Exploitation of pepper EST–SSRs and an SSR-based linkage map

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F₂ individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.Gibum Yi and Je Min Lee equally contributed to this work.     

Constructing a linkage–linkage disequilibrium map using dominant-segregating markers

The relationship between linkage disequilibrium (LD) and recombination fraction can be used to infer the pattern of genetic variation and evolutionary process in humans and other systems. We described a computational framework to construct a linkage–LD map from commonly used biallelic, single-nucleotide polymorphism (SNP) markers for outcrossing plants by which the decline of LD is visualized with genetic distance. The framework was derived from an open-pollinated (OP) design composed of plants randomly sampled from a natural population and seeds from each sampled plant, enabling simultaneous estimation of the LD in the natural population and recombination fraction due to allelic co-segregation during meiosis. We modified the framework to infer evolutionary pasts of natural populations using those marker types that are segregating in a dominant manner, given their role in creating and maintaining population genetic diversity. A sophisticated two-level EM algorithm was implemented to estimate and retrieve the missing information of segregation characterized by dominant-segregating markers such as single methylation polymorphisms. The model was applied to study the relationship between linkage and LD for a non-model outcrossing species, a gymnosperm species, Torreya grandis, naturally distributed in mountains of the southeastern China. The linkage–LD map constructed from various types of molecular markers opens a powerful gateway for studying the history of plant evolution.     

Development of Capsicum EST–SSR markers for species identification and in silico mapping onto the tomato genome sequence

Capsicum spp. are widely cultivated for use as vegetables and spices. The Kihara Institute for Biological Research, Yokohama City University, Japan, has stocks of approximately 800 lines of Capsicum spp. collected from various regions of Central and South America, the regions of origin for Capsicum spp. In this study, 5,751 primer pairs for simple sequence repeat markers, based on 118,060 publicly available sequences of expressed sequence tags of Capsicum annuum, were designed and subjected to a similarity search against the genomic sequence of tomato, a model Solanaceae species. Nucleotide sequences spanning 2,245 C. annuum markers were successfully mapped onto the tomato genome, and 96 of these, which spanned the entire tomato genome, were selected for further analysis. In genotyping analysis, 60 out of the 77 markers that produced specific DNA amplicons showed polymorphism among the Capsicum lines examined. On the basis of the resulting data, the 192 tested lines were grouped into five main clusters. The additional sequencing analysis of the plastid genes, matK and rbcL, divided the resources into three groups. As a result, 19 marker loci exhibited genotypes specific to species and cluster, suggesting that the DNA markers are useful for species identification. Information on the DNA markers will contribute to Capsicum genetics, genomics, and breeding.     

A linkage map of 243 DNA markers in an intercross of Göttingen miniature and Meishan pigs

A resource family of pigs has been constructed by using a boar of Göttingen miniature pig and two sows of Meishan pig as parents. In the construction of the family, two F1 males and 18 F1 females were intercrossed to generate 143 F2 offspring. The members of the family were genotyped using 243 genetic markers including 26 markers developed in our laboratory in order to generate a linkage map of markers for use in detecting quantitative trait loci (QTLs) in the family. The markers consisted of 237 microsatellites, five PRE-1 markers, and one RFLP marker. The linkage map was revealed to cover all 18 autosomes and the X chromosome; and the total length of the sex-averaged linkage map was calculated to be 2561 ·9 c m . Four out of the 26 markers developed in our laboratory ex-ended the current linkage map at the termini of chromosomes 1p, 5p, 11p, and Xq. The linkage maps of all the chromosomes except for chromosome 1 were found to be longer in females than in males. Concerning chromosome 1, the length of the linkage map showed no difference between females and males, which was attributed to low recombination rates between markers localized in the centromeric region in females. The average ratio of female-to-male recombination was calculated to be 1 ·55.     

Mining and comparative survey of EST–SSR markers among members of Euphorbiaceae family

Euphorbiaceae represents flowering plants family of tropical and sub-tropical region rich in secondary metabolites of economic importance. To understand and assess the genetic makeup among the members, this study was undertaken to characterize and compare SSR markers from publicly available ESTs and GSSs of nine selected species of the family. Mining of SSRs was performed by MISA, primer designing by Primer3, while functional annotation, gene ontology (GO) and enrichment analysis were performed by Blast2GO. A total 12,878 number of SSRs were detected from 101,701 number of EST sequences. SSR density ranged from 1 SSR/3.22 kb to 1 SSR/15.65 kb. A total of 1873 primer pairs were designed for the annotated SSR-Contigs. About 77.07% SSR–ESTs could be assigned a significant match to the protein database. 3037 unique SSR–FDM were assigned and IPR003657 (WRKY Domain) was found to be the most dominant FDM among the members. 1810 unique GO terms obtained were further subjected to enrichment analysis to obtain 513 statistically significant GO terms mapped to the SSR containing ESTs. Most frequent enriched GO terms were, GO:0003824 for molecular function, GO:0006350 for biological process and GO:0005886 for cellular component, justifying the richness of defensive secondary metabolites and phytomedicine within the family. The results from this study provides tangible insight to genetic make-up and distribution of SSRs. Functional annotation corresponded many genes of unknown functions which may be considered as novel genes or genes responsible for stress specific secondary metabolites. Further studies are required to understand stress specific genes accountable for leveraging the synthesis of secondary metabolites.     

Construction of a SNP and SSR linkage map in autotetraploid blueberry using genotyping by sequencing

The construction of the first genetic map in autotetraploid blueberry has been made possible by the development of new SNP markers developed using genotyping by sequencing in a mapping population created from a cross between two key highbush blueberry cultivars, Draper × Jewel (Vaccinium corymbosum). The novel SNP markers were supplemented with existing SSR markers to enable the alignment of parental maps.  In total, 1794 single nucleotide polymorphic (SNP) markers and 233 simple sequence repeat (SSR) markers exhibited segregation patterns consistent with a random chromosomal segregation model for meiosis in an autotetraploid. Of these, 700 SNPs and 85 SSRs were utilized for construction of the ‘Draper’ genetic map, and 450 SNPs and 86 SSRs for the ‘Jewel’ map.  The ‘Draper’ map comprises 12  linkage groups (LG), associated with the haploid chromosome number for blueberry, and totals 1621 cM while the ‘Jewel’ map comprises 20 linkage groups totalling 1610 cM. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents.     

Saccharum spontaneum L. ‘SES 208’ genetic linkage map combining RFLP- and PCR-based markers

A 527 marker linkage map ofSaccharum spontaneum L. SES 208 (2n = 64) was established by analyzing 208 single-dose (SD) arbitrarily primed PCR polymorphisms, 234 SD RFLPs, 41 double-dose (DD) and one triple-dose (TD) polymorphisms. A map hypothesis constructed using these markers (minimum LOD = 4.00, = 0.25 M) had 64 linkage groups with 13 SD, nine DD, and one TD markers unlinked. Eight chromosome homology groups were identified by using DD fragments as well as SD RFLPs that identified more than one linkage group. Linkages in repulsion phase were absent from the map, as found in two previous genetic studies of this species. Together, these data demonstrate that SES 208 displayed polysomic segregation, a genetic behavior typical of autopolyploid species. As with previous studies, it was concluded that SES 208 behaved like an auto-octoploid, which was also in agreement with the number of homology groups observed. A ² was used to test whether the 527 markers were randomly distributed throughout the genome: both arbitrarily primed PCR markers and RFLPs had a distribution that was statistically indistinguishable from random. The integrated arbitrarily primed PCR-RFLP map had a predicted genomic coverage of 93% (considering only 442 SD polymorphisms) and an average interval between markers of 6 cM. SD markers were used to estimate the genome size of SES 208 at ca. 33 00 cM.     

High-density genetic map of durum wheat × wild emmer wheat based on SSR and DArT markers

A genetic linkage map of tetraploid wheat was constructed based on a cross between durum wheat [Triticum turgidum ssp. durum (Desf.) MacKey] cultivar Langdon and wild emmer wheat [T. turgidum ssp. dicoccoides (K?rn.) Thell.] accession G18-16. One hundred and fifty-two single-seed descent derived F(6) recombinant inbred lines (RILs) were analyzed with a total of 690 loci, including 197 microsatellite and 493 DArT markers. Linkage analysis defined 14 linkage groups. Most markers were mapped to the B-genome (60%), with an average of 57 markers per chromosome and the remaining 40% mapped to the A-genome, with an average of 39 markers per chromosome. To construct a stabilized (skeleton) map, markers interfering with map stability were removed. The skeleton map consisted of 307 markers with a total length of 2,317 cM and average distance of 7.5 cM between adjacent markers. The length of individual chromosomes ranged between 112 cM for chromosome 4B to 217 cM for chromosome 3B. A fraction (30.1%) of the markers deviated significantly from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on chromosomes 1A, 1BL, 2BS, 3B, and 4B. DArT markers showed high proportion of clustering, which may be indicative of gene-rich regions. Three hundred and fifty-two new DArT markers were mapped for the first time on the current map. This map provides a useful groundwork for further genetic analyses of important quantitative traits, positional cloning, and marker-assisted selection, as well as for genome comparative genomics and genome organization studies in wheat and other cereals.     

An interspecific (Capsicum annuum ×C. chinese) F2 linkage map in pepper using RFLP and AFLP markers

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F₂ population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206–60.3 cM) and 5 small (32.6–10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones, and these markers were evenly distributed across the genome. By using 30 primer combinations, we were able to generate 444 AFLP markers in the F₂ population. The majority of the AFLP markers clustered in each linkage group, although PstI/MseI markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. Genes for the biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper. Received: 20 October 1999 / Accepted: 3 July 2000     

Development and validation of an HPLC–FLD method for milbemectin quantification in dog plasma

Milbemectin is a widely used veterinary antiparasitic agent. A high-performance liquid chromatography with fluorescent detection (HPLC–FLD) method is described for the determination of milbemectin in dog plasma. The derivative procedure included mixing 1-methylimizole [MI, MI-ACN (1:1, v/v), 100 μL], trifluoroacetic anhydride [TFAA, TFAA-ACN (1:2, v/v), 150 μL] with a subsequent incubation for 3 s at the room temperature to obtain a fluorescent derivative, which is reproducible in different blood samples and the derivatives proved to be stable for at least 80 h at room temperature. HPLC method was developed on C18 column with FLD detection at an excitation wavelength of 365 nm and emission wavelength of 475 nm, with the mobile phase consisting of methanol and water in the ratio of 98:2 (v/v). The assay lower limit of quantification was 1 ng/mL. The calibration curve was linear over concentration range of 1–200 ng/mL. The intra- and inter-day accuracy was >94% and precision expressed as % coefficient of variation was <5%. This method is specific, simple, accurate, precise and easily adaptable to measure milbemycin in blood of other animals.     

Genetical and physical assignments of equine microsatellites—first integration of anchored markers in horse genome mapping

Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map. Received: 24 September 1996 / Accepted: 1 December 1996     

A population ‘consensus’, partial linkage map of Picea abies Karst. based on RAPD markers

 We built a “consensus” partial linkage map based on RAPD markers using 48 sibships of eight megagametophytes each from a natural population of Norway spruce. A RAPD linkage map for a single individual from the same population had previously been constructed. Using 30 random decamers that had yielded 83 RAPD markers in the single-tree map, eight megagametophytes for each of the 48 sibships were screened. The linkage relationship among markers was estimated considering each family of eight megagametophytes as a progeny of a phase-unknown backcross mating between a heterozygous mother and a fictitious ‘recessive’ father. Markers were assigned to windows using LOD=2.0 and θ=0.4 as thresholds, and ordered using a criterion of interval support ≥2.0. For eight “windows” of recombination selected on the single-tree map, we investigated the consistency of marker order in the two maps. We adopted restrictive criteria for rejecting co-linearity between the two locus orders. For each window we imposed the most likely locus order obtained from one data set to the other (and vice versa), obtaining two symmetrical log-likelihood differences. We considered the hypothesis of co-linearity rejected when both symmetrical differences were significant (ΔLOD>3.0). By bootstrapping a subset of markers for each window (highly informative, ‘framework’ loci chosen on the previous single-tree map using a matrix correlation method) the sampling variability of the single-tree and population maps was estimated. As expected the population map was affected by a larger variability than the single-tree map. Heterogeneity in pairwise recombination fractions among groups of sibship revealed a (possible) alternative genomic arrangement detected within a single recombination window. Received : 4 January 1997 / Accepted : 24 January 1997     

A high-density genetic map of Sorghum bicolor (L.) Moench based on 2926 AFLP®, RFLP and SSR markers

Using AFLP technology and a recombinant inbred line population derived from the sorghum cross of BTx623 × IS3620C, a high-density genetic map of the sorghum genome was constructed. The 1713 cM map encompassed 2926 loci distributed on ten linkage groups; 2454 of those loci are AFLP products generated from either the EcoRI/MseI or PstI/MseI enzyme combinations. Among the non-AFLP markers, 136 are SSRs previously mapped in sorghum, and 203 are cDNA and genomic clones from rice, barley, oat, and maize. This latter group of markers has been mapped in various grass species and, as such, can serve as reference markers in comparative mapping. Of the nearly 3000 markers mapped, 692 comprised a LOD 3.0 framework map on which the remaining markers were placed with lower resolution (LOD <3.0). By comparing the map positions of the common grass markers in all sorghum maps reported to date, it was determined that these reference markers were essentially collinear in all published maps. Some clustering of the EcoRI/MseI AFLP markers was observed, possibly in centromeric regions. In general, however, the AFLP markers filled most of the gaps left by the RFLP/SSR markers demonstrating that AFLP technology is effective in providing excellent genome coverage. A web site, http://SorghumGenome.tamu.edu, has been created to provide all the necessary information to facilitate the use of this map and the 2590 PCR-based markers. Finally, we discuss how the information contained in this map is being integrated into a sorghum physical map for map-based gene isolation, comparative genome analysis, and as a source of sequence-ready clones for genome sequencing projects.     

Construction of a saturated linkage map for Prunus using an almond×peach F2 progeny

 A map with 246 markers (11 isozymes and 235 RFLPs) was constructed using an interspecific F₂ population between almond (cv Texas) and peach (cv Earlygold). RFLPs were obtained using 213 probes from the genomic and cDNA libraries of different species (almond, peach, P. ferganensis, cherry, plum and apple), including 16 almond probes which correspond to known genes. All markers were distributed in eight linkage groups, the same as the basic chromosome number of the genus, covering a total distance of 491 cM. The average map density was 2.0 cM/marker and only four gaps of 10 cM or more were found; the two largest gaps were 12cM each. This map was compared with one constructed previously with an intraspecific almond population sharing 67 anchor loci. Locus order was nearly identical and distances were not significantly different. A large proportion of the mapped loci (46%) had skewed segregations; in approximately half of them, the distortion was due to an excess of heterozygotes. One of the distorted regions could be associated with the position of the self-incompatibility gene of almond. Received: 6 November 1997 / Accepted: 26 May 1998     

Estimation and test for linkage between markers: a comparison of lod score and χ 2 test in a linkage study of maritime pine (Pinus pinaster Ait.)

The first step in the construction of a linkage map involves the estimation and test for linkage between all possible pairs of markers. The lod score method is used in many linkage studies for the latter purpose. In contrast with classical statistical tests, this method does not rely on the choice of a first-type error level. We thus provide a comparison between the lod score and a ² test on linkage data from a gymnosperm, the maritime pine. The lod score appears to be a very conservative test with the usual thresholds. Its severity depends on the type of data used.     

An expanded genetic linkage map of an intervarietal Agaricus bisporus var. bisporus × A. bisporus var. burnettii hybrid based on AFLP,SSR and CAPS markers sheds light on the recombination behaviour of the species

A genetic linkage map for the edible basidiomycete Agaricus bisporus was constructed from 118 haploid homokaryons derived from an intervarietal A. bisporus var. bisporus × A. bisporus var. burnettii hybrid. Two hundred and thirty-one AFLP, 21 SSR, 68 CAPS markers together with the MAT, BSN, PPC1 loci and one allozyme locus (ADH) were evenly spread over 13 linkage groups corresponding to the chromosomes of A. bisporus. The map covers 1156 cM, with an average marker spacing of 3.9 cM and encompasses nearly the whole genome. The average number of crossovers per chromosome per individual is 0.86. Normal recombination over the entire genome occurs in the heterothallic variety, burnettii, contrary to the homothallic variety, bisporus, which showed adaptive genome-wide suppressed recombination. This first comprehensive genetic linkage map for A. bisporus provides foundations for quantitative trait analyses and breeding programme monitoring, as well as genome organisation studies.     

Development of STS markers for Verticillium wilt resistance in cotton based on RGA–AFLP analysis

Verticillium wilt (VW) is one of the most destructive diseases of cotton that decreases yield and quality. Identification of resistance gene analogs (RGAs) can provide potential candidate gene markers for marker-assisted selection (MAS) for VW resistance and cloning of VW resistance genes. The objective of this study was to convert RGA-targeted RGA–AFLP (amplified fragment length polymorphism) markers into sequence-tagged site (STS) markers. A total of 54 RGA–AFLP markers, including 28 from a backcross inbred line (BIL) population and 26 from a recombinant inbred line (RIL) population, were cloned and sequenced. Of the resulted 86 unique sequences, the majority were found to be homologous to genes, some of which showed similarity to genes encoding resistance-related products. A total of 163 STS primer pairs were designed and screened in the BIL population, 72 of which were also screened in the RIL population, resulting in 21 polymorphic STS markers in the BIL population and seven in the RIL population. Twelve STS markers were mapped onto eight chromosomes or linkage groups in the BIL population, six of which were mapped on the same chromosomes with their original RGA–AFLP markers including two STS markers on c4 flanking two VW resistance quantitative trait loci. These STS markers will be useful in MAS in breeding cotton for VW resistance. This study represents the first attempt to successfully convert RGA–AFLP markers into STS for mapping resistance genes in cotton.     

Development and characterization of an oat TILLING-population and identification of mutations in lignin and β-glucan biosynthesis genes

Background  

Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved β-glucan-, antioxidant- and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda.