Power analysis of QTL detection in half-sib families using selective DNA pooling

Individual loci of economic importance (QTL) can be detected by comparing the inheritance of a trait and the inheritance of loci with alleles readily identifiable by laboratory methods (genetic markers). Data on allele segregation at the individual level are costly and alternatives have been proposed that make use of allele frequencies among progeny, rather than individual genotypes. Among the factors that may affect the power of the set up, the most important are those intrinsic to the QTL: the additive effect of the QTL, and its dominance, and distance between markers and QTL. Other factors are relative to the choice of animals and markers, such as the frequency of the QTL and marker alleles among dams and sires. Data collection may affect the detection power through the size of half-sib families, selection rate within families, and the technical error incurred when estimating genetic frequencies. We present results for a sensitivity analysis for QTL detection using pools of DNA from selected half-sibs. Simulations showed that conclusive detection may be achieved with families of at least 500 half-sibs if sires are chosen on the criteria that most of their marker alleles are either both missing, or one is fixed, among dams.     

Identification of an ovulation rate QTL in cattle on BTA14 using selective DNA pooling and interval mapping

Increased twinning incidence in beef cattle has the potential to improve production efficiency. However, phenotypic selection for twinning rate is difficult because of the trait's low heritability and the long time interval necessary to collect phenotypic records. Therefore, this trait and the correlated trait of ovulation rate are ideal candidates for marker-assisted selection. The objective of this study was to complete a genome-wide search for ovulation rate quantitative trait loci (QTL) in two related sire families. The families (paternal halfsib sires 839802 and 839803) were from a population of cattle selected for ovulation rate at the USDA Meat Animal Research Center, Clay Center, Nebraska. Putative ovulation rate QTL have previously been identified in the 839802 family on chromosomes 7 and 19; however, marker coverage in the original scan was not complete. This study fills the gaps in marker coverage of the earlier study by adding approximately 60 informative microsatellites to each sire family. Each family was genotyped using selective DNA pooling. Sons and daughters were included in either the high or low pool based on their estimated breeding value deviations from the mid-parent average (EBVMD) for ovulation rate. Approximately 40% (839802) and 26% (839803) of available progeny comprised the high and low pools combined. Pooled typing revealed possible associations (nominal P < 0.05) between ovulation rate and marker genotype for 11 and 15 microsatellites in the 839802 and 839803 families, respectively. Subsequent interval mapping strengthened support for the presence of an ovulation rate QTL on BTA14 (chromosome-wise P < 0.02).     

A genome‐wide association study using selective DNA pooling identifies candidate markers for fertility in Holstein cattle

The decline in the reproductive efficiency of dairy cows, especially those with high producing potential, has become a challenging problem. In this study, a selective DNA pooling approach was applied to a cow population whose oocytes were fertilized and cultured to obtain phenotypic records of fertilization rate and blastocyst rate. Using a stringent 5% genome‐wide significance level, 22 and five single nucleotide polymorphisms (SNPs) were found to be associated with fertilization rate and blastocyst rate, respectively. SNPs that showed significant association in selective DNA pooling were further evaluated by individual genotyping. Interestingly, the majority of the SNP associations were confirmed by individual genotyping, testifying to the effectiveness of selective DNA pooling using a high‐density SNP genotyping array. This study is the first application of the selective DNA pooling approach using the BovineSNP50 array in cattle.     

Detection of QTL for growth rate in the blacklip abalone (Haliotis rubra Leach) using selective DNA pooling

The objective of this study was to identify QTL for growth rate in the blacklip abalone Haliotis rubra using selective DNA pooling. Three full-sibling families of H. rubra derived from crosses of wild broodstock were used. DNA was extracted from the largest and smallest 10% of progeny and combined into two pools for each phenotypic tail. The DNA pools were typed with 139 microsatellites, and markers showing significant differences between the peak height ratios of alleles inherited from the parents were individually genotyped and analysed by interval mapping. A strong correlation (r = 0.94, P < 0.001) was found between the t-values from the analysis of pools and the t-values from the analysis of individual genotypes. Based on the interval mapping analysis, QTL were detected on nine linkage groups at a chromosome-wide P < 0.01 and one linkage group at a chromosome-wide P < 0.05. The study demonstrated that selective DNA pooling is efficient and effective as a first-pass screen for the discovery of QTL in an aquaculture species.     

Mapping economic trait loci for somatic cell score in Holstein cattle using microsatellite markers and selective genotyping

Marker-assisted selection (MAS) uses genetic marker genotypes to predict an animal's production potential and will provide additional selection information for progeny testing. With the discovery of highly polymorphic microsatellite markers, the tools now exist to begin the search for economic trait loci (ETL), which is the first step toward MAS. The objective of this study was to identify ETL for somatic cell score in an existing Holstein population. Using the granddaughter design, sons from seven grandsire families were genotyped with 20 autosomal microsatellites from five chromosomes (4, 8, 13, 17, 23), with an emphasis on chromosome 23, which is the location of the bovine major histocompatibility complex (BoLA). Selective genotyping was used to reduce the number of genotypes required, in which the 10 highest and 10 lowest sons from the phenotypic distribution curve were tested (140 sons in seven families). One marker (513), located near BoLA, showed evidence of an ETL in three of five polymorphic families. Additional sons were genotyped from the five families to estimate the effect and to compare selective and ‘complete’ genotyping. Both methods detected an ETL at marker 513, but in different families. This study provides evidence of the usefulness of microsatellite markers and the granddaughter design in the detection of ETL; however, additional markers need to be evaluated to determine the usefulness of selective genotyping. Based on the results from the 20 studied markers, the most likely position of a somatic cell score ETL lies near marker 513, located on chromosome 23.     

基于DNA池测序法筛选奶牛高信息量SNP标记的可行性

首先选择139个牛SNP标记, 利用DNA池测序法, 根据测序峰图中不同碱基信号峰高的比值确定了92个SNP为高信息量标记(比值>1/2); 为了进一步验证筛选的准确性, 对其中59个标记采用基质辅助激光解析电离飞行时间质谱(Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, MALDI-TOF MS)技术检测了122头荷斯坦牛的基因型。结果显示, 检出率高于85%的标记有56个, 其平均最小等位基因频率(Minor allele frequency, MAF)为0.41, 最小值为0.27, 最大值为0.5; MAF>0.3的标记有54个, 占96.4%(54/56)。文章结果表明, 采用DNA池测序法筛选高信息量SNP标记是可行和可信的。     

Quantitative trait loci mapping for conjugated linoleic acid,vaccenic acid and ∆9‐desaturase in Italian Brown Swiss dairy cattle using selective DNA pooling

A selective DNA pooling approach was applied to identify QTL for conjugated linoleic acid (CLA), vaccenic acid (VA) and Δ⁹‐desaturase (D9D) milk content in Italian Brown Swiss dairy cattle. Milk samples from 60 animals with higher values (after correction for environmental factors) and 60 animals with lower values for each of these traits from each of five half‐sib families were pooled separately. The pools were genotyped using the Illumina BovineSNP50 BeadChip. Sire allele frequencies were compared between high and low tails at the sire and marker level for SNPs for which the sires were heterozygous. An r procedure was implemented to perform data analysis in a selective DNA pooling design. A correction for multiple tests was applied using the proportion of false positives among all test results. BTA 19 showed the largest number of markers in association with CLA. Associations between SNPs and the VA and Δ⁹‐desaturase traits were found on several chromosomes. A bioinformatics survey identified genes with an important role in pathways for milk fat and fatty acids metabolism within 1 Mb of SNP markers associated with fatty acids contents.     

Quantitative trait locus mapping in dairy cattle by means of selective milk DNA pooling using dinucleotide microsatellite markers: analysis of milk protein percentage.

"Selective DNA pooling" accomplishes quantitative trait locus (QTL) mapping through densitometric estimates of marker allele frequencies in pooled DNA samples of phenotypically extreme individuals. With poly(TG) microsatellites, such estimates are confounded by "shadow" ("stutter") bands. A correction procedure was developed on the basis of an observed linear regression between shadow band intensity and allele TG repeat number. Using this procedure, a selective DNA pooling study with respect to milk protein percentage was implemented in Israel-Holstein dairy cattle. Pools were prepared from milk samples of high and low daughters of each of seven sires and genotyped with respect to 11 markers. Highly significant associations with milk protein percentage were found for 5 of the markers; 4 of these markers confirmed previous reports. Selective DNA pooling accessed 80.6 and 48.3%, respectively, of the information that would have been available through individual selective genotyping or total population genotyping. In effect, the statistical power of 45,600 individual genotypings was obtained from 328 pool genotypings. This methodology can make genome-wide mapping of QTL accessible to moderately sized breeding organizations.     

A whole genome scan for QTL affecting milk protein percentage in Italian Holstein cattle, applying selective milk DNA pooling and multiple marker mapping in a daughter design

We report on a complete genome scan for quantitative trait loci (QTL) affecting milk protein percentage (PP) in the Italian Holstein-Friesian cattle population, applying a selective DNA pooling strategy in a daughter design. Ten Holstein-Friesian sires were chosen, and for each sire, about 200 daughters, each from the high and low tails of estimated breeding value for PP, were used to construct milk DNA pools. Sires and pools were genotyped for 181 dinucleotide microsatellites covering all cattle autosomes. Sire marker allele frequencies in the pools were obtained by shadow correction of peak height in the electropherograms. After quality control, pool data from eight sires were used for all subsequent analyses. The QTL heterozygosity estimate was lower than that of similar studies in other cattle populations. Multiple marker mapping identified 19 QTL located on 14 chromosomes (BTA1, 2, 3, 4, 5, 6, 8, 9, 12, 14, 17, 20, 23 and 27). The sires were also genotyped for seven polymorphic sites in six candidate genes (ABCG2, SPP1, casein kappa, DGAT1, GHR and PRLR) located within QTL regions of BTA6, 14 and 20 found in this study. The results confirmed or excluded the involvement of some of the analysed markers as the causative polymorphic sites of the identified QTL. The QTL identified, combined with genotype data of these candidate genes, will help to identify other quantitative trait genes and clarify the complex QTL patterns observed for a few chromosomes. Overall, the results are consistent with the Italian Holstein population having been under long-term selection for high PP.     

Identification of loci governing eight agronomic traits using a GBS‐GWAS approach and validation by QTL mapping in soya bean

Soya bean is a major source of edible oil and protein for human consumption as well as animal feed. Understanding the genetic basis of different traits in soya bean will provide important insights for improving breeding strategies for this crop. A genome‐wide association study (GWAS) was conducted to accelerate molecular breeding for the improvement of agronomic traits in soya bean. A genotyping‐by‐sequencing (GBS) approach was used to provide dense genome‐wide marker coverage (>47 000 SNPs) for a panel of 304 short‐season soya bean lines. A subset of 139 lines, representative of the diversity among these, was characterized phenotypically for eight traits under six environments (3 sites × 2 years). Marker coverage proved sufficient to ensure highly significant associations between the genes known to control simple traits (flower, hilum and pubescence colour) and flanking SNPs. Between one and eight genomic loci associated with more complex traits (maturity, plant height, seed weight, seed oil and protein) were also identified. Importantly, most of these GWAS loci were located within genomic regions identified by previously reported quantitative trait locus (QTL) for these traits. In some cases, the reported QTLs were also successfully validated by additional QTL mapping in a biparental population. This study demonstrates that integrating GBS and GWAS can be used as a powerful complementary approach to classical biparental mapping for dissecting complex traits in soya bean.     

Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by real-time PCR and culture: a comparison of the two assays

AIMS: To compare the two different diagnostic assays for the detection of Mycobacterium avium ssp. paratuberculosis, the aetiological agent of paratuberculosis. METHODS AND RESULTS: Faecal samples were derived from 310 cows, representing 13 commercial dairy herds in various locations in Switzerland with expected increased risk because of a past history of disease. Detection assays for M. avium ssp. paratuberculosis were culture (gold standard) and a newly designed real-time PCR. Real-time PCR identified 31 of 310 animals as positive within this risk population whereas culture identified 20 positive animals. The specificity of real-time PCR was confirmed by DNA sequencing of the PCR product. Depending on the test used, the paratuberculosis prevalence in our tested risk population ranged from 6.5 to 10%. CONCLUSIONS: Real-time PCR and culture data were in good agreement, and real-time PCR generates data in a short time in contrast to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: We consider real-time PCR as a suitable alternative method to culture for the detection of M. avium ssp. paratuberculosis in a national surveillance programme.     

A whole genome scan for quantitative trait loci affecting milk protein percentage in Israeli-Holstein cattle, by means of selective milk DNA pooling in a daughter design, using an adjusted false discovery rate criterion

Selective DNA pooling was employed in a daughter design to screen all bovine autosomes for quantitative trait loci (QTL) affecting estimated breeding value for milk protein percentage (EBVP%). Milk pools prepared from high and low daughters of each of seven sires were genotyped for 138 dinucleotide microsatellites. Shadow-corrected estimates of sire allele frequencies were compared between high and low pools. An adjusted false discovery rate (FDR) method was employed to calculate experimentwise significance levels and empirical power. Significant associations with milk protein percentage were found for 61 of the markers (adjusted FDR = 0.10; estimated power, 0.68). The significant markers appear to be linked to 19--28 QTL. Mean allele substitution effects of the putative QTL averaged 0.016 (0.009--0.028) in units of the within-sire family standard deviation of EBVP% and summed to 0.460 EBVP%. Overall QTL heterozygosity was 0.40. The identified QTL appear to account for all of the variation in EBVP% in the population. Through use of selective DNA pooling, 4400 pool data points provided the statistical power of 600,000 individual data points.