Genomic organization of the mouse T-cell receptor β-chain gene family

We have combined three different methods, deletion mapping of T-cell lines, field-inversion gel electrophoresis, and the restriction mapping of a cosmid clone, to construct a physical map of the murine T-cell receptor β-chain gene family. We have mapped 19 variable (Vβ) gene segments and the two clusters of diversity (Dβ) and joining (Jβ) gene segments and constant (Cβ) genes. These members of the β-chain gene family span ~450 kilobases of DNA, excluding one potential gap in the DNA fragment alignments

A universal method for automated gene mapping

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost

Transposon variants and their effects on gene expression in arabidopsis

Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA–mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs, and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24-nt-long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA–targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants

A framework for gene mapping in wheat demonstrated using the Yr7 yellow rust resistance gene

We used three approaches to map the yellow rust resistance gene Yr7 and identify associated SNPs in wheat. First, we used a traditional QTL mapping approach using a double haploid (DH) population and mapped Yr7 to a low-recombination region of chromosome 2B. To fine map the QTL, we then used an association mapping panel. Both populations were SNP array genotyped allowing alignment of QTL and genome-wide association scans based on common segregating SNPs. Analysis of the association panel spanning the QTL interval, narrowed the interval down to a single haplotype block. Finally, we used mapping-by-sequencing of resistant and susceptible DH bulks to identify a candidate gene in the interval showing high homology to a previously suggested Yr7 candidate and to populate the Yr7 interval with a higher density of polymorphisms. We highlight the power of combining mapping-by-sequencing, delivering a complete list of gene-based segregating polymorphisms in the interval with the high recombination, low LD precision of the association mapping panel. Our mapping-by-sequencing methodology is applicable to any trait and our results validate the approach in wheat, where with a near complete reference genome sequence, we are able to define a small interval containing the causative gene

Molecular genetics of chicken egg quality

Faultless quality in eggs is important in all production steps, from chicken to packaging, transportation, storage, and finally to the consumer. The egg industry (specifically transportation and packing) is interested in robustness, the consumer in safety and taste, and the chicken itself in the reproductive performance of the egg. High quality is commercially profitable, and egg quality is currently one of the key traits in breeding goals. In conventional breeding schemes, the more traits that are included in a selection index, the slower the rate of genetic progress for all the traits will be. The unveiling of the genes underlying the traits, and subsequent utilization of this genomic information in practical breeding, would enhance the selection progress, especially with traits of low inheritance, genderconfined traits, or traits which are difficult to assess. In this study, two experimental mapping populations were used to identify quantitative trait loci (QTL) of egg quality traits. A whole genome scan was conducted in both populations with different sets of microsatellite markers. Phenotypic observations of albumen quality, internal inclusions, egg taint, egg shell quality traits, and production traits during the entire production period were collected. To study the presence of QTL, a multiple marker linear regression was used. Polymorphisms found in candidate genes were used as SNP (single nucleotide polymorphism) markers to refine the map position of QTL by linkage and association. Furthermore, independent commercial egg layer lines were utilized to confirm some of the associations. Albumen quality, the incidence of internal inclusions, and egg taint were first mapped with the whole genome scan and fine-mapped with subsequent analyses. In albumen quality, two distinct QTL areas were found on chromosome 2. Vimentin, a gene maintaining the mechanical integrity of the cells, was studied as a candidate gene. Neither sequencing nor subsequent analysis using SNP within the gene in the QTL analysis suggested that variation in this gene could explain the effect on albumen thinning. The same mapping approach was used to study the incidence of internal inclusions, specifically, blood and meat spots. Linkage analysis revealed one genome-wide significant region on chromosome Z. Fine-mapping exposed that the QTL overlapped with a tight junction protein gene ZO-2, and a microsatellite marker inside the gene. Sequencing of a fragment of the gene revealed several SNPs. Two novel SNPs were found to be located in a miRNA (gga-mir-1556) within the ZO-2. MicroRNA-SNP and an exonic synonymous SNP were genotyped in the populations and showed significant association to blood and meat spots. A good congruence between the experimental population and commercial breeds was achieved both in QTL locations and in association results. As a conclusion, ZO-2 and gga-mir-1556 remained candidates for having a role in susceptibility to blood and meat spot defects across populations. This is the first report of QTL affecting blood and meat spot frequency in chicken eggs, albeit the effect explained only 2 % of the phenotypic variance. Fishy taint is a disorder, which is a characteristic of brown layer lines. Marker-trait association analyses of pooled samples indicated that egg-taint and the FMO3 gene map to chicken chromosome 8 and that the variation found by sequencing in the chicken FMO3 gene was associated with the TMA content of the egg. The missense mutation in the FMO3 changes an evolutionary, highly conserved amino acid within the FMO-characteristic motif (FATGY). In conclusion, several QTL regions affecting egg quality traits were successfully detected. Some of the QTL findings, such as albumen quality, remained at the level of wide chromosomal regions. For some QTL, a putative causative gene was indicated: miRNA gga-mir-1556 and/or its host gene ZO-2 might have a role in susceptibility to blood and meat spot defects across populations. Nonetheless, fishy taint in chicken eggs was found to be caused with a substitution within a conserved motif of the FMO3 gene. This variation has been used in a breeding program to eliminate fishy-taint defects from commercial egg layer lines. Objective The objective of this thesis was to map loci affecting economically important egg quality traits in chickens and to increase knowledge of the molecular genetics of these complex traits. The aim was to find markers linked to the egg quality traits, and finally unravel the variation in the genes underlying the phenotypic variation of internal egg quality. QTL mapping methodology was used to identify chromosomal regions affecting various production and egg quality traits (I, III, IV). Three internal egg quality traits were selected for fine-mapping (II, III, IV). Some of the results were verified in independent mapping populations and present-day commercial lines (III, IV). The ultimate objective was to find markers to be applied in commercial selection programs

A Novel Genome-Wide Association Study Approach Using Genotyping by Exome Sequencing Leads to the Identification of a Primary Open Angle Glaucoma Associated Inversion Disrupting ADAMTS17

Closed breeding populations in the dog in conjunction with advances in gene mapping and sequencing techniques facilitate mapping of autosomal recessive diseases and identification of novel disease-causing variants, often using unorthodox experimental designs. In our investigation we demonstrate successful mapping of the locus for primary open angle glaucoma in the Petit Basset Griffon Vendéen dog breed with 12 cases and 12 controls, using a novel genotyping by exome sequencing approach. The resulting genome-wide association signal was followed up by genome sequencing of an individual case, leading to the identification of an inversion with a breakpoint disrupting the ADAMTS17 gene. Genotyping of additional controls and expression analysis provide strong evidence that the inversion is disease causing. Evidence of cryptic splicing resulting in novel exon transcription as a consequence of the inversion in ADAMTS17 is identified through RNAseq experiments. This investigation demonstrates how a novel genotyping by exome sequencing approach can be used to map an autosomal recessive disorder in the dog, with the use of genome sequencing to facilitate identification of a disease-associated variant

Polymorphism identification and improved genome annotation of Brassica rapa through Deep RNA sequencing.

The mapping and functional analysis of quantitative traits in Brassica rapa can be greatly improved with the availability of physically positioned, gene-based genetic markers and accurate genome annotation. In this study, deep transcriptome RNA sequencing (RNA-Seq) of Brassica rapa was undertaken with two objectives: SNP detection and improved transcriptome annotation. We performed SNP detection on two varieties that are parents of a mapping population to aid in development of a marker system for this population and subsequent development of high-resolution genetic map. An improved Brassica rapa transcriptome was constructed to detect novel transcripts and to improve the current genome annotation. This is useful for accurate mRNA abundance and detection of expression QTL (eQTLs) in mapping populations. Deep RNA-Seq of two Brassica rapa genotypes-R500 (var. trilocularis, Yellow Sarson) and IMB211 (a rapid cycling variety)-using eight different tissues (root, internode, leaf, petiole, apical meristem, floral meristem, silique, and seedling) grown across three different environments (growth chamber, greenhouse and field) and under two different treatments (simulated sun and simulated shade) generated 2.3 billion high-quality Illumina reads. A total of 330,995 SNPs were identified in transcribed regions between the two genotypes with an average frequency of one SNP in every 200 bases. The deep RNA-Seq reassembled Brassica rapa transcriptome identified 44,239 protein-coding genes. Compared with current gene models of B. rapa, we detected 3537 novel transcripts, 23,754 gene models had structural modifications, and 3655 annotated proteins changed. Gaps in the current genome assembly of B. rapa are highlighted by our identification of 780 unmapped transcripts. All the SNPs, annotations, and predicted transcripts can be viewed at http://phytonetworks.ucdavis.edu/

Identification and Mapping of a Gene for Rice Slender Kernel Using Oryza Glumaepatula Introgression Lines

World demand for superior rice grain quality tends to increase. One of the criteria of appearance quality of rice grain is grain shape. Rice consumers exhibit wide preferences for grain shape, but most Indonesian rice consumers prefer long and slender grain. The objectives of this study were to identify and map a gene for rice slender kernel trait using Oryza glumaepatula introgression lines with O. sativa cv. Taichung 65 genetic background. A segregation analysis of BC4F2 population derived from backcrosses of a donor parent O. glumaepatula into a recurrent parent Taichung 65 showed that the slender kernel was controlled by a single recessive gene. This new identified gene was designated as sk1 (slender kernel 1). Moreover, based on the RFLP analyses using 14 RFLP markers located on chromosomes 2, 8, 9, and 10 in which the O. glumaepatula chromosomal segments were retained in BC4F2 population, the sk1 was located between RFLP markers C679 and C560 on the long arm of chromosome 2, with map distances of 2.8 and 1.5 cM, respectively. The wild rice O. glumaepatula carried a recessive allele for slender kernel. This allele may be useful in breeding of rice with slender kernel types. In addition, the development of plant materials and RFLP map associated with slender kernel in this study is the preliminary works in the effort to isolate this important grain shape gene