratite. In addition, the 13 orthologous loci identified in this study will be advantageous to the construction of a comparative genetic map between chicken and ostrich.     

Y-chromosomal markers for the European brown hare (<Emphasis Type="Italic">Lepus europaeus</Emphasis>, Pallas 1778)

Studies on demographic population history and gene flow among populations often rely exclusively on matrilinearly inherited mitochondrial DNA markers. However, by excluding patrilines, such approach introduces an analytical bias into the study. To overcome this bias, we established a set of ten Y-chromosomal markers for the European brown hare (Lepus europaeus), which comprises of three overlapping fragments spanning over the sex-determining region Y, five microsatellite loci (LeMS-Y), and two introns of the Y-linked zinc finger protein (LeZFY). Besides the generation of male specific fragments, both the ZFY and the LeMS-Y01 primer pairs also generated amplification products in females, which are visible in standard agarose gels. These polymerase chain reaction (PCR) products were easily distinguishable from the Y-specific amplicons and thus can function as internal positive PCR control in molecular sexing.     

Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the septoria tritici blotch pathogen Mycosphaerella graminicola

A database of 30,137 EST sequences from Mycosphaerella graminicola, the septoria tritici blotch fungus of wheat, was scanned with a custom software pipeline for di- and trinucleotide units repeated tandemly six or more times. The bioinformatics analysis identified 109 putative SSR loci, and for 99 of them, flanking primers were developed successfully and tested for amplification and polymorphism by PCR on five field isolates of diverse origin, including the parents of the standard M. graminicola mapping population. Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested. Among these 51 loci, 23 were polymorphic between the parents of the mapping population. Twenty-one of these as well as two previously published microsatellite loci were positioned on the existing genetic linkage map of M. graminicola on 13 of the 24 linkage groups. Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested. A subset of the primer pairs also revealed polymorphisms when tested with DNA from the related banana black leaf streak (Black Sigatoka) pathogen, M. fijiensis. The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be multiplexed for high-throughput genetic analyses of M. graminicola and related species.     

Development, characterisation, inheritance, and cross-species utility of American lobster (Homarus americanus) microsatellite and mtDNA PCR-RFLP markers.

Effective management of exploited species demands contemporary knowledge of population structure and mating patterns. Genetic markers can prove useful in providing this knowledge. Despite its commercial importance, genetic markers for American lobster (Homarus americanus) are limited. We developed 12 tetra- and 1 trinucleotide microsatellite loci for American lobster that exhibit little stuttering after PCR amplification. Gene diversity of these loci ranged from 0.516 to 0.929. A four-locus multiplex permits rapid genotyping of progeny in parentage experiments with a paternity exclusion probability over the four loci of 97.8%. We examined the loci for conformity to Hardy-Weinberg expectations (HWE) and linkage using individuals from one location and found that four loci deviated from HWE. We also tested inheritance and pairwise linkage using 48 embryos from each of two females. With the exception of two loci that were derived from the same clone and separated by 72 bp, no evidence of linkage was found. We, for the first time, demonstrate the occurrence of multiple paternity in American lobster. We also observed an apparent occurrence of dispermic androgenesis, possibly the first documentation of such an event within a species. Ten of the loci amplified in European lobster (Homarus gammarus), although two were monomorphic and one deviated significantly from HWE. We quantified mitochondrial DNA (mtDNA) sequence variation through the use of PCR amplification of two DNA fragments, followed by digestion with restriction enzymes; eight haplotypes were detected. One of the two fragments amplified in European lobster. Both sets of markers should prove useful for population discrimination purposes, and the microsatellites, in particular the four-locus multiplex, should prove highly amenable to rapidly addressing questions about mating patterns.     

Isolation, characterization, inheritance and linkage of microsatellite markers in Tetranychus kanzawai (Acari: Tetranychidae)

We isolated and characterized seven microsatellite markers in Tetranychus kanzawai (Acari: Tetranychidae). We also examined the conformity of the isolated markers to Mendelian laws and analyzed linkage among the microsatellite loci. All microsatellite markers fit expected 1:1 disomic segregation ratio and hence were inherited in a Mendelian manner. Significant pairwise linkage was detected in three pairs of microsatellite loci. These isolated microsatellite markers may become a powerful tool for the study of behavioral ecology, population genetics, and genome mapping of T. kanzawai.     

Isolation and characterization of polymorphic microsatellite markers from Primula nutans (Primulaceae)

Seven polymorphic microsatellite loci were developed for a perennial seashore plant, Primula nutans. Degenerate oligonucleotide‐primed (DOP)–polymerase chain reaction (PCR)‐amplified DNA was ligated to TOPO TA vector and screened with radioactively labelled dinucleotide repeat probes. A sample of 378 individuals from Finland, Norway and Russia were used to characterize those loci, which exhibited two to four alleles per locus with observed heterozygosity of 0.003–0.229 and expected heterozygosity of 0.016–0.527. No linkage disequilibrium was found between these seven loci. These are the first microsatellite markers reported for P. nutans.     

Isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by B1 repetitive sequence-representational difference analysis

Mapping of genetic suppressors, modifiers, and quantitative trait loci (QTLs) requires genetic markers that can be efficiently and inexpensively genotyped for a large number of individuals. To isolate rat genetic markers suitable for this purpose, representational difference analysis (RDA) was performed with amplicons prepared by PCR with the B1 repetitive sequence used as the primer (B1-amplicons). In total, 48 polymorphic DNA fragments were isolated by five series of RDA, subtracting the B1-amplicons prepared from an ACI/N (ACI) rat from those prepared from BUF/Nac (BUF), and vice versa. All the polymorphic fragments detected ``presence-or-absence' polymorphisms with B1-amplicons prepared from ACI, BUF, and their F₂ progeny, and each fragment was linkage mapped. Dot-blotting amplicons onto filters at a high density and hybridization of the filters with these B1-RDA markers made it possible to genotype a large number of rats simultaneously for multiple loci. These B1-RDA markers were polymorphic between two given inbred strains of rat at frequencies between 30% and 70%. This is the first report on the isolation of B1-RDA markers among inbred strains of rats. Received: 15 July 1998 / Accepted: 18 August 1998