BAC-derived markers converted from RFLP linked to Phytophthora capsici resistance in pepper (Capsicum annuum L.)

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.     

Comparative mapping of bovine chromosome 13 by fluorescence in situ hybridization

We present chromosomal fluorescence in situ hybridization (FISH) results that both extend the HSA20/BTA13 comparative map as well as cytogenetically anchor two microsatellite markers. A bovine bacterial artificial chromosome (BAC) library was screened for conserved genes (type I loci) previously assigned to HSA10 or HSA20 and BTA13, and for microsatellites selected from two published BTA13 linkage maps. Clones from six out of nine comparative loci and both microsatellites were found represented in the BAC library. These BAC clones were used as probes in single colour FISH to determine the chromosome band position of each locus. As predicted by the human/bovine comparative map, all type I loci mapped to BTA13. Because single colour FISH analysis revealed that the loci were clustered within the distal half of BTA13, dual colour FISH was used to confirm the locus order. Established order was centromere- PRNP-(SODIL/AVP/OXT)-(BL42/GNAS1)-HCK-CSSM30 . The findings confirm the presence of a conserved HSA20 homologous synteny group on BTA13 distal of a HSA10 homologous segment.     

Comparative analysis and development of microsatellite markers on swine (Sus scrofa) chromosome 1qter

Several quantitative trait loci (QTL) have been detected on SSC1qter (Sus scrofa chromosome 1qter), including QTL for the number of vertebrae, as reported in our previous study. To provide the tools for analysis of QTLs on SSC1qter, we constructed a comparative map of swine and human. In addition, we identified 26 swine STSs and mapped 16 of them on SSC1qter using the INRA - University of Minnesota porcine radiation hybrid (IMpRH) panel. We screened a BAC library using these swine STSs and developed 35 new polymorphic microsatellite markers from the BAC clones, of which 26 were informative in our reference family. We also mapped nine microsatellite markers we had isolated previously. Consequently a total of 44 new polymorphic microsatellite markers were located within a 60-cM region of SSC1qter, spanning from SW1092 to the telomere.     

Characterization and RH mapping of bovine microsatellites generated from a microdissected BTA20-specific DNA library

Bovine chromosome 20 (BTA20) is associated with several quantitative trait loci (QTL) for meat tenderness, birth weight, milk yield and composition. Fine mapping of these QTL requires the development of additional informative markers to increase the resolution of the BTA20 genetic and physical maps. A BTA20-specific library was constructed by means of microdissection and microcloning, and screened for dinucleotide repeats with (CA)16 and (GT)16 oligos. A total of 60 new microsatellites (MS) were developed and characterized for polymorphism using the U.S. Department of Agriculture (USDA)/Meat Animal Research Center (MARC) bovine reference family, of which 53 markers were informative in this family. The number of alleles for these loci varied from 1 to 14, with an average of 6.5. Thirty-three of these MSs, together with 105 markers previously mapped to BTA20, were scored on a 7000-rad cattle-hamster whole-genome radiation hybrid panel (SUNbRH), resulting in a high-resolution RH7000 rad map for BTA20.     

High-resolution genetic map of bovine chromosome 29 through focused marker development

Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome.     

Radiation hybrid and genetic linkage mapping of two genes related to fat metabolism in cattle: fatty acid synthase (FASN) and glycerol-3-phosphate acyltransferase mitochondrial (GPAM)

Fatness traits, such as fat deposition, carcass composition, fat content, and the percentage of fat in milk, are economically relevant to cattle production. Fatty acid synthase (FASN) and glycerol-3-phosphate acyltransferase mitochondrial (GPAM) are two enzymes that play a central role in de novo lipogenesis. Both could be putative candidate genes for quantitative trait loci (QTL). Several clones containing the fatty acid synthase (FASN) and glycerol-3-phosphate acyltransferase mitochondrial (GPAM) genes were isolated after screening the INRA bovine bacterial artificial chromosome (BAC) library using PCR. Five microsatellite loci were derived from the BAC clones containing the genes of interest with heterozygosity values ranging from 27 to 78%, using DNA samples from the International Bovine Reference Panel (IBRP). The newly developed markers were genotyped on the IBRP animals and on a radiation hybrid panel to compare the obtained linkage and RH maps. Radiation hybrid maps were developed for chromosome BTA19 and BTA26 regions containing FASN and GPAM genes, respectively. The two genes and their associated microsatellite markers were located on the genetic or RH maps or on both. These microsatellite markers could be useful to study the QTL effect on fat synthesis in reference population.     

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.     

Physical assignment of six type I anchor loci to bovine chromosome 19 by fluorescence in situ hybridization

A bovine bacterial artificial chromosome (BAC) library was screened for the presence of eight type I anchor loci previously used within hybrid somatic cells and an interspecies hybrid backcross to construct a genome map of bovine chromosome 19 (BTA19). Six out of eight loci were identified in the BAC library ( NF1, CRYB1, CHRNB1, TP53, GH1 and P4HB ). The BACs were then used in single-colour fluorescence in situ hybridization (FISH) to assign these genes to BTA19 band locations. Gene order was determined by single-colour FISH, and was confirmed by dual-colour FISH to mitotic and meiotic chromosomes. The order, centromere- NF1-CRYB1-CHRNB1-TP53-GH1-P4HB , was in agreement with the order determined by linkage analyses. In addition, the order of CHRNB1 and TP53 , previously unresolved by linkage analyses, was established. These data provide high-resolution cytogenetic anchorage of the BTA19 genome map from chromosome bands 14–22.     

Mapping of a milk production quantitative trait locus to a 420-kb region on bovine chromosome 6

A QTL affecting milk production traits was previously mapped to an interval of 7.5 cM on chromosome 6 in Norwegian dairy cattle. This article aimed to refine this position by increasing the map density in the region by a set of single-nucleotide polymorphisms and analyzing the data with a combined linkage and linkage disequilibrium approach. Through a series of single- and multitrait and single- and multipoint analyses, the QTL was positioned to an interval surrounded by the genes ABCG2 and LAP3. As no recombinations were detected in this interval, physical mapping was required for further refining. By using radiation hybrid mapping as well as BAC clones, the bovine and human comparative maps in the region are resolved, and the QTL is mapped within a distance of 420 kb.     

High-resolution physical mapping and construction of a porcine contig spanning the intramuscular fat content QTL

We previously mapped a locus for porcine intramuscular fat content (IMF) by linkage analysis to a 17.1-cM chromosome interval on Sus scrofa chromosome 7 (SSC7) flanked by microsatellite markers SW1083 and SW581. In this study, we identified 34 microsatellite markers and 14 STSs from the 17.1-cM IMF quantitative trait loci (QTL) region corresponding to HSA14q and aligned those loci using the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel. We then constructed a 5.2-Mb porcine bacterial artificial chromosome (BAC) contig of this region that was aligned using the RH panel. Finally, the IMF QTL was fine-mapped to 12.6 cM between SJ169 and MM70 at the 0.1% chromosome-wise significance level by genotyping the previously studied F2 resource family with 17 additional microsatellites. We also demonstrated that the SJ169-MM70 interval spans approximately 3.0 Mb and contains at least 12 genes: GALC, GPR65, KCNK10, SPATA7, PTPN21, FLJ11806, EML5, TTC8, CHES1, CAP2P1, CHORDC2P and C14orf143.     

Genetic linkage maps of two apricot cultivars ( Prunus armeniaca L.), and mapping of PPV (sharka) resistance

Genetic linkage maps for two apricot cultivars have been constructed using AFLP, RAPD, RFLP and SSR markers in 81 F1 individuals from the cross 'Goldrich' x 'Valenciano'. This family segregated for resistance to 'plum pox virus' (PPV), the most-important virus affecting Prunus species. Of the 160 RAPD arbitrary primers screened a total of 44 were selected. Sixty one polymorphic RAPD markers were scored on the mapping population: 30 heterozygous in 'Goldrich', 19 heterozygous in 'Valenciano', segregating 1:1, and 12 markers heterozygous in both parents, segregating 3:1. A total of 33 and 19 RAPD markers were mapped on the 'Goldrich' and 'Valenciano' maps respectively. Forteen primer combinations were used for AFLPs and all of them detected polymorphism. Ninety five markers segregating 1:1 were identified, of which 62 were heterozygous in the female parent 'Goldrich' and 33 in the male parent 'Valenciano'. Forty five markers were present in both parents and segregated 3:1. A total of 82 and 48 AFLP markers were mapped on the 'Goldrich' and 'Valenciano' maps. Twelve RFLPs probes were screened in the population, resulting in five loci segregating in the family, one locus heterozygous for 'Valenciano' and four heterozygous for both, segregating 1:2:1. Of the 45 SSRs screened 17 segregated in the mapping family, resulting in seven loci heterozygous for the maternal parent and ten heterozygous for both, segregating 1:2:1 or 1:1:1:1. A total of 16 and 13 co-dominant markers were mapped in the female and male parent maps respectively. A total of 132 markers were placed into eight linkage groups on the 'Goldrich' map, defining 511 cM of the total map-length. The average distance between adjacent markers was 3.9 cM. A total of 80 markers were placed into seven linkage groups on the 'Valenciano' map, defining 467.2 cM of the total map-distance, with an average interval of 5.8 cM between adjacent markers. Thirty six marker loci heterozygous in both parents revealed straightforward homologies between five linkage groups in both maps. The sharka resistance trait mapped on linkage group 2. The region containing sharka resistance is flanked by two co-dominant markers that will be used for targeted SSR development employing a recently constructed complete apricot BAC library. SSRs tightly linked to sharka resistance will facilitate MAS in breeding for resistance in apricot.     

Fine mapping of quantitative trait loci for mastitis resistance on bovine chromosome 11

Quantitative trait loci (QTL) affecting clinical mastitis (CM) and somatic cell score (SCS) were mapped on bovine chromosome 11. The mapping population consisted of 14 grandsire families belonging to three Nordic red cattle breeds: Finnish Ayrshire (FA), Swedish Red and White (SRB) and Danish Red. The families had previously been shown to segregate for udder health QTL. A total of 524 progeny tested bulls were included in the analysis. A linkage map including 33 microsatellite and five SNP markers was constructed. We performed combined linkage disequilibrium and linkage analysis (LDLA) using the whole data set. Further analyses were performed for FA and SRB separately to study the origin of the identified QTL/haplotype and to examine if it was common in both populations. Finally, different two-trait models were fitted. These postulated either a pleiotropic QTL affecting both traits; two linked QTL, each affecting one trait; or one QTL affecting a single trait. A QTL affecting CM was fine-mapped. In FA, a haplotype having a strong association with a high negative effect on mastitis resistance was identified. The mapping precision of an earlier detected SCS-QTL was not improved by the LDLA analysis because of lack of linkage disequilibrium between the markers used and the QTL in the region.     

A comparative map of chicken chromosome 24 and human chromosome 11

To improve the physical and comparative map of chicken chromosome 24 (GGA24; former linkage group E49C20W21) bacterial artificial chromosome (BAC) contigs were constructed around loci previously mapped on this chromosome by linkage analysis. The BAC clones were used for both sample sequencing and BAC end sequencing. Sequence tagged site (STS) markers derived from the BAC end sequences were used for chromosome walking. In total 191 BAC clones were isolated, covering almost 30% of GGA24, and 76 STS were developed (65 STS derived from BAC end sequences and 11 STS derived within genes). The partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases, and revealed matches to 19 genes, expressed sequence tags (ESTs) and genomic clones located on human chromosome 11q22-q24 and mouse chromosome 9. Furthermore, 11 chicken orthologues of human genes located on HSA11q22-q24 were directly mapped within BAC contigs of GGA24. These results provide a better alignment of GGA24 with the corresponding regions in human and mouse and identify several intrachromosomal rearrangements between chicken and mammals.     

Linkage of bovine erythrocyte antigen loci B, C, L, S, Z, R' and T' and the serum protein loci post-transferrin 2 (PTF 2), vitamin D binding protein (GC) and albumin (ALB) to DNA microsatellite markers

Seven bovine erythrocyte antigen loci and three serum protein loci were tentatively assigned to chromosomes or synteny groups by linkage analysis to previously assigned microsatellite DNA markers. The erythrocyte antigen locus EAB was mapped to synteny group U27; EAC to chromosome 18, synteny group U9; EAL to chromosome 3, synteny group U6; EAS to chromosome 21, synteny group U4; EAZ to chromosome 10, synteny group U5; EAR' to chromosome 16, synteny group U1; and EAT' to chromosome 19, synteny group U21. The vitamin D binding protein (GC) and albumin (ALB) loci were assigned to chromosome 6, synteny group U15 and post-transferrin 2 (PTF 2) to chromosome 19, synteny group U21.     

Linkage mapping of the ovine alpha-inhibin (INHA) beta(A)-inhibin/activin (INHBA) and beta(B)-inhibin/activin (INHBB) genes

A full-sib mapping population was typed for Taql Southern blot RFLPs at the ovine alpha-inhibin (INHA), beta(A)-inhibin/activin (INHBA), and beta(B)-inhibin/activin (INHBB) loci. The most likely positions were found by genetic linkage analysis. INHA was placed on ovine chromosome 2q, between microsatellite markers BM6444 and INRA135, while INHBB was located 39 cM proximal to INHA between markers BMS1126 and BMS2626. The INHBA gene, a comparative mapping anchor locus, mapped to chromosome 4, between microsatellites OARCP26 and BMS719. These data confirm and refine previous chromosomal assignments by in situ hybridization, provide comparative mapping information, and identify microsatellite markers suitable for QTL association studies with inhibin/activin genes in ruminants.     

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.     

A refined radiation hybrid map of the telomeric region of bovine chromosome 18q25-q26 compared with human chromosome 19q13

Genome-wide scans have mapped economically important quantitative trait loci (QTL) for mastitis susceptibility in dairy cattle at the telomeric end of bovine chromosome 18 (BTA18). In order to increase the density of markers in this chromosomal region and to improve breakpoint resolution in the human-bovine comparative map, this study describes the chromosomal assignment of seven newly developed gene-associated markers and five microsatellites and eight previously mapped sequence tagged site markers near these QTL. The orientation of KCNJ14, BAX, CD37, NKG7, LIM2, PRKCG, TNNT1, MGC2705, RPL28, EPN1, ZNF582, ZIM2, STK13, ZNF132 and SLC27A5 on the 3000-rad radiation hybrid (RH) map of BTA18 is homologous to the organization found on the corresponding 10 Mbp of human chromosome 19q (HSA19q). The resulting bovine RH map with a length of 20.9 cR spans over about 11 cM on the bovine linkage map. The location of KCNJ14 and SLC27A5 flanking the RH map on BTA18q25-26 has been confirmed by fluorescence in situ hybridization. The data of this refined human-bovine comparative map should improve selection of candidate genes for mastitis susceptibility in dairy cattle.     

Conformation polymorphisms and targeted marker development

Genetic markers were developed for three targeted bovine loci using both single-stranded and double-stranded DNA conformation polymorphisms. Eight of nine DNA fragments exhibited single-stranded conformation polymorphisms while only one of nine exhibited a double-strand conformation polymorphism. All but one of the polymorphic fragments exhibited two allelic forms, with the exception being a single-stranded conformation polymorphism with three alleles. Utility of conformation polymorphisms relative to microsatellite markers for linkage map development and quantitative trait loci (QTL) mapping was assessed by comparing frequency of heterozygote parents from reference and resource families. Heterozygosity was greater for microsatellite markers (P < 0.01) and reference family parents (P < 0.01), though the disparity between marker types tended to be less dramatic for the reference family parents (P < 0.09). These results suggest conformation polymorphisms will be a useful tool for targeted marker development in linkage map and comparative map development provided genetically diverse families are studied.     

Functional genes mapped on the chicken genome

Microsatellite polymorphisms are finding increasing use in genetics. In addition to the random isolation of microsatellite markers, such markers can also be developed from sequences already present in public domain databases. An advantage of public domain databases is that these microsatellites are known to be located within or close to identified functional genes. In this study the GenBank and EMBL databases were screened for microsatellite markers and primers were defined for amplification. Subsequently, these markers were tested on a panel of five different birds from layer and broiler stocks and on the international reference families: the East Lansing reference family and the Compton reference family. Of the 33 loci tested, 25 were polymorphic on the test panel and from these 25, 14 were polymorphic in one or both reference families. Twelve of the 14 loci that could be mapped fell into previously defined linkage groups. The other two markers were not linked. Because three of the loci had previously been mapped to specific chromosomes by in situ hybridization, linkage groups E6 and C3 could be assigned to chromosome 6, E5 and C17 to chromosome 4 and E21 to one of the microchromosomes.