Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1–8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species.     

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     

Sequence similarity based identification of abiotic stress responsive genes in chickpea

Chickpea (Cicer arietinum L.) is an important food legume crop, particularly for the arid regions including Indian subcontinent. Considering the detrimental effect of drought, temperature and salt stress on crop yield, efforts have been initiated in the direction of developing improved varieties and designing alternate strategies to sustain chickpea production in adverse environmental conditions. Identification of genes that confer abiotic stress tolerance in plants remains a challenge in contemporary plant breeding. The present study focused on the identification of abiotic stress responsive genes in chickpea based on sequence similarity approach exploiting known abiotic stress responsive genes from model crops or other plant species. Ten abiotic stress responsive genes identified in other plants were partially amplified from eight chickpea genotypes and their presence in chickpea was confirmed after sequencing the PCR products. These genes have been functionally validated and reported to play significant role in stress response in model plants like Arabidopsis, rice and other legume crops. Chickpea EST sequences available at NCBI EST database were used for the identification of abiotic stress responsive genes. A total of 8,536 unique coding long sequences were used for identification of chickpea homologues of these abiotic stress responsive genes by sequence similarity search (BLASTN and BLASTX). These genes can be further explored towards achieving the goal of developing superior chickpea varieties providing improved yields under stress conditions using modern molecular breeding approaches.     

Development of SNP markers and haplotype analysis of the candidate gene for rhg1, which confers resistance to soybean cyst nematode in soybean

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is one of the most destructive pests in the cultivation of soybean (Glycine max (L.) Merr.) worldwide. Markers based on the SCN resistance gene will enable efficient marker-assisted selection (MAS). We sequenced the candidate gene rhg1 in six resistant and two susceptible soybean genotypes and identified 37 SNPs (single nucleotide polymorphisms) among the sequences, of which 11 were in the coding region. Seven of these 11 SNPs led to changes in the amino acid sequence of the gene. The amino acid sequence we obtained differs from the previously published one by a stretch of 26–27 amino acids. Six codominant allele-specific SNP markers based on agarose gel detection were developed and tested in 70 genotypes, among which occurred only nine different haplotypes. Two neutrality tests (Tajima’s D and Fu and Li’s F) were significant for the six SNP loci in the 70 genotypes, which is consistent with intensive directional selection. A strong LD pattern was detected among five SNPs except 2868T > C. Two SNPs (689C > A and 757C > T) formed one haplotype (689C-757C) that was perfectly associated with SCN resistance. The new allele-specific PCR markers located in the alleged sequence of the rhg1 candidate gene, combined with the microsatellite marker BACR-Satt309, will significantly improve the efficiency of MAS during the development of SCN-resistant cultivars.     

Identification and annotation of abiotic stress responsive candidate genes in peanut ESTs

Peanut (Arachis hypogaea L.) ranks fifth among the world oil crops and is widely grown in India and neighbouring countries. Due to its large and unknown genome size, studies on genomics and genetic modification of peanut are still scanty as compared to other model crops like Arabidopsis, rice, cotton and soybean. Because of its favourable cultivation in semi-arid regions, study on abiotic stress responsive genes and its regulation in peanut is very much important. Therefore, we aim to identify and annotate the abiotic stress responsive candidate genes in peanut ESTs. Expression data of drought stress responsive corresponding genes and EST sequences were screened from dot blot experiments shown as heat maps and supplementary tables, respectively as reported by Govind et al. (2009). Some of the screened genes having no information about their ESTs in above mentioned supplementary tables were retrieved from NCBI. A phylogenetic analysis was performed to find a group of utmost similar ESTs for each selected gene. Individual EST of the said group were further searched in peanut ESTs (1,78,490 whole EST sequences) using stand alone BLAST. For the prediction as well as annotation of abiotic stress responsive selected genes, various tools (like Vec-Screen, Repeat Masker, EST-Trimmer, DNA Baser, WISE2 and I-TASSER) were used. Here we report the predicted result of Contigs, domain as well as 3D structure for HSP 17.3KDa protein, DnaJ protein and Type 2 Metallothionein protein.     

A chicken linkage map based on microsatellite markers genotyped on a Japanese Large Game and White Leghorn cross

A detailed linkage map is necessary for efficient detection of quantitative trait loci (QTL) in chicken resource populations. In this study, microsatellite markers isolated from a (CA)n-enriched library (designated as ABR Markers) were mapped using a population developed from a cross between Japanese Game and White Leghorn chickens. In total, 296 markers including 193 ABR, 43 MCW, 31 ADL, 22 LEI, 3 HUJ, 2 GCT, 1 UMA and 1 ROS were mapped by linkage to chicken chromosomes 1-14, 17-21, 23, 24, 26-28 and Z. In addition, five markers were assigned to the map based on the chicken draft genomic sequence, bringing the total number of markers on the map to 301. The resulting linkage map will contribute to QTL mapping in chicken.     

The linkage maps of Dendrobium species based on RAPD and SRAP markers

Dendrobium plants are used commonly as tonic herbs and health food in many Asian countries,especially in China.Here we report the genetic map construction of two Dendrobium species with a double pseudo-testcross strategy using random amplified polymorphic DNA(RAPD) and sequence-related amplified polymorphism(SRAP) markers.A F1 mapping population of 90 individuals was developed from a cross between D.officinale and D.hercoglossum.A total of 307 markers,including 209 RAPD and 98 SRAP,were identified and used ...     

Detection of loci controlling seed dormancy on group 4 chromosomes of wheat and comparative mapping with rice and barley genomes

Three quantitative trait loci (QTLs) controlling seed dormancy were detected on group 4 chromosomes of wheat (Triticum aestivum L.) using 119 doubled haploid lines (DHLs) derived from a cross between AC Domain and Haruyutaka. A major QTL, designated QPhs.ocs-4A.1, was identified within the marker interval between Xcdo795 and Xpsr115 in the proximal region of the long arm of chromosome 4A. Two minor QTLs, QPhs.ocs-4B.2 on 4B and QPhs.ocs-4D.2 on 4D, were flanked by common markers, Xbcd1431.1 and Xbcd1431.2 in the terminal region of the long arms, suggesting a homoeologous relationship. These three QTLs explained more than 80% of the total phenotypic variance in seed dormancy of DHLs grown in the field and under glasshouse conditions. The AC Domain alleles at the three QTLs contributed to increasing seed dormancy. Comparative maps across wheat, barley and rice demonstrated the possibility of a homoeologous relationship between QPhs.ocs-4A.1 and the barley gene SD4, while no significant effects of the chromosome regions of wheat and barley orthologous to rice chromosome 3 region carrying a major seed dormancy QTL were detected. Received: 5 June 2000 / Accepted: 31 August 2000     

Genetic linkage map of the guppy,Poecilia reticulata,and quantitative trait loci analysis of male size and colour variation

We report construction of a genetic linkage map of the guppy genome using 790 single nucleotide polymorphism markers, integrated from six mapping crosses. The markers define 23 linkage groups (LGs), corresponding to the known haploid number of guppy chromosomes. The map, which spans a genetic length of 899 cM, includes 276 markers linked to expressed genes (expressed sequence tag), which have been used to derive broad syntenic relationships of guppy LGs with medaka chromosomes. This combined linkage map should facilitate the advancement of genetic studies for a wide variety of complex adaptive phenotypes relevant to natural and sexual selection in this species. We have used the linkage data to predict quantitative trait loci for a set of variable male traits including size and colour pattern. Contributing loci map to the sex LG for many of these traits.     

Construction of a genetic linkage map of Japanese quail (Coturnix japonica) based on AFLP and microsatellite markers

The Japanese quail (Coturnix japonica) is a notably valuable egg and meat producer but has also been used as a laboratory animal. In the present study, we constructed a Japanese quail linkage map with 1735 polymorphic amplified fragment length polymorphisms markers, and nine chicken microsatellite (MS) markers, as well as sex and phenotypes of two genetic diseases; a muscular disorder (LWC) and neurofilament-deficient mutant (Quv). Linkage analysis revealed 578 independent loci. The resulting linkage map contained 44 multipoint linkage groups covering 2597.8 cM and an additional 218.2 cM was contained in 21 two-point linkage groups. The total map was 2816 cM in length with an average marker interval of 5.5 cM. The Quv locus was located on linkage group 5, but linkage was not found between the LWC locus and any of the markers. Comparative mapping with chicken using orthologous markers revealed chromosomal assignments of the quail linkage group 1 to chicken chromosome 2 (GGA2), 5 to GGA22, 2 to GGA5, 8 to GGA7, 27 to GGA11, 29 to GGA1 and 45 to GGA4.     

SSR based linkage and mapping analysis of C,a yellow cocoon gene in the silkworm,Bombyx moil

The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y （Yellow haemolymph）, 1 （Yellow inhibitor） and C （ Outer-layer yellow cocoon）, which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 （BC1） progeny were used for linkage analysis and mapping of the C gene using silkworm strains C 108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat （SSR） markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross （BC1F） showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross （BC1M）, we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.     

SSR based linkage and mapping analysis of C,a yellow cocoon gene in the silkworm,Bombyx mori

The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y (Yellow haemolymph), I (Yellow inhibitor) and C (Outer‐layer yellow cocoon), which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F₁ (BC₁) progeny were used for linkage analysis and mapping of the C gene using silkworm strains C108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat (SSR) markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross (BC₁ F) showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross (BC₁ M), we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.     

A comparative map of macrochromosomes between chicken and Japanese quail based on orthologous genes

In order to develop a comparative map between chicken and quail, we identified orthologous gene markers based on chicken genomic sequences and localized them on the Japanese quail Kobe-NIBS linkage map, which had previously been constructed with amplified fragment length polymorphisms. After sequencing the intronic regions of 168 genes located on chicken chromosomes 1-8, polymorphisms among Kobe-NIBS quail family parents were detected in 51 genes. These orthologous markers were mapped on eight Japanese quail linkage groups (JQG), and they allowed the comparison of JQG to chicken macrochromosomes. The locations of the genes and their orders were quite similar between the two species except within a previously reported inversion on quail chromosome 2. Therefore, we propose that the respective quail linkage groups are macrochromosomes and designated as quail chromosomes CJA 1-8.     

SNP analysis of AMY2 and CTSL genes in Litopenaeus vannamei and Penaeus monodon shrimp

Genetic studies in shrimp have focused on disease, with production traits such as growth left unexamined. Two shrimp species, Litopenaeus vannamei and Penaeus monodon, which represent the majority of US shrimp imports, were selected for single nucleotide polymorphism (SNP) discovery in alpha-amylase (AMY2) and cathepsin-l (CTSL), both candidate genes for growth. In L. vannamei, four SNPs were found in AMY2 and one SNP was found in CTSL. In P. monodon, one SNP was identified in CTSL. The CTSL gene was mapped to linkage group 28 of P. monodon using the female map developed with the Australian P. monodon mapping population. Association analyses for the AMY2 and CTSL genes with body weight (BW) were performed in two L. vannamei populations. While neither gene was found to be significantly associated with BW in these populations, there was a trend in one population towards higher BW for allele G of CTSL SNP C681G.