A functional genomics approach to evaluate candidate genes located in a QTL interval for milk production traits on BTA6

The potential genetic and economic advantage of marker-assisted selection for enhanced production in dairy cattle has provided an impetus to conduct numerous genome scans in order to identify associations between DNA markers and future productive potential. One area of focus has been a quantitative trait locus on bovine chromosome 6 (BTA6) found to be associated with milk yield, milk protein and fat percentage, which has been subsequently fine-mapped to six positional candidate genes. Subsequent investigations have yet to resolve which of the potential positional candidate genes is responsible for the observed associations with productive performance. In this study, we analysed candidate gene expression and the effects of gene knockdown on expression of β- and κ-casein mRNA in a small interfering RNA transfected bovine in vitro mammosphere model. From our expression studies in vivo , we observed that four of the six candidates ( ABCG2 , SPP1 , PKD2 and LAP3 ) exhibited differential expression in bovine mammary tissue over the lactation cycle, but in vitro functional studies indicate that inhibition of only one gene, SPP1 , had a significant impact on milk protein gene expression. These data suggest that the gene product of SPP1 (also known as osteopontin) has a significant role in the modulation of milk protein gene expression. While these findings do not exclude other positional candidates from influencing lactation, they support the hypothesis that the gene product of SPP1 is a significant lactational regulatory molecule.     

Systems genetics approach reveals candidate genes for parasite resistance from quantitative trait loci studies in agricultural species

A systems genetics approach combining pathway analysis of quantitative trait loci (QTL) and gene expression information has provided strong evidence for common pathways associated with genetic resistance to internal parasites. Gene data, collected from published QTL regions in sheep, cattle, mice, rats and humans, and microarray data from sheep, were converted to human Entrez Gene IDs and compared to the KEGG pathway database. Selection of pathways from QTL data was based on a selection index that ensured that the selected pathways were in all species and the majority of the projects overall and within species. Pathways with either up- and down-regulated genes, primarily up-regulated genes or primarily down-regulated genes, were selected from gene expression data. After comparing the data sets independently, the pathways from each data set were compared and the common set of pathways and genes was identified. Comparisons within data sets identified 21 pathways from QTL data and 66 pathways from gene expression data. Both selected sets were enriched with pathways involved in immune functions, disease and cell responses to signals. The analysis identified 14 pathways that were common between QTL and gene expression data, and four directly associated with IFNγ or MHCII, with 31 common genes, including three MHCII genes. In conclusion, a systems genetics approach combining data from multiple QTL and gene expression projects led to the discovery of common pathways associated with genetic resistance to internal parasites. This systems genetics approach may prove significant for the discovery of candidate genes for many other multifactorial, economically important traits.     

Expression quantitative trait loci: present and future

The last few years have seen the development of large efforts for the analysis of genome function, especially in the context of genome variation. One of the most prominent directions has been the extensive set of studies on expression quantitative trait loci (eQTLs), namely, the discovery of genetic variants that explain variation in gene expression levels. Such studies have offered promise not just for the characterization of functional sequence variation but also for the understanding of basic processes of gene regulation and interpretation of genome-wide association studies. In this review, we discuss some of the key directions of eQTL research and its implications.     

Identification and reciprocal introgression of a QTL affecting body mass in mice

The aim of this study was to examine the effects of a QTL in different genetic backgrounds. A QTL affecting body mass on chromosome 6 was identified in an F₂ cross between two lines of mice that have been divergently selected for this trait. The effect of the QTL on mass increased between 6 and 10 weeks of age and was not sex-specific. Body composition analysis showed effects on fat-free dry body mass and fat mass. To examine the effect of this QTL in different genetic backgrounds, the high body mass sixth chromosome was introgressed into the low body mass genetic background and vice versa by repeated marker-assisted backcrossing. After three generations of backcrossing, new F₂ populations were established within each of the introgression lines by crossing individuals that were heterozygous across the sixth chromosome. The estimated additive effect of the QTL on 10-week body mass was similar in both genetic backgrounds and in the original F₂ population (i.e., ~0.4 phenotypic standard deviations); no evidence of epistatic interaction with the genetic background was found. The 95% confidence interval for the location of the QTL was refined to a region of approximately 7 cM between D6Mit268 and D6Mit123.     

QTL and QTL x environment effects on agronomic and nitrogen acquisition traits in rice

Agricultural environments deteriorate due to excess nitrogen application.Breeding for low nitrogen responsive genotypes can reduce soil nitrogen input.Rice genotypes respond variably to soil available nitrogen.The present study attempted quantification of genotype x nitrogen level interaction and mapping of quantitative trait loci (QTLs) associated with nitrogen use efficiency (NUE) and other associated agronomic traits.Twelve parameters were observed across a set of 82 double haploid (DH) lines derived from IR64/Azucena.Three nitrogen regimes namely,native (0 kg/ha; no nitrogen applied),optimum (100 kg/ha) and high (200 kg/ha) replicated thrice were the environments.The parents and DH lines were significantly varying for all traits under different nitrogen regimes.All traits except plant height recorded significant genotype x environment interaction.Individual plant yield was positively correlated with nitrogen use efficiency and nitrogen uptake.Sixteen QTLs were detected by composite interval mapping.Eleven QTLs showed significant QTL x environment interactions.On chromosome 3,seven QTLs were detected associated with nitrogen use,plant yield and associated traits.A QTL region between markers RZ678,RZ574 and RZ284 was associated with nitrogen use and yield.This chromosomal region was enriched with expressed gene sequences of known key nitrogen assimilation genes.     

植物抗病遗传育种中的QTL定位和克隆(综述)

对植物抗病遗传育种中QTL定位与克隆研究进行综述。主要阐述了数量抗性的遗传学基础、作物抗病性QTL的定位作图、QTL作图的可靠性及应对措施、QTLs候选基因的证实和定位克隆等,并对植物抗病遗传育种未来的研究方向予以讨论。     

Identification of candidate genes and mutations in QTL regions for immune responses in chicken

There are two categories of immune responses – innate and adaptive immunity – both having polygenic backgrounds and a significant environmental component. In our study, adaptive immunity was represented by the specific antibody response toward keyhole limpet hemocyanin (KLH); innate immunity was represented by natural antibodies toward lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Defining genetic bases of immune responses leads from defining quantitative trait loci (QTL) toward a single mutation responsible for variation in the phenotypic trait. The goal of the reported study was to define candidate genes and mutations for the immune traits of interest in chicken by performing an association study of SNPs located in candidate genes defined in QTL regions. Candidate genes and SNPs in QTL regions were selected in silico. SNP association was based on a custom SNP panel, GoldenGate genotyping assay (Illumina) and two statistical models: random mixed model and CAR score. The most significant SNP for immune response toward KLH was located in the JMJD6 gene located on GGA18. Four SNPs in candidate genes FOXJ1 (GGA18), EPHB1 (GGA9), PTGER4 (GGAZ) and PRKCB (GGA14) showed association with natural antibodies for LPS. A single SNP in ITGB4 (GGA18) was associated with natural antibodies for LTA. All associated SNPs mentioned above showed additive effects.     

Genome‐wide SNP analysis identifies major QTL for Salmonella colonization in the chicken

Salmonella‐infected poultry products are a major source of human Salmonella infection. The prophylactic use of antimicrobials in poultry production was recently banned in the EU, increasing the need for alternative methods to control Salmonella infections in poultry flocks. Genetic selection of chickens more resistant to Salmonella colonization provides an attractive means of sustainably controlling the pathogen in commercial poultry flocks and its subsequent entry into the food chain. Analysis of different inbred chickens has shown that individual lines are consistently either susceptible or resistant to the many serovars of Salmonella that have been tested. In this study, two inbred chicken lines with differential susceptibility to Salmonella colonization (6₁^(R) and N^(S)) were used in a backcross experimental design. Unlike previous studies that used a candidate gene approach or low‐density genome‐wide screens, we have exploited a high‐density marker set of 1255 SNPs covering the whole genome to identify quantitative trait loci (QTL). Analysis of log‐transformed caecal bacterial levels between the parental lines revealed a significant difference at 1, 2, 3 and 4 days post‐infection (P < 0.05). Analysis of the genotypes of the backcross (F₁ × N) population (n = 288) revealed four QTL on chromosomes 2, 3, 12 and 25 for the two traits examined in this study: log‐transformed bacterial counts in the caeca and presence of a hardened caseous caecal core. These included one genome‐wide significant QTL on chromosome 2 at 20 Mb and three additional QTL, on chromosomes 3, 12 and 25 at 96, 15 and 1 Mb, respectively, which were significant at the chromosome‐wide level (P < 0.05). The results generated in this study will inform future breeding strategies to control these pathogens in commercial poultry flocks.     

Novel integrative genomics strategies to identify genes for complex traits

Forward genetics is a common approach to dissecting complex traits like common human diseases. The ultimate aim of this approach was the identification of genes that are causal for disease or other phenotypes of interest. However, the forward genetics approach is by definition restricted to the identification of genes that have incurred mutations over the course of evolution or that incurred mutations as a result of chemical mutagenesis, and that as a result lead to disease or to variations in other phenotypes of interest. Genes that harbour no such mutations, but that play key roles in parts of the biological network that lead to disease, are systematically missed by this class of approaches. Recently, a class of novel integrative genomics approaches has been devised to elucidate the complexity of common human diseases by intersecting genotypic, molecular profiling, and clinical data in segregating populations. These novel approaches take a more holistic view of biological systems and leverage the vast network of gene–gene interactions, in combination with DNA variation data, to establish causal relationships among molecular profiling traits and Fbetween molecular profiling and disease (or other classic phenotypes). A number of novel genes for disease phenotypes have been identified as a result of these approaches, highlighting the utility of integrating orthogonal sources of data to get at the underlying causes of disease.     

Moving from QTL experimental results to the utilization of QTL in breeding programmes

Results from quantitative trait loci studies cannot be readily implemented into breeding schemes through marker assisted selection because of uncertainty about whether the quantitative trait loci identified are real and whether the identified quantitative trait loci are segregating in the breeding population. The present paper outlines and discusses strategies to reduce uncertainty in the results from quantitative trait loci studies. One strategy to confirm results from quantitative trait loci studies is to combine P -values from many quantitative trait loci experiments, while another is to establish a confirmation study. The power of a confirmation study must be high to ensure that the postulated quantitative trait loci can be verified. In the calculation of the experimental power, there are many issues that have to be addressed: size of the quantitative trait loci to be detected, significance level required, experimental design and expected heterozygosity for the design. To ensure marker assisted selection can be quickly implemented once quantitative trait loci are confirmed, DNA samples should be retained from daughters, and the sires and dams of elite sires.     

An Expression QTL of Closely Linked Candidate Genes Affects pH of Meat in Chickens

Genetical genomics has been suggested as a powerful approach to study the genotype–phenotype gap. However, the relatively low power of these experiments (usually related to the high cost) has hindered fulfillment of its promise, especially for loci (QTL) of moderate effects.One strategy with which to overcome the issue is to use a targeted approach. It has two clear advantages: (i) it reduces the problem to a simple comparison between different genotypic groups at the QTL and (ii) it is a good starting point from which to investigate downstream effects of the QTL. In this study, from 698 F2 birds used for QTL mapping, gene expression profiles of 24 birds with divergent homozygous QTL genotypes were investigated. The targeted QTL was on chromosome 1 and affected initial pH of breast muscle. The biological mechanisms controlling this trait can be similar to those affecting malignant hyperthermia or muscle fatigue in humans. The gene expression study identified 10 strong local signals that were markedly more significant compared to any genes on the rest of the genome. The differentially expressed genes all mapped to a region <1 Mb, suggesting a remarkable reduction of the QTL interval. These results, combined with analysis of downstream effect of the QTL using gene network analysis, suggest that the QTL is controlling pH by governing oxidative stress. The results were reproducible with use of as few as four microarrays on pooled samples (with lower significance level). The results demonstrate that this cost-effective approach is promising for characterization of QTL.     

Genome-wide scan of plasma cholecystokinin in baboons shows linkage to human chromosome 17

Objective: Cholecystokinin (CCK) is known to inhibit food intake and is an important signal for controlling meal volume, indicating a possible role in weight regulation. Our objective was to investigate genetic influences on plasma CCK in baboons. Research Methods and Procedures: Subjects were 376 baboons (males = 113, females = 263) from the Southwest National Primate Research Center, housed at the Southwest Foundation for Biomedical Research, San Antonio, Texas. Anthropometric and biochemical parameters were analyzed. Genetic effects on plasma CCK were estimated by the maximum likelihood‐based variance components method implemented in the software program SOLAR (Sequential Oligogenic Linkage Analysis Routines). Results: Male baboons (32.7 ± 6 kg) were much heavier than females (20.2 ± 4 kg). Similarly, mean (± standard deviation) plasma CCK values were also higher in male baboons (13.8 ± 6 pM) than female baboons (12.5 ± 4 pM). Significant heritabilities were observed for plasma CCK (0.14 ± 0.1, p < 0.05), body weight (h² = 0.62 ± 0.15, p < 10^?8), and glucose (h² = 0.68 ± 0.17, p < 10^?7). A genome‐wide scan of plasma CCK detected a strong signal for a quantitative trait locus (QTL) on chromosome 17p12–13 [logarithm of the odds (LOD) = 3.1] near marker D17S804. Suggestive evidence of a second QTL was observed on chromosome 4q34–35 (LOD = 2.3) near marker D4S2374. Discussion: A substantial contribution of additive genetic effects to the variation in plasma levels of CCK was demonstrated in baboons. The identification of a QTL for plasma CCK on chromosome 17p is significant, as several obesity‐related traits such as BMI, leptin, adiponectin, and acylation stimulating protein have already been mapped to this region.     

家禽数量性状基因座定位的研究进展

近二十年来,各种DNA标记技术及相关生物技术的发展和完善为高密度、覆盖面广的连锁图谱的构建及QTL的定位奠定了基础,本就目前世界上建立的几个较有影响的资源家系、各种DNA标记技术、家禽中定位的QTL及存在问题等方面作一综述。     

Saturation mapping of QTL regions and identification of putative candidate genes for drought tolerance in rice

We have developed 85 new markers (50 RFLPs, 5 SSRs, 12 DD cDNAs, 9 ESTs, 8 HSP-encoding cDNAs and one BSA-derived AFLP marker) for saturation mapping of QTL regions for drought tolerance in rice, in our efforts to identify putative candidate genes. Thirteen of the markers were localized in the close vicinity of the targeted QTL regions. Fifteen of the additional markers mapped, respectively, inside one QTL region controlling osmotic adjustment on chromosome 3 ( oa3.1) and 14 regions that affect root traits on chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10 and 12. Differential display was used to identify more putative candidate genes and to saturate the QTL regions of the genetic map. Eleven of the isolated cDNA clones were found to be derived from drought-inducible genes. Two of them were unique and did not match any genes in the GenBank, while nine were highly similar to cDNAs encoding known proteins, including a DnaJ-related protein, a zinc-finger protein, a protease inhibitor, a glutathione-S-transferase, a DNA recombinase, and a protease. Twelve new cDNA fragments were mapped onto the genetic linkage map; seven of these mapped inside, or in close proximity to, the targeted QTL regions determining root thickness and osmotic adjustment capacity. The gene I12A1, which codes for a UDP-glucose 4-epimerase homolog, was identified as a putative target gene within the prt7.1/brt7.1 QTL region, as it is involved in the cell wall biogenesis pathway and hence may be implicated in modulating the ability of rice roots to penetrate further into the substratum when exposed to drought conditions. RNAs encoding elongation factor 1, a DnaJ-related protein, and a homolog of wheat zinc-finger protein were more prominently induced in the leaves of IR62266 (the lowland rice parent of the mapping materials used) than in those of CT9993 (the upland rice parent) under drought conditions. Homologs of 18S ribosomal RNA, and mRNAs for a multiple-stress induced zinc-finger protein, a protease inhibitor, and a glutathione-S-transferase were expressed at significantly higher levels in CT9993 than in IR62266. Thus several genes involved in the regulation of DNA structure and mRNA translation were found to be drought-regulated, and may be implicated in drought resistance.Communicated by R. Hagemann     

Candidate genes for plasma triglyceride, FFA, and glucose revealed from an intercross between inbred mouse strains NZB/B1NJ and NZW/LacJ

To identify the genes controlling plasma concentrations of triglycerides (TGs), FFAs, and glucose, we carried out a quantitative trait loci (QTL) analysis of the closely related mouse strains New Zealand Black (NZB/B1NJ) and New Zealand White (NZW/LacJ), which share 63% of their genomes. The NZB x NZW F(2) progeny were genotyped and phenotyped to detect QTL, and then comparative genomics, bioinformatics, and sequencing were used to narrow the QTL and reduce the number of candidate genes. Triglyceride concentrations were linked to loci on chromosomes (Chr) 4, 7, 8, 10, and 18. FFA concentrations were affected by a significant locus on Chr 4, a suggestive locus on Chr 16, and two interacting loci on Chr 2 and 15. Plasma glucose concentrations were affected by QTL on Chr 2, 4, 7, 8, 10, 15, 17, and 18. Comparative genomics narrowed the QTL by 31% to 86%; haplotype analysis was usually able to further narrow it by 80%. We suggest several candidate genes: Gba2 on Chr 4, Irs2 on Chr 8, and Ppargc1b on Chr 18 for TG; A2bp1 on Chr 16 for FFA; and G6pc2 on Chr 2 and Timp3 on Chr 10 for glucose.     

A genome-wide survey of maize lipid-related genes: candidate genes mining,digital gene expression profiling and co-location with QTL for maize kernel oil

Lipids play an important role in plants due to their abundance and their extensive participation in many metabolic processes. Genes involved in lipid metabolism have been extensively studied in Arabidopsis and other plant species. In this study, a total of 1003 maize lipid-related genes were cloned and annotated, including 42 genes with experimental validation, 732 genes with full-length cDNA and protein sequences in public databases and 229 newly cloned genes. Ninety-seven maize lipid-related genes with tissue-preferential expression were discovered by in silico gene expression profiling based on 1984483 maize Expressed Sequence Tags collected from 182 cDNA libraries. Meanwhile, 70 QTL clusters for maize kernel oil were identified, covering 34.5% of the maize genome. Fifty-nine (84%) QTL clusters co-located with at least one lipid-related gene, and the total number of these genes amounted to 147. Interestingly, thirteen genes with kernel-preferential expression profiles fell within QTL clusters for maize kernel oil content. All the maize lipid-related genes identified here may provide good targets for maize kernel oil QTL cloning and thus help us to better understand the molecular mechanism of maize kernel oil accumulation.     

In the presence of population structure: From genomics to candidate genes underlying local adaptation

Understanding the genomic signatures, genes, and traits underlying local adaptation of organisms to heterogeneous environments is of central importance to the field evolutionary biology. To identify loci underlying local adaptation, models that combine allelic and environmental variation while controlling for the effects of population structure have emerged as the method of choice. Despite being evaluated in simulation studies, there has not been a thorough investigation of empirical evidence supporting local adaptation across these alleles. To evaluate these methods, we use 875 Arabidopsis thaliana Eurasian accessions and two mixed models (GEMMA and LFMM) to identify candidate SNPs underlying local adaptation to climate. Subsequently, to assess evidence of local adaptation and function among significant SNPs, we examine allele frequency differentiation and recent selection across Eurasian populations, in addition to their distribution along quantitative trait loci (QTL) explaining fitness variation between Italy and Sweden populations and cis‐regulatory/nonsynonymous sites showing significant selective constraint. Our results indicate that significant LFMM/GEMMA SNPs show low allele frequency differentiation and linkage disequilibrium across locally adapted Italy and Sweden populations, in addition to a poor association with fitness QTL peaks (highest logarithm of odds score). Furthermore, when examining derived allele frequencies across the Eurasian range, we find that these SNPs are enriched in low‐frequency variants that show very large climatic differentiation but low levels of linkage disequilibrium. These results suggest that their enrichment along putative functional sites most likely represents deleterious variation that is independent of local adaptation. Among all the genomic signatures examined, only SNPs showing high absolute allele frequency differentiation (AFD) and linkage disequilibrium (LD) between Italy and Sweden populations showed a strong association with fitness QTL peaks and were enriched along selectively constrained cis‐regulatory/nonsynonymous sites. Using these SNPs, we find strong evidence linking flowering time, freezing tolerance, and the abscisic‐acid pathway to local adaptation.     

Whole-genome scan to detect QTL for milk production, conformation, fertility and functional traits in two US Holstein families

A genome scan was conducted in two US Holstein half-sib families to identify quantitative trait loci (QTL) affecting milk production and conformation traits using the granddaughter design. The sires of the two studied families were related as sire and son and had 96 and 212 sons respectively. A total of 221 microsatellite loci were scored in both families. Statistical analysis was performed using two different analytical methods; half-sib least squares regression and Bayesian Monte Carlo Markov Chain. Traits analysed included five traditional milk production traits, somatic cell count, daughter pregnancy rate, male fertility and 20 conformation traits. A total of 47 tests achieved at least genome-wise significance. However, results from the two methods of analysis were only concordant for QTL location and level of significance in eight instances.