Detection of genomic regions that differentiate Bos indicus from Bos taurus ancestral breeds for milk yield in Indian crossbred cows.

Introduction: In India, crossbred cows incorporate the high production of B. taurus dairy breeds and the environmental adaptation of local B. indicus cattle. Adaptation to different environments and selection in milk production have shaped the genetic differences between B. indicus and B. taurus cattle. The aim of this paper was to detect, for milk yield of crossbred cows, quantitative trait loci (QTL) that differentiate B. indicus from B. taurus ancestry, as well as QTL that are segregating within the ancestral breeds. Methods: A total of 123,042 test-day milk records for 4,968 crossbred cows, genotyped with real and imputed 770 K SNP, were used. Breed origins were assigned to haplotypes of crossbred cows, and from that, were assigned to SNP alleles. Results: At a false discovery rate (FDR) of 30%, a large number of genomic regions showed significant effects of B. indicus versus B. taurus origin on milk yield, with positive effects coming from both ancestors. No significant regions were detected for Holstein Friesian (HF) versus Jersey effects on milk yield. Additionally, no regions for SNP alleles segregating within indigenous, within HF, and within Jersey were detected. The most significant effects, at FDR 5%, were found in a region on BTA5 (43.98-49.44 Mbp) that differentiates B. indicus from B. taurus, with an estimated difference between homozygotes of approximately 10% of average yield, in favour of B. indicus origin. Discussion: Our results indicate that evolutionary differences between B. indicus and B. taurus cattle for milk yield, as expressed in crossbred cows, occur at many causative loci across the genome. Although subject to the usual first estimation bias, some of the loci appear to have large effects that might make them useful for genomic selection in crossbreds, if confirmed in subsequent studies.

Genomic evaluation of milk yield in a smallholder crossbred dairy production system in India.

BACKGROUND: India is the largest milk producer globally, with the largest proportion of cattle milk production coming from smallholder farms with an average herd size of less than two milking cows. These cows are mainly undefined multi-generation crosses between exotic dairy breeds and indigenous Indian cattle, with no performance or pedigree recording. Therefore, implementing genetic improvement based on genetic evaluation has not yet been possible. We present the first results from a large smallholder performance recording program in India, using single nucleotide polymorphism (SNP) genotypes to estimate genetic parameters for monthly test-day (TD) milk records and to obtain and validate genomic estimated breeding values (GEBV). RESULTS: The average TD milk yield under the high, medium, and low production environments were 9.64, 6.88, and 4.61 kg, respectively. In the high production environment, the usual profile of a lactation curve was evident, whereas it was less evident in low and medium production environments. There was a clear trend of an increasing milk yield with an increasing Holstein Friesian (HF) proportion in the high production environment, but no increase above intermediate grades in the medium and low production environments. Trends for Jersey were small but yield estimates had a higher standard error than HF. Heritability estimates for TD yield across the lactation ranged from 0.193 to 0.250, with an average of 0.230. The additive genetic correlations between TD yield at different times in lactation were high, ranging from 0.846 to 0.998. The accuracy of phenotypic validation of GEBV from the method that is believed to be the least biased was 0.420, which was very similar to the accuracy obtained from the average prediction error variance of the GEBV. CONCLUSIONS: The results indicate strong potential for genomic selection to improve milk production of smallholder crossbred cows in India. The performance of cows with different breed compositions can be determined in different Indian environments, which makes it possible to provide better advice to smallholder farmers on optimum breed composition for their environment.

Genetic diversity and effective population sizes of thirteen Indian cattle breeds.

BACKGROUND: The genetic structure of a diverse set of 15 Indian indigenous breeds and non-descript indigenous cattle sampled from eight states was examined, based on 777 k single nucleotide polymorphism (SNP) genotypes obtained on 699 animals, with sample sizes ranging from 17 to 140 animals per breed. To date, this is the largest and most detailed assessment of the genetic diversity of Indian cattle breeds. RESULTS: Admixture analyses revealed that 109 of the indigenous animals analyzed had more than 1% Bos taurus admixture of relatively recent origin. Pure indigenous animals were defined as having more than 99% Bos indicus ancestry. Assessment of the genetic diversity within and between breeds using principal component analyses, F statistics, runs of homozygosity, the genomic relationship matrix, and maximum likelihood clustering based on allele frequencies revealed a low level of genetic diversity among the indigenous breeds compared to that of Bos taurus breeds. Correlations of SNP allele frequencies between breeds indicated that the genetic variation among the Bos indicus breeds was remarkably low. In addition, the variance in allele frequencies represented less than 1.5% between the Indian indigenous breeds compared to about 40% between Bos taurus dairy breeds. Effective population sizes (Ne) increased during a period post-domestication, notably for Ongole cattle, and then declined during the last 100 generations. Although we found that most of the identified runs of homozygosity are short in the Indian indigenous breeds, indicating no recent inbreeding, the high FROH coefficients and low FIS values point towards small population sizes. Nonetheless, the Ne of the Indian indigenous breeds is currently still larger than that of Bos taurus dairy breeds. CONCLUSIONS: The changes in the estimates of effective population size are consistent with domestication from a large native population followed by consolidation into breeds with a more limited population size. The surprisingly low genetic diversity among Indian indigenous cattle breeds might be due to their large Ne since their domestication, which started to decline only 100 generations ago, compared to approximately 250 to 500 generations for Bos taurus dairy cattle.

Inference of Ancestries and Heterozygosity Proportion and Genotype Imputation in West African Cattle Populations.

Several studies have evaluated computational methods that infer the haplotypes from population genotype data in European cattle populations. However, little is known about how well they perform in African indigenous and crossbred populations. This study investigates: (1) global and local ancestry inference; (2) heterozygosity proportion estimation; and (3) genotype imputation in West African indigenous and crossbred cattle populations. Principal component analysis (PCA), ADMIXTURE, and LAMP-LD were used to analyse a medium-density single nucleotide polymorphism (SNP) dataset from Senegalese crossbred cattle. Reference SNP data of East and West African indigenous and crossbred cattle populations were used to investigate the accuracy of imputation from low to medium-density and from medium to high-density SNP datasets using Minimac v3. The first two principal components differentiated Bos indicus from European Bos taurus and African Bos taurus from other breeds. Irrespective of assuming two or three ancestral breeds for the Senegalese crossbreds, breed proportion estimates from ADMIXTURE and LAMP-LD showed a high correlation (r ≥ 0.981). The observed ancestral origin heterozygosity proportion in putative F1 crosses was close to the expected value of 1.0, and clearly differentiated F1 from all other crosses. The imputation accuracies (estimated as correlation) between imputed and the real data in crossbred animals ranged from 0.142 to 0.717 when imputing from low to medium-density, and from 0.478 to 0.899 for imputation from medium to high-density. The imputation accuracy was generally higher when the reference data came from the same geographical region as the target population, and when crossbred reference data was used to impute crossbred genotypes. The lowest imputation accuracies were observed for indigenous breed genotypes. This study shows that ancestral origin heterozygosity can be estimated with high accuracy and will be far superior to the use of observed individual heterozygosity for estimating heterosis in African crossbred populations. It was not possible to achieve high imputation accuracy in West African crossbred or indigenous populations based on reference data sets from East Africa, and population-specific genotyping with high-density SNP assays is required to improve imputation.

Small SNP panels for breed proportion estimation in Indian crossbred dairy cattle.

Reliably identifying breed proportions in crossbred cattle in smallholder farms is a crucial step to improve mating decisions and optimizing management in these systems. High-density genotype information is able to estimate higher-order breed proportions accurately, but, are too expensive for mass application in smallholder systems. We used high-density genotype information (777 k SNPs) of 623 crossbred cattle from India that had Holstein-Friesian (HFX) and/or Jersey and indigenous breeds in their ancestry to select a smaller number of SNPs for breed proportion estimation. The accuracy of estimates obtained from panels with 100-500 SNP was compared to estimates based on all SNPs. Panels were selected for highest absolute allele frequency difference between exotic dairy versus indigenous Bos indicus, or between HFX versus Jersey breeds. A step-wise pruning approach was developed showing that and increased physical distances between markers of 8.5 Mb improved breed proportion estimation compared to a standard 1 Mb distance. A panel of 500 SNPs optimized to estimate HFX versus Jersey versus indicine ancestry was able to estimate indicine breed proportions with r2  = .991, HFX proportions with r2  = .979 and Jersey proportions with r2  = .949. The number of markers was a deciding factor in estimation accuracy, together with the distribution of markers across the genome.

SNP panels for the estimation of dairy breed proportion and parentage assignment in African crossbred dairy cattle.

BACKGROUND: Understanding the relationship between genetic admixture and phenotypic performance is crucial for the optimization of crossbreeding programs. The use of small sets of informative ancestry markers can be a cost-effective option for the estimation of breed composition and for parentage assignment in situations where pedigree recording is difficult. The objectives of this study were to develop small single nucleotide polymorphism (SNP) panels that can accurately estimate the total dairy proportion and assign parentage in both West and East African crossbred dairy cows. METHODS: Medium- and high-density SNP genotype data (Illumina BovineSNP50 and BovineHD Beadchip) for 4231 animals sampled from African crossbreds, African Bos taurus, European Bos taurus, Bos indicus, and African indigenous populations were used. For estimating breed composition, the absolute differences in allele frequency were calculated between pure ancestral breeds to identify SNPs with the highest discriminating power, and different combinations of SNPs weighted by ancestral origin were tested against estimates based on all available SNPs. For parentage assignment, informative SNPs were selected based on the highest minor allele frequency (MAF) in African crossbred populations assuming two Scenarios: (1) parents were selected among all the animals with known genotypes, and (2) parents were selected only among the animals known to be a parent of at least one progeny. RESULTS: For the medium-density genotype data, SNPs selected for the largest differences in allele frequency between West African indigenous and European Bos taurus breeds performed best for most African crossbred populations and achieved a prediction accuracy (r2) for breed composition of 0.926 to 0.961 with 200 SNPs. For the high-density dataset, a panel with 70% of the SNPs selected on their largest difference in allele frequency between African and European Bos taurus performed best or very near best across all crossbred populations with r2 ranging from 0.978 to 0.984 with 200 SNPs. In all African crossbred populations, unambiguous parentage assignment was possible with ≥ 300 SNPs for the majority of the panels for Scenario 1 and ≥ 200 SNPs for Scenario 2. CONCLUSIONS: The identified low-cost SNP assays could overcome incomplete or inaccurate pedigree records in African smallholder systems and allow effective breeding decisions to produce progeny of desired breed composition.

Ancestral Haplotype Mapping for GWAS and Detection of Signatures of Selection in Admixed Dairy Cattle of Kenya.

Understanding the genetic structure of adaptation and productivity in challenging environments is necessary for designing breeding programs that suit such conditions. Crossbred dairy cattle in East Africa resulting from over 60 years of crossing exotic dairy breeds with indigenous cattle plus inter se matings form a highly variable admixed population. This population has been subject to natural selection in response to environmental stresses, such as harsh climate, low-quality feeds, poor management, and strong disease challenge. Here, we combine two complementary sets of analyses, genome-wide association (GWA) and signatures of selection (SoS), to identify genomic regions that contribute to variation in milk yield and/or contribute to adaptation in admixed dairy cattle of Kenya. Our GWA separates SNP effects due to ancestral origin of alleles from effects due to within-population linkage disequilibrium. The results indicate that many genomic regions contributed to the high milk production potential of modern dairy breeds with no region having an exceptional effect. For SoS, we used two haplotype-based tests to compare haplotype length variation within admixed and between admixed and East African Shorthorn Zebu cattle populations. The integrated haplotype score (iHS) analysis identified 16 candidate regions for positive selection in the admixed cattle while the between population Rsb test detected 24 divergently selected regions in the admixed cattle compared to East African Shorthorn Zebu. We compare the results from GWA and SoS in an attempt to validate the most significant SoS results. Only four candidate regions for SoS intersect with GWA regions using a low stringency test. The identified SoS candidate regions harbored genes in several enriched annotation clusters and overlapped with previously found QTLs and associations for different traits in cattle. If validated, the GWA and SoS results indicate potential for SNP-based genomic selection for genetic improvement of smallholder crossbred cattle.

Livestock Genomics for Developing Countries - African Examples in Practice.

African livestock breeds are numerous and diverse, and typically well adapted to the harsh environment conditions under which they perform. They have been used over centuries to provide livelihoods as well as food and nutritional security. However, African livestock systems are dynamic, with many small- and medium-scale systems transforming, to varying degrees, to become more profitable. In these systems the women and men livestock keepers are often seeking new livestock breeds or genotypes - typically those that increase household income through having enhanced productivity in comparison to traditional breeds while maintaining adaptedness. In recent years genomic approaches have started to be utilized in the identification and development of such breeds, and in this article we describe a number of examples to this end from sub-Saharan Africa. These comprise case studies on: (a) dairy cattle in Kenya and Senegal, as well as sheep in Ethiopia, where genomic approaches aided the identification of the most appropriate breed-type for the local productions systems; (b) a cross-breeding program for dairy cattle in East Africa incorporating genomic selection as well as other applications of genomics; (c) ongoing work toward creating a new cattle breed for East Africa that is both productive and resistant to trypanosomiasis; and (d) the use of African cattle as resource populations to identify genomic variants of economic or ecological significance, including a specific case where the discovery data was from a community based breeding program for small ruminants in Ethiopia. Lessons learnt from the various case studies are highlighted, and the concluding section of the paper gives recommendations for African livestock systems to increasingly capitalize on genomic technologies.

Genetic tests for estimating dairy breed proportion and parentage assignment in East African crossbred cattle.

BACKGROUND: Smallholder dairy farming in much of the developing world is based on the use of crossbred cows that combine local adaptation traits of indigenous breeds with high milk yield potential of exotic dairy breeds. Pedigree recording is rare in such systems which means that it is impossible to make informed breeding decisions. High-density single nucleotide polymorphism (SNP) assays allow accurate estimation of breed composition and parentage assignment but are too expensive for routine application. Our aim was to determine the level of accuracy achieved with low-density SNP assays. METHODS: We constructed subsets of 100 to 1500 SNPs from the 735k-SNP Illumina panel by selecting: (a) on high minor allele frequencies (MAF) in a crossbred population; (b) on large differences in allele frequency between ancestral breeds; (c) at random; or (d) with a differential evolution algorithm. These panels were tested on a dataset of 1933 crossbred dairy cattle from Kenya/Uganda and on crossbred populations from Ethiopia (N = 545) and Tanzania (N = 462). Dairy breed proportions were estimated by using the ADMIXTURE program, a regression approach, and SNP-best linear unbiased prediction, and tested against estimates obtained by ADMIXTURE based on the 735k-SNP panel. Performance for parentage assignment was based on opposing homozygotes which were used to calculate the separation value (sv) between true and false assignments. RESULTS: Panels of SNPs based on the largest differences in allele frequency between European dairy breeds and a combined Nelore/N'Dama population gave the best predictions of dairy breed proportion (r2 = 0.962 to 0.994 for 100 to 1500 SNPs) with an average absolute bias of 0.026. Panels of SNPs based on the highest MAF in the crossbred population (Kenya/Uganda) gave the most accurate parentage assignments (sv = -1 to 15 for 100 to 1500 SNPs). CONCLUSIONS: Due to the different required properties of SNPs, panels that did well for breed composition did poorly for parentage assignment and vice versa. A combined panel of 400 SNPs was not able to assign parentages correctly, thus we recommend the use of 200 SNPs either for breed proportion prediction or parentage assignment, independently.

Genetic and economic benefits of selection based on performance recording and genotyping in lower tiers of multi-tiered sheep breeding schemes.

BACKGROUND: Performance recording and genotyping in the multiplier tier of multi-tiered sheep breeding schemes could potentially reduce the difference in the average genetic merit between nucleus and commercial flocks, and create additional economic benefits for the breeding structure. METHODS: The genetic change in a multiple-trait breeding objective was predicted for various selection strategies that included performance recording, parentage testing and genomic selection. A deterministic simulation model was used to predict selection differentials and the flow of genetic superiority through the different tiers. Cumulative discounted economic benefits were calculated based on trait gains achieved in each of the tiers and considering the extra revenue and associated costs of applying recording, genotyping and selection practices in the multiplier tier of the breeding scheme. RESULTS: Performance recording combined with genomic or parentage information in the multiplier tier reduced the genetic lag between the nucleus and commercial flock by 2 to 3 years. The overall economic benefits of improved performance in the commercial tier offset the costs of recording the multiplier. However, it took more than 18 years before the cumulative net present value of benefits offset the costs at current test prices. Strategies in which recorded multiplier ewes were selected as replacements for the nucleus flock did modestly increase profitability when compared to a closed nucleus structure. Applying genomic selection is the most beneficial strategy if testing costs can be reduced or by genotyping only a proportion of the selection candidates. When the cost of genotyping was reduced, scenarios that combine performance recording with genomic selection were more profitable and reached breakeven point about 10 years earlier. CONCLUSIONS: Economic benefits can be generated in multiplier flocks by implementing performance recording in conjunction with either DNA pedigree recording or genomic technology. These recording practices reduce the long genetic lag between the nucleus and commercial flocks in multi-tiered breeding programs. Under current genotyping costs, the time to breakeven was found to be generally very long, although this varied between strategies. Strategies using either genomic selection or DNA pedigree verification were found to be economically viable provided the price paid for the tests is lower than current prices, in the long-term.

Characterization and Profiling of Liver microRNAs by RNA-sequencing in Cattle Divergently Selected for Residual Feed Intake.

MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate expression of mRNAs in many biological pathways. Liver plays an important role in the feed efficiency of animals and high and low efficient cattle demonstrated different gene expression profiles by microarray. Here we report comprehensive miRNAs profiles by next-gen deep sequencing in Angus cattle divergently selected for residual feed intake (RFI) and identify miRNAs related to feed efficiency in beef cattle. Two microRNA libraries were constructed from pooled RNA extracted from livers of low and high RFI cattle, and sequenced by Illumina genome analyser. In total, 23,628,103 high quality short sequence reads were obtained and more than half of these reads were matched to the bovine genome (UMD 3.1). We identified 305 known bovine miRNAs. Bta-miR-143, bta-miR-30, bta-miR-122, bta-miR-378, and bta-let-7 were the top five most abundant miRNAs families expressed in liver, representing more than 63% of expressed miRNAs. We also identified 52 homologous miRNAs and 10 novel putative bovine-specific miRNAs, based on precursor sequence and the secondary structure and utilizing the miRBase (v. 21). We compared the miRNAs profile between high and low RFI animals and ranked the most differentially expressed bovine known miRNAs. Bovine miR-143 was the most abundant miRNA in the bovine liver and comprised 20% of total expressed mapped miRNAs. The most highly expressed miRNA in liver of mice and humans, miR-122, was the third most abundant in our cattle liver samples. We also identified 10 putative novel bovine-specific miRNA candidates. Differentially expressed miRNAs between high and low RFI cattle were identified with 18 miRNAs being up-regulated and 7 other miRNAs down-regulated in low RFI cattle. Our study has identified comprehensive miRNAs expressed in bovine liver. Some of the expressed miRNAs are novel in cattle. The differentially expressed miRNAs between high and low RFI give some insights into liver miRNAs regulating physiological pathways underlying variation in this measure of feed efficiency in bovines.

Identification of novel loci associated with gastrointestinal parasite resistance in a Red Maasai x Dorper backcross population.

Gastrointestinal (GI) parasitic infection is the main health constraint for small ruminant production, causing loss of weight and/or death. Red Maasai sheep have adapted to a tropical environment where extreme parasite exposure is a constant, especially with highly pathogenic Haemonchus contortus. This breed has been reported to be resistant to gastrointestinal parasite infection, hence it is considered an invaluable resource to study associations between host genetics and resistance. The aim of this study was to identify polymorphisms strongly associated with host resistance in a double backcross population derived from Red Maasai and Dorper sheep using a SNP-based GWAS analysis. The animals that were genotyped represented the most resistant and susceptible individuals based on the tails of phenotypic distribution (10% each) for average faecal egg counts (AVFEC). AVFEC, packed cell volume (AVPCV), and live weight (AVLWT) were adjusted for fixed effects and co-variables, and an association analysis was run using EMMAX. Revised significance levels were calculated using 100,000 permutation tests. The top five significant SNP markers with - log10 p-values >3.794 were observed on five different chromosomes for AVFEC, and BLUPPf90/PostGSf90 results confirmed EMMAX significant regions for this trait. One of these regions included a cluster of significant SNP on chromosome (Chr) 6 not in linkage disequilibrium to each other. This genomic location contains annotated genes involved in cytokine signalling, haemostasis and mucus biosynthesis. Only one association detected on Chr 7 was significant for both AVPCV and AVLWT. The results generated here reveal candidate immune variants for genes involved in differential response to infection and provide additional SNP marker information that has potential to aid selection of resistance to gastrointestinal parasites in sheep of a similar genetic background to the double backcross population.

Characterization of a novel melt curve by use of the Roche LightCycler HSV 1/2 analyte-specific reagent real-time PCR assay: frequencies of this novel (low) melt curve and commonly encountered (intermediate) melt curves.

We characterize a novel probe binding-site polymorphism detectable solely by melt curve analysis using the Roche LightCycler HSV 1/2 analyte-specific reagent real-time PCR assay. The frequencies of this novel (47°C) and previously described intermediate (60 to 62°C) melt curves were 0.016% and 4.9%, respectively.

Trypanotolerance in N'Dama x Boran crosses under natural trypanosome challenge: effect of test-year environment, gender, and breed composition.

BACKGROUND: Trypanosomosis, a protozoal disease affecting livestock, transmitted by Glossina (tsetse) flies is a major constraint to agricultural production in Sub-Saharan Africa. It is accepted that utilization of the native trypanotolerance exhibited in some of the African cattle breeds to improve trypanotolerance of more productive but susceptible breeds, will offer a cost effective and sustainable solution to the problem. The success of this approach is based on the premise that quantitative trait loci previously identified under relatively controlled situations confer useful trypanotolerance under natural field situations. As part of a study to authenticate this hypothesis, a population of 192 cattle, consisting of six batches of N'Dama and Kenya-Boran backcross animals [(N'Dama x Kenya-Boran) x Kenya-Boran] born over the period 2002 to 2006 was constructed. Some of the batches also included pure Kenya-Boran cattle, or N'Dama x Kenya- Boran F1 animals. Each batch was exposed as yearlings to natural field trypanosomosis challenge over a period of about one year; the entire challenge period extending from December 2003 to June 2007. Performance of the animals was evaluated by weekly or biweekly measurements of body weight, packed blood cell volume (PCV), parasitemia score, and number of trypanocide treatments. From these basic data, 49 phenotypes were constructed reflecting dynamics of body weight, packed cell volume (PCV) and parasitemia under challenge. RESULTS: Females were distinctly more trypanotolerant than males. F1, backcross and pure Kenya- Boran animals ranked in that order with respect to trypanotolerance. Overall batch effects were highly significant (p<0.001) for most traits, and were generally more significant than the gender or genetic type effects. The superior trypanotolerance of the F1 animals was expressed in all three components of animal defense strategies against pathogens: Avoidance resistance, and tolerance. CONCLUSIONS: The results show that trypanotolerance derived from the N'Dama is expressed under field conditions; and that the trait is primarily additive in nature, being expressed in heterozygous condition and in a three-quarters Boran genetic background. The results further, underscore the complexity of the trait in the field manifesting all three host disease-control strategies, and show the importance of gender and local environmental conditions in determining response to challenge.

Use of a bovine genome array to identify new biological pathways for beef marbling in Hanwoo (Korean Cattle).

BACKGROUND: Marbling (intramuscular fat) is a valuable trait that impacts on meat quality and an important factor determining price of beef in the Korean beef market. Animals that are destined for this high marbling market are fed a high concentrate ration for approximately 30 months in the Korean finishing farms. However, this feeding strategy leads to inefficiencies and excessive fat production. This study aimed to identify candidate genes and pathways associated with intramuscular fat deposition on highly divergent marbling phenotypes in adult Hanwoo cattle. RESULTS: Bovine genome array analysis was conducted to detect differentially expressed genes (DEGs) in m. longissimus with divergent marbling phenotype (marbling score 2 to 7). Three data-processing methods (MAS5.0, GCRMA and RMA) were used to test for differential expression (DE). Statistical analysis identified 21 significant transcripts from at least two data-processing methods (P < 0.01). All 21 differentially expressed genes were validated by real-time PCR. Results showed a high concordance in the gene expression fold change between the microarrays and the real time PCR data. Gene Ontology (GO) and pathway analysis demonstrated that some genes (ADAMTS4, CYP51A and SQLE) over expressed in high marbled animals are involved in a protein catabolic process and a cholesterol biosynthesis process. In addition, pathway analysis also revealed that ADAMTS4 is activated by three regulators (IL-17A, TNFα and TGFß1). QRT-PCR was used to investigate gene expression of these regulators in muscle with divergent intramuscular fat contents. The results demonstrate that ADAMTS4 and TGFß1 are associated with increasing marbling fat. An ADAMTS4/TGFß1 pathway seems to be associated with the phenotypic differences between high and low marbled groups. CONCLUSIONS: Marbling differences are possibly a function of complex signaling pathway interactions between muscle and fat. These results suggest that ADAMTS4, which is involved in connective tissue degradation, could play a role in an important biological pathway for building up marbling in cattle. Moreover, ADAMTS4 and TGFß1could potentially be used as an early biological marker for marbling fat content in the early stages of growth.

Construction of an almond linkage map in an Australian population Nonpareil x Lauranne.

BACKGROUND: Despite a high genetic similarity to peach, almonds (Prunus dulcis) have a fleshless fruit and edible kernel, produced as a crop for human consumption. While the release of peach genome v1.0 provides an excellent opportunity for almond genetic and genomic studies, well-assessed segregating populations and the respective saturated genetic linkage maps lay the foundation for such studies to be completed in almond. RESULTS: Using an almond intraspecific cross between 'Nonpareil' and 'Lauranne' (N x L), we constructed a moderately saturated map with SSRs, SNPs, ISSRs and RAPDs. The N x L map covered 591.4 cM of the genome with 157 loci. The average marker distance of the map was 4.0 cM. The map displayed high synteny and colinearity with the Prunus T x E reference map in all eight linkage groups (G1-G8). The positions of 14 mapped gene-anchored SNPs corresponded approximately with the positions of homologous sequences in the peach genome v1.0. Analysis of Mendelian segregation ratios showed that 17.9% of markers had significantly skewed genotype ratios at the level of P < 0.05. Due to the large number of skewed markers in the linkage group 7, the potential existence of deleterious gene(s) was assessed in the group. Integrated maps produced by two different mapping methods using JoinMap® 3 were compared, and their high degree of similarity was evident despite the positional inconsistency of a few markers. CONCLUSIONS: We presented a moderately saturated Australian almond map, which is highly syntenic and collinear with the Prunus reference map and peach genome V1.0. Therefore, the well-assessed almond population reported here can be used to investigate the traits of interest under Australian growing conditions, and provides more information on the almond genome for the international community.

Mapping SNP-anchored genes using high-resolution melting analysis in almond.

Peach and almond have been considered as model species for the family Rosaceae and other woody plants. Consequently, mapping and characterisation of genes in these species has important implications. High-resolution melting (HRM) analysis is a recent development in the detection of SNPs and other markers, and proved to be an efficient and cost-effective approach. In this study, we aimed to map genes corresponding to known proteins in other species using the HRM approach. Prunus unigenes were searched and compared with known proteins in the public databases. We developed single-nucleotide polymorphism (SNP) markers, polymorphic in a mapping population produced from a cross between the cloned cultivars Nonpareil and Lauranne. A total of 12 SNP-anchored putative genes were genotyped in the population using HRM, and mapped to an existing linkage map. These genes were mapped on six linkage groups, and the predicted proteins were compared to putative orthologs in other species. Amongst those genes, four were abiotic stress-responsive genes, which can provide a starting point for construction of an abiotic resistance map. Two allergy and detoxification related genes, respectively, were also mapped and analysed. Most of the investigated genes had high similarities to sequences from closely related species such as apricot, apple and other eudicots, and these are putatively orthologous. In addition, it was shown that HRM can be an effective means of genotyping populations for the purpose of constructing a linkage map. Our work provides basic genomic information for the 12 genes, which can be used for further genetic and functional studies.

High resolution melting analysis of almond SNPs derived from ESTs.

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.

Cytokine mRNA profiling of peripheral blood mononuclear cells from trypanotolerant and trypanosusceptible cattle infected with Trypanosoma congolense.

To examine differences in cytokine profiles that may confer tolerance/susceptibility to bovine African trypanosomiasis, N'Dama (trypanotolerant, n = 8) and Boran (trypanosusceptible, n = 8) cattle were experimentally challenged with Trypanosoma congolense. Blood samples were collected over a 34-day period, and RNA was extracted from peripheral blood mononuclear cells. The expression levels of a panel of 14 cytokines were profiled over the time course of infection and between breeds. Messenger RNA (mRNA) transcript levels for the IL2, IL8, and IL1RN genes were significantly downregulated across the time course of infection in both breeds. There was an early increase in transcripts for genes encoding proinflammatory mediators (IFNG, IL1A, TNF, and IL12) in N'Dama by 14 days postinfection (dpi) compared with preinfection levels that was not detected in the susceptible Boran breed. By the time of peak parasitemia, a type 2 helper T cells (T(H)2)-like cytokine environment was prevalent that was particularly evident in the Boran. Increases in transcripts for the IL6 (29 and 34 dpi) and IL10 (21, 25, and 29 dpi) genes were detected that were higher in the Boran compared with N'Dama. These findings highlight the implications for using murine models to study the bovine immune response to trypanosomiasis, where in some cases cytokine expression patterns differ. Overall, these data suggest that the trypanotolerant N'Dama are more capable of responding very early in infection with proinflammatory and T(H)1 type cytokines than the trypanosusceptible Boran and may explain why N'Dama control parasitemia more efficiently than Boran during the early stages of infection.

High resolution mapping of chromosomal regions controlling resistance to gastrointestinal nematode infections in an advanced intercross line of mice.

Fine mapping of quantitative trait loci (QTL) associated with resistance to the gastrointestinal parasite Heligmosomoides polygyrus was achieved on F(6)/F(7) offspring (1076 mice) from resistant (SWR) and susceptible (CBA) mouse strains by selective genotyping (top and bottom 20% selected on total worm count in week 6). Fecal egg counts were recorded at weeks 2, 4, and 6, and the average was also analyzed. Blood packed cell volume in weeks 3 and 6 and five immunological traits (mucosal mast cell protease 1, granuloma score, IgG1 against adult worm, IgG1, and IgE to L4 antigen) were also recorded. On Chromosome 1 single-trait analyses identified a QTL with effects on eight traits located at about 24 cM on the F(2) mouse genome database (MGD) linkage map, with a 95% confidence interval (CI) of 20-32 cM established from a multitrait analysis. On Chromosome 17 a QTL with effects on nine traits was located at about 18 cM on the MGD map (CI 17.9-18.4 cM). Strong candidate genes for the QTL position on Chromosome 1 include genes known to be involved in regulating immune responses and on Chromosome 17 genes within the MHC, notably the Class II molecules and tumor necrosis factor.