The SNPs that were significantly associated with the phenotypic traits were identified using both simple (GLM) and compressed mixed linear (MLM) models. For nine of ten body composition traits studied, SNPs showing genome wide significance (P<2.59E−6) have been identified. A consistent region on chicken  chromosome 4 (GGA4), including seven significant SNPs and four candidate genes , were found to be associated with CW and EW. Another 0.65 Mb region on GGA3 for BrW and BrP was identified. After measuring the mRNA content in beast muscle for five genes located in this region, the changes in GJA1 expression were found to be consistent with that of breast muscle weight across development. It is highly possible that GJA1 is a functional gene for breast muscle development in chickens. For meat quality traits, several SNPs reaching suggestive association were identified and possible candidate genes with their functions were discussed.Body composition and meat quality traits are important economic traits of chickens. The development of high-throughput genotyping platforms and relevant statistical methods have enabled genome-wide association studies in chickens. In order to identify molecular markers and candidate genes associated with body composition and meat quality traits, genome-wide association studies were conducted using the Illumina 60 K SNP Beadchip to genotype 724 Beijing-You chickens. For each bird, a total of 16 traits were measured, including carcass weight (CW), eviscerated weight (EW), dressing percentage, breast muscle weight (BrW) and percentage (BrP), thigh muscle weight and percentage, abdominal fat weight and percentage, dry matter and intramuscular fat contents of breast and thigh muscle, ultimate pH, and shear force of the  With the advance of high-throughput genotyping platforms, much effort has been spent on identifying molecular markers and genes related to complex traits using genome-wide association studies (GWAS) in several species.In the field of animal breeding, loci or narrow regions affecting milk production, fertility and growth traits in cattle http://www.animalgenome.org/cgi-bin/QTLdb/GG/index,July,2012), which associated with eight body composition traits: carcass weight, dressed percentage, weight and percentage of breast muscle, thigh muscle and abdominal fat. Similar studies on meat quality traits are rare except for 10 QTLs reported for pH value. Application of these QTL results in broiler breeding remains impracticable because of the low precision of mapping.Body composition and meat quality in broilers are important economic traits. Body composition traits have been analyzed using QTL techniques in F2 crosses between various lines of chickens. A total of 146 QTLs reaching significance have been reported via genome scans based on marker-QTL linkage analyses  were used in GWAS studies. A total of 16 body composition and meat quality traits were measured or derived.The animal component of this study was conducted in accordance with the Guidelines for the Experimental Animals, established by the Ministry of Science and Technology . Blood was collected from the brachial vein of the chickens at 80 d by venipuncture, using citrated syringes during a routine health inspection at the experimental station of the Chinese Academy of Agricultural Sciences (CAAS). Animal experiments were approved by the Science Research Department  of the Institute of Animal Sciences, CAAS .Br, DMTh, IMFBr and IMFTh), ultimate pH (pHu), and shear force (SF) of the pectoralis major muscle. The measurements and data were obtained as follows:The 728 male Beijing-You chickens were generated from 50 half-sib families. The 50 sires and 250 dams were chosen from conservation stock for this breed and were unrelated. The Beijing-You chickens maintained by the Institute of Animal Sciences, CAAS and have not been subjected to any systematic selection for any trait. All birds were reared in stair-step caging under the same recommended nutritional and environmental conditions. A total of 16 carcass and meat quality traits were measured for the GWAS studies: carcass weight , eviscerated weight (EW), breast weight (BrW), thigh muscle weight (ThW), abdominal fat weight (AbFW), dressed percentage (DP), eviscerated yield, as a percentage of CW (EWP), BrW, ThW and AbFW as percentages of EW , dry matter and intramuscular fat contents of breast and thigh muscle  and slaughtered at d 100 by standard commercial procedures and CW was recorded. The removable adipose tissues surrounding the proventriculus and gizzard along with those located around the cloaca were weighed as AbFW The data for CW and EW were also expressed as percentages on the basis of LW and CW, respectively (DP and EWP). The pHu was the mean of three measurements taken from the left breast muscle after 24-h storage at 4°C . Shear force was determined on breast muscles following Li’s method One-day-old hatchlings with similar genetic background were reared in the same conditions as above. On each of weeks 4, 8, 10, 12, and 14, six to eight birds of similar weight were randomly selected, stunned, and euthanized using approved procedures. Breast muscle was rapidly dissected, weighed, snap-frozen in liquid nitrogen, and stored at −80°C for mRNA extraction.Genomic DNA was extracted from the blood samples using phenol-chloroform and was diluted to 50 ng/µl. Each chicken was genotyped using the Illumina 60 K Chicken SNP Beadchip . Of the total of 728 chickens, four were excluded because sample call rate was <95%. Approximately 20%  of the SNPs were removed for one or more of the following: low call rate (<95%), minor allelic frequency (<0.01) and Hardy-Weinberg equilibrium (HWE) test (p<1E−06), or chromosomal location was unknown. After imposing these constraints, 724 individuals and 45,548 SNP markers that distributed on 30 autosomes and the Z chromosome  were useI) and compressed mixed linear  models Y is the phenotypic value, X is the genotype , F is the family, and K is the relative kinship matrix; Xα were regarded as fixed effects, while Fβ and Kμ were random effects, and e is the random error.The SNPs that were significantly associated with the phenotypic traits were identified using both simple  was constructed from 8,006 independent SNP markers, acquired using Plink v1.07 software 2 threshold of 0.2. The analyses were performed using TASSEL 3.0 software The relative kinship matrix  deviated from normality and Box-Cox or Johnson transformations were applied using Minitab 15 (http://www.minitab.com). Significance thresholds were established from the estimated number of independent SNP markers and LD blocks, defined as a set of contiguous SNPs having pairwise r2 values >0.40. Using this approach, the estimated total number of independent SNP markers and LD blocks was 19,284. The two threshold P-values were therefore set at 5.19E−05  for suggestive significance, and 2.59E−06  for genome-wide significance The raw data for some traits .Quantile-quantile (Q-Q) plots for each trait and Manhattan plots of genome-wide association analyses were produced with R 2.13.2 software  from the GenBank sequences  or suggestive significance (P<5.19E−05) are presented in full in LCORL), leucineamino peptidase 3 (LAP3), quinoid dihydro pteridine reductase (QDPR); LIM domain-binding protein 2 (LDB2), transmembrane anterior posterior transformation1 (TAPT1). The two SNPs on GGA1 are located within dachshund homolog 1 (Drosophila) (DACH1).As detailed in DACH1, was also the same as that associated with CW. The remaining two SNPs were on GGA7 and GGA19, neither was associated with any other trait.In the case of EW, nine significant SNPs were identified by compressed MLM, of which five were of genome-wide significance by GLM analysis. The six SNPs on GGA4 are the same as those found for CW but there were slight differences in which of them were of greater significance . The oneUCHL1) and neuronal acetylcholine receptor subunit alpha-9 (CHRNA9). The SNP on GGA17 was in close proximity (8 kb) to F-box/WD repeat-containing protein 5 (FBXW5). No SNPs associated with the percentage of eviscerated yield (EWP) were found.As seen from TRMT11), nuclear receptor coactivator (NCOA7), tumor protein D53 (TPD52L1), fatty acid binding protein 7 (FABP7) and gap junction protein, alpha 1 (GJA1) genes. Noteworthy are the SNPs with extreme signals (P = 2.37E−15 and 9.78E−13 for BrW and 1.98E−18 and 6.12E−15 for BrP) located near FABP7 and GJA1, accounting for more than 8% of the phenotypic variance of BrW and BrP (Associations identified with these breast muscle traits are shown in  and BrP .NUTF2) and this locus was also associated with ThP. The other SNP for ThP was of genome-wide significance and was on GGA2, just 2 kb from forkhead box N4 (FOXN4).Four SNPs, significantly associated with ThW were identified by compressed MLM and occurred on GGA3, GGA11, GGA2 and GGA16; those on GGA3 and GGA11 were of genome-wide significance . The SNPSNRNP40) and the other was 11.7 kb from pumilio homolog 1 (PUM1). Of the two SNPs identified by compressed MLM to be associated with AbFP, the one on GGA2 was of genome-wide significance by GLM and was 209.8 kb from a human reference gene, human zinc finger protein 521 (ZNF521), with 86.4% sequence identity with the chicken genome. The other detected SNP, on GGA17, had proximity (within 51.6 kb) to ankyrin repeat and SOCS box protein 6 (ASB6)  from small nuclear ribonucleoprotein 40 kDa (U5) (6 (ASB6) .Th) and shear force (SF) of breast muscle.The SNPs associated with four traits related to meat quality are provided in ANGPT1). The SNPs on GGA27 have some proximity to FtsJ homolog 3 ; SNP Gga_rs16205470 was the only SNP related to meat quality shown to have genome-wide significance.Five SNPs of significance were identified, located on GGA2 and GGA27. The three SNPs on GGA2 are in the vicinity of or within angiopoietin 1 (CCK). The SNP on GGA5 is 159.7 kb from Toll interacting protein (TOLLIP).Two significant SNPs were identified and located on GGA2 and GGA5. The SNP on GGA2 is 120.9 kb away from cholecystokinin . The SNP on GGA8 is 8.5 kb from UMP-CMP kinase (CMPK1).The four SNPs on GGA1 are clustered within a 1.30 Mb region with no annotated genes nearby.Six significant SNPs associated with DMGALNT1) is in the vicinity. Two SNPs on GGA4 located within protocadherin 19 (PCDH19) or 286.3 kb away from diaphanous homolog 1 (DIAPH1). The SNP on GGA7 located 143.9 kb from the secreted phosphoprotein 2 (SPP2).Eleven significant SNPs were identified and located on GGA2, GGA4 and GGA7. The eight SNPs on GGA2 are distributed within a 2.06 Mb region and only one gene  located in a 0.65 Mb region for BrW and BrP, the change in GJA1 expression was found to be consistent with that of the breast muscle weight across development  (GJA1 mRNA and BrW was moderate (r = 0.554) and significant at the 0.001 level. It is highly possible that GJA1 is a functional gene for breast muscle development in chickens.Expression of candidate genes detected near associated signals for BrW and BrP in the GWAS analysis was further tested by real-time quantitative PCR (Q-PCR) in breast muscle. After measuring the mRNA content of five genes (14 week) . The corTo identify potential loci and candidate genes affecting chicken body composition and meat quality traits, GWAS studies were performed using a conservation population of Beijing-You (BJY) chickens. The BJY chicken is one representative indigenous breed in China Two statistical methods, compressed mixed linear model (MLM) and generalized linear model (GLM), were implemented to analyze association between SNPs and phenotypes. Emphasis is placed on the associations revealed by the compressed MLM analyses because population structure effect could be controlled and false positives could be reduced with this approach, as shown in Q-Q plots . HoweverTRMT11), nuclear receptor coactivator (NCOA7), tumor protein D53 (TPD52L1), fatty acid binding protein 7 (FABP7) and gap junction protein, alpha 1(GJA1) genes. Of these five genes, only the change of GJA1  expression was consistent with that of BrW across development  and the correlation between mRNA level and BrW was significant (p<0.001). Thus, it is highly possible that GJA1 is a functional gene for chicken breast muscle development. This result might supply a novel functional gene for breast muscle development and extent the known function of GJA1 which has been found to play a role in skeletal form GJA1 for BrW and BrP.Breast muscle yield is the most important carcass component in meat-type chickens because of the high premium paid by consumers. Of special interest, one important region (61.83 Mb–68.57 Mb) on GGA3 was identified as being associated with BrW and BrP. Most of the SNPs were located within or near tRNA methyltransferase 11 . Polymorphisms in LCORL have been detected to associate with human skeletal frame size, linear growth LDB2) has been associated with chicken body weight (7–12 wk) and average daily gain (6–12 wk) in another GWAS study For CW and EW, one consistent region was identified (about 78.47 Mb to 79.53 Mb) on GGA4, which corresponded to QTL regions previously reported DACH1 and this gene located near a QTL region known to be related to CW DACH1 is a target of FGF signaling during limb skeletal development On GGA1, significant SNPs associated with both CW and EW were found within MYCN. These two associations are consistent with the relatively high correlation (r2 = 0.75) found between two traits, which possibly reflects their being controlled by common loci.The SNP Gga_rs14402759 on GGA3 was found to associate with both ThW and LW, and this locus was near Br and were located in the vicinity of or within ANGPT1. Angiopoietin-1 (ANGPT1) is an angiogenesis factor that is also an important modulator of skeletal muscle function FTSJ3 (a putative ortholog of yeast Spb1p) and conditional knockdown revealed that depletion of FTSJ3 affects HEK293 cell proliferation and causes pre-rRNA processing defects Three significant SNPs on GGA2 were found to associate with DMTh, four SNPs of suggestive significance on GGA1 were clustered within a 1.3 Mb region, currently lacking any annotated gene. Another SNP on GGA2 was located near SOCS6, which is involved in the regulation of glucose metabolism and plays an important role in regulating insulin action in vivoFor DMBr and cholecystokinin (CCK) was found nearby. The gut hormone CCK plays a multiplicity of roles, including those influencing digestion of fat A SNP on GGA2 was associated with IMFGALNT1) has been found near this region. The encoded protein catalyzes the transfer of N-acetylgalactosamine  from UDP-GalNAc to the hydroxyl group of a serine or threonine residues on proteins with O-linked glycosylation, a common post-translational modification. Two significant SNPs were found on GGA4, within PCDH19 and near DIAPH1, respectively, and another on GGA7 near SPP2. Protocadherin-19 (PCDH19) plays a role as an adhesion protein in optic nerve fiber bundling, optic nerve targeting, and/or synapse formation DIAPH1 encodes a protein that may have a role in the regulation of actin polymerization in hair cells of the inner ear. The SPP2 gene encodes a secreted phosphoprotein that is a member of the cystatin superfamily. These proteins play important roles in tumorigenesis, stabilization of matrix metalloproteinases, glomerular filtration rate, immunomodulation, and neurodegenerative diseases.For ultimate pH, eight SNPs within a 2.06 Mb region on GGA2 were identified although only one known gene  plots of the GLM (red dots) and compressed models (blue dots) for carcass and meat quality traits. Plotted on the x-axis are the expected p-values under the null hypothesis and on the y-axis are the observed p-values. A: CW, carcass weight; B, EW, eviscerated weight; C, DP, dressed percentage; D, EWP, percentage of eviscerated yield; E, BrW, breast muscle weight; F, BrP, percentage of breast muscle; G, ThW, thigh muscle weight; H, ThP, Percentage of thigh muscle; I, AbFW, weight of abdominal fat; J, AbFP, percentage of abdominal fat; K, DMBr, dry matter content in breast; L, IMFBr, intramuscular fat in breast; M, DMTh, dry matter content in thigh; N, IMFTh, intramuscular fat in thigh; O, pHu, ultimate pH, P, SF, shear force of breast muscle.(TIF)Click here for additional data file.Table S1Information for all significant SNPs related to body composition and meat quality traits.(XLSX)Click here for additional data file.


A recent, large genome-wide association study (GWAS) of European ancestry individuals has identified multiple genetic variants influencing serum lipids. Studies of the transferability of these associations to African Americans remain few, an important limitation given interethnic differences in serum lipids and the disproportionate burden of lipid-associated metabolic diseases among African Americans.We attempted to evaluate the transferability of 95 lipid-associated loci recently identified in European ancestry individuals to 887 non-diabetic, unrelated African Americans from a population-based sample in the Washington, DC area. Additionally, we took advantage of the generally reduced linkage disequilibrium among African ancestry populations in comparison to European ancestry populations to fine-map replicated GWAS signals.CILP2/SF4, STARD3, LPL, CYP7A1, DOCK7/ANGPTL3, APOE, SORT1, IRS1, CETP, and UBASH3B). Through trans-ethnic fine-mapping, we were able to reduce associated regions around 75% of the loci that replicated.We successfully replicated reported associations for 10 loci (Between this study and previous work in African Americans, 40 of the 95 loci reported in a large GWAS of European ancestry individuals also influence lipid levels in African Americans. While there is now evidence that the lipid-influencing role of a number of genetic variants is observed in both European and African ancestry populations, the still considerable lack of concordance highlights the importance of continued ancestry-specific studies to elucidate the genetic underpinnings of these traits. Given the generally greater linkage disequilibrium (LD) among European-ancestry individuals, it is expected that index SNPs tag larger regions than they would among African ancestry individuals. Therefore, the functional variant tagged by the index SNP in European ancestry individuals might not be in LD with the same SNP in African-ancestry individuals, motivating different analytical strategies . In the present study, we used more robust analytic strategies to investigate the transferability of reported genetic associations for serum lipids to African Americans. We also exploited these interethnic differences in LD to conduct fine-mapping of the replicated loci.The distribution of serum lipid concentrations has well-established clinical utility as a risk factor for a range of metabolic diseases, including cardiovascular disease (CVD) and type 2 diabetes (T2D). As such, great effort has gone into uncovering the genetic epidemiology of serum lipids, including a recent meta-analysis of 46 genome-wide studies comprising more than 100,000 individuals of European ancestry  and T2D Ethical approval for this study was obtained from the Howard University Internal Review Board. Written informed consent was provided by all participants. The Howard University Family Study (HUFS) is a population-based study of African Americans in the Washington, DC metropolitan area . Unrelatp-value <1 × 10-3 . A total of 808,465 autosomal SNPs passed these filters. The average call rate for this set of SNPs was 99.55%, with concordance of blind duplicates of 99.74%. A check for population stratification was conducted using non-parametric clustering of genotypes as previously described [Genome-wide genotyping was performed using the Affymetrix® Genome-Wide Human SNP Array 6.0. Genotype calls were made using Birdseed, version 2 . SNPs wehttp://www.1000genomes.org/). SNPs were excluded if they had a missingness rate ≥10%, Hardy-Weinberg test p-value <1 × 10-3, or a minor allele frequency <0.01. Imputed SNPs for which no rsid is currently available are described with a “chromosome:position” nomenclature (position refers to NCBI36 build). After quality control filters, 5,396,780 markers were included in the analysis.Power calculations were performed using QUANTO . When thp-value was ≤0.05 [2 ≥0.30 . P-valurted SNP , and an http://csg.sph.umich.edu/locuszoom/) and comparison of haplotype block structure between the CEU and YRI for SNPs of interest . Finally, we examined the other SNPs on the array and the imputed SNPs for any association with a genome-wide significant p < 2.5 × 10-8[To take advantage of the generally decreased haplotype size in African ancestry populations, fine-mapping of replicated signals was attempted using the following strategies: inspection of regional plots of association to identify SNPs with a stronger signal than the index SNP in HUFS , with a mean age of 46 years and a mean BMI of 28 kg/mIRS1, CETP, and UBASH3B , stronger association was observed for LPL intronic SNP rs12679834 (p = 0.001). While these two SNPs are in the same 53 kb haplotype block among the CEU, rs12678919 is not associated with a haplotype block in the YRI and rs12679834 is associated with a much smaller haplotype block (8 kb). This result suggests that the causal SNP is more closely linked with rs12679834 than rs12678919, and dramatically reduces the region for further investigation from 53 kb to 8 kb  was not associated with TC in HUFS (p = 0.10), but rs6589939, which was in the same 40 kb haplotype block in the CEU, was (p = 0.005). Among the YRI, neither rs6589939 nor rs7941030 was in a haplotype block. In this study sample, an intronic SNP in NSMAF, rs10088541, had a lower p value than index SNP rs2081687 . While these SNPs are correlated among the CEU (r2 = 0.75), they are not correlated in the HUFS samples (r2 = 0.03): the causal SNP may be more closely linked to rs10088541  in HUFS significantly reduces the region of interest for this signal. While this SNP is in a 16 kb haplotype block in the CEU, the block is reduced to less than 500 bp among the YRI , rs711794 near ZAK , and rs1047163, a 3’ UTR variant near HS1BP3 .A search for other hits in the full set of genotyped and imputed SNPs showed that no SNPs reached genome-wide significance  with TG, rs2081687 (CYP7A1) with LDL, rs2972146 (IRS1) with HDL, and rs4420638 (APOE) with HDL (this association replicated in PAGE). CARe, PAGE, and HUFS all support the association of two loci with HDL in African Americans: rs3764261 (CETP) and rs4420638 (APOE).We identified 10 loci that influence lipid levels in this cohort of African Americans. Of these, 7 were identified through testing the reported SNP while an additional 3 loci were identified using an LD-based strategy employed to account for the potential non-transfer of association signals across populations with different ancestral background . Teslovim [et al.). The PAPossible explanations for the lack of transferability of findings include differences in allele frequencies . ApoE, associated with HDL, is a main lipoprotein of the chylomicron and is involved in the catabolism of triglyceride-rich lipoprotein constituents; defects in the gene encoding this protein result in familial dysbetalipoproteinemia . CETP, associated with HDL, plays multiple roles in HDL metabolism and in the reverse cholesterol transport pathway [CETP SNP (rs247617) that was unlinked with the replicated SNP was one of the top hits for HDL in our discovery GWAS  – HDL, which was replicated in the CARe consortium analysis, was not genotyped or imputed in this sample, but a local SNP, rs6601299 (r2 = 0.86) was associated in the same direction, but just above the significance level (p = 0.07). As the previous publication is a meta-analysis with a very large sample size, it was able to detect small effect sizes, which would be difficult to replicate in a GWAS with a more limited sample size. As a result, more accurate estimates of transferability will await the aggregation of African ancestry GWAS into a suitably large meta-analysis. Of the 10 replicated loci in this study, only 1 had been previously identified in an individual GWAS  of the European ancestry-identified loci to African Americans. Notably, conclusive inferences about the transferability of all of the previous findings are precluded by the limitations in replication attempts conducted in African Americans thus far in terms of relative sample size and coverage of African ancestry genetic diversity by currently available GWAS chips. Further work in African ancestry populations will be necessary to completely evaluate these loci.The authors declare that they have no competing interests.AA and ARB analyzed the data, prepared figures and tables, and drafted the manuscript. KGM also conducted analyses. APD and HH assayed serum lipids. GC prepared the genome-wide genotype data. GC, DS, and AA conducted the genome-wide imputation of genotypes. AH, NPG, and MFC genotyped the samples. CNR, AA, ARB, and KGM contributed to the conception and design of the study. All authors read, provided important feedback, and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/13/88/prepubAllele Frequency Comparison for Tested Loci that were not Transferable.Click here for fileTop SNPs for GWAS of Total Cholesterol.Click here for fileTop SNPs for GWAS of HDL.Click here for fileTop SNPs for GWAS of LDL.Click here for fileTop SNPs for GWAS of TG.Click here for file


Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between extracellular materials.The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber  Echinoderms have unique collagenous structures called catch connective tissues or mutable collagenous tissues. The tissues can extensively change their mechanical properties, such as elasticity and viscosity, within a few minutes under the regulation of their nervous systems Actinopyga mauritiana revealed that the tissue can adopt three different states 2+ (ASW) is in the standard state, that stimulated with acetylcholine or artificial sea water with a high concentration of K+ (KASW) is in the stiff state, and that in the Ca2+-free ASW (CFASW) is in the soft state.The holothurian body wall dermis is a typical catch connective tissue that shows rapid and reversible changes in its mechanical properties in response to various stimuli. Extensive studies on the dynamic mechanical properties of the dermis of the sea cucumber C-tensilin), first isolated from body-wall dermis of the dendrochirotid sea cucumber Cucumaria frondosaH-tensilin) from the dermis of the aspidochirotid sea cucumber Holothuria leucospilotaH. leucospilotaStiffeners and softeners of sea-cucumber dermis have been isolated from holothurian body walls. Three stiffeners have been found. One is pentapeptide NGIWYamide Apostichopus japonicusStudies on softeners have lagged behind those of stiffeners. The presence of some softening factors has been shown in the coelomic fluid The current hypothesis for the stiffness-change mechanism of catch connective tissue is as follows: some chemicals, which affect the interacting forces between macromolecules in the extracellular materials, are secreted from certain cells whose secretory activities are controlled through nerves Stichopus chloronotus was used, because its softening response is so intense that it “melts” into a slimy mass The aim of the present study was to find a softener that works directly on the extracellular materials of the sea-cucumber dermis. For this purpose we used “Triton models” of dermis in which cells have been disrupted while the extracellular components remain almost intact. We developed two kinds of Triton models, one corresponding to the standard-state dermis and the other to the soft-state dermis. Extracts from sea-cucumber body wall were fractionated through a series of columns to isolate proteins that softened the standard-state model. For the extraction, the dermis of the aspidochirotid sea cucumber We succeeded in purifying a novel protein. It softened Triton models in dynamic mechanical tests, and so it is the first softener shown to work on extracellular materials directly.Stichopus chloronotus, Holothuria leucospilota) are invertebrates, thus no specific permits were required for the present study. The field studies were performed in a location that was neither privately owned nor protected. The field studies did not involve endangered or protected species.Sea cucumbers used in present study . We checked whether the Triton treatment had disrupted the cellular functions by using artificial sea water with a high concentration of potassium (KASW) that had the following composition: 0.4 mol l−1 NaCl, 110 mmol l−1 KCl, 50 mmol l−1 MgCl2, 10 mmol l−1 CaCl2 and 20 mM Tris-HCl, pH 8.0. The Triton-treated dermis did not change stiffness in response to KASW. Because KASW is a potent stiffening agent that affects mechanical properties through effects on cellular activities H. leucospilota which were isolated from live individuals without Triton treatment were also used for mechanical tests. We did not use fresh or TFT dermis of S. chloronotus because they showed such intensive strain softening that they “melted” soon after the dynamic mechanical tests had begun.Tissue samples used for the mechanical tests were Triton-treated dermis whose cells had been disrupted by Triton X-100. Triton-treatment was performed as described previously Dynamic mechanical tests were performed as described previously Tensile force was monitored by a load cell. The maximal stiffness of the sample in one stretch-compression cycle was proportional to the maximal tensile stress which was used as a measure of stiffness. Otherwise stated, it was expressed as a relative value in percent, normalized by the peak value in the steady state as 100% (see RESULTS).H-tensilin was introduced during the steady phase that will be defined in RESULTS. ASW in the trough was replaced with the test solution whose ionic composition was adjusted to the same one as that of ASW. The final concentration of the purified softener was adjusted to 60 µg ml−1. The mechanical tests were performed at room temperature (20–24°C), which did not change by more than 1°C during any one mechanical test.After the dermal sample was fixed in the experimental trough, 50 µl of ASW was introduced. We started measurements 10 min after ASW introduction. A test solution that contained softener or S. chloronotus to find out fractions containing the softener. When relative stiffness decreased to less than 80% of that just before the application of a fraction in 10 min, we regarded that the fraction contained the softener.We tested each fraction eluted from chromatography columns by dynamic mechanical test on the Triton-treated dermis of −1 NaCl solution containing 20 mmol l−1 Tris-HCl (pH 8.0) at 4°C and stirred with a stainless steel bar for two hours. This procedure caused the dermis to “melt” into a homogenate, which very probably corresponded to the melting of body walls when this sea cucumber species was subjected to strong mechanical stimuli −1 Tris-HCl, pH 8.0 and filtered through the micro-filtration device Vivaspin 2  to remove viscous materials. The filtrate thus obtained was the crude extract, which was applied to a Mono-Q chromatography column  equilibrated with the same buffer as the dialysis solution. The column was washed with the same buffer containing no NaCl, and eluted with the buffer containing increasing concentration of NaCl (the stepwise elution 0 and 0.1 mol l−1 followed by the gradient elution 0.1–1.0 mol l−1). The fractions from the stepwise elution were pooled and applied on two serially-attached gel-filtration columns of Superose 12 10/300 GL (Pharmacia Biotech) equilibrated with 0.5 mol l−1 NaCl and 20 mmol l−1 Tris-HCl, pH 8.0. The fractions with softening activities were collected, and concentrated with ultra-filtration device Vivaspin 500 . Concentrated sample was applied on a Superdex Peptide 10/300 GL gel-filtration column (Pharmacia Biotech) equilibrated with the same solution as used in Superose 12 column, and the purified softener fraction was collected. The concentration of the softening protein was measured with Qubit protein assay kit  using bovine serum albumin as a standard.Twenty gram dermal pieces were placed in the same volume of 5 mol lwww.ncbi.nlm.nih.gov).Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Schägger and von Jagow H-tensilin was purified from the dermis of H. leucospilota as described previously −1 NaCl, 10 mmol l−1 EGTA and 20 mmol l−1 Tris-HCl, pH 8.0. Its supernatant after centrifugation was precipitated with 60% saturated (NH4)2SO4. The precipitate was dissolved in 0.5 mol l−1 NaCl and 20 mmol l−1 Tris-HCl, pH 8.0, and dialyzed against the same solution. The crude extract was then applied to a Mono-Q chromatography column equilibrated with the same solution. Stepwise elution fractionation between 0.65 mol l−1 and 1.0 mol l−1 NaCl was used for purification of H-tensilin. This fraction was further applied on a Superose 6HR 10/30 gel-filtration column (Pharmacia Biotech) equilibrated with 0.5 mol l−1 NaCl and 20 mmol l−1 Tris-HCl, pH 8.0, and the H-tensilin fraction was collected. H-tensilin concentration was measured with Qubit protein assay kit.H. leucospilota, and the aggregation assay was performed in buffer without Ca2+  as described previously H-tensilin. In control samples, buffer without Ca2+ was added to the suspending solution instead of the softener. The final concentrations of the softener and H-tensilin were 11.8 µg ml−1 and 20 µg ml−1 respectively which were adjusted to the same number of molecules of these two agents in the suspending solution. We also studied the effect of repetitive freeze-thawing on the tensilin-induced aggregation. In test samples, the solution with the aggregate was frozen to −20°C, and then thawed under room temperature; the freeze-thawing was repeated three times. In control samples, solution with the aggregate was stored at 4°C while test samples were under freeze-thaw treatment which took about 24 hours.Collagen fibrils were isolated from the dermis of −1 NaCl , nuclear hormone receptors-153, mitochondrial 2-oxoglutarate/malate carrier protein and two amyloid-like proteins, all of which had higher bit scores than those of echinoderms. Proteins with the highest bit scores among whole organism and among echinoderms were compared to the softener by alignment those sequences , minichromosome maintenance 3-associated protein, HES family transcription factor of starfish, tumor necrosis factor receptor, and 4-hydroxyphenylpyruvate dioxygenase. All proteins but HES family transcription factor were derived from sea urchin equences . The ide−1 caused rapid decrease in stiffness. −1. The relative stiffness 10 min after the application of the softener was 34.7% of that just before the application of the softener . Stiffness recovered partially when the softener was washed out with ASW  stiffness reached a peak that was several times larger than the stiffness value just after the test had begun . Subsequwith ASW . The recwith ASW ; the relH. leucospilota. The Triton-treated H-dermis also showed two phases, the early rapidly increasing phase and the later steady phase, in dynamic tests; the peak value of the latter phase was held for more than an hour  caused marked stiffening. The increased stiffness was maintained or stiffness decreased only slowly and slightly when tensilin was washed out by ASW. −1) and tensilin (10 µg ml−1) were applied successively. We first showed that the softener had no effect on the TFT dermis. Then the dermis was stiffened by tensilin. The introduction of the softener to this stiffened dermis caused a rapid decrease in stiffness to the level similar to that before tensilin application. The second application of tensilin after having washed out the softener increased stiffness again to the level comparable to that attained by the first application of tensilin . Thus we regarded that the softener is novel protein. We coin the name “softenin” after the function of this new protein. Because softenin of S. chloronotus was also effective on H. leucospilota, softenin and its homologues are likely common softeners in sea cucumbers.We purified a protein from the sea cucumber We used Triton-treated dermis whose cells had been disrupted by the detergent Triton X-100 for bioassay in order to find chemicals that exerted their effects on the mechanical properties of extracellular materials. As the stiffness value of Triton-treated dermis was not different from that of fresh dermis in the standard state, it is very likely that Triton-treated dermis corresponded to the dermis in the standard state; it was not in the stiff state, which is much stiffer We tried to make a cell-disrupted “model” dermis that corresponded to the dermis in the soft state. We added freeze-thaw treatment to Triton-treated dermis expecting that this extra freeze-thaw treatment might disrupt some mechanical interactions between macromolecules to make the dermis softer. TFT dermis had lower stiffness as was expected. We inferred that TFT dermis corresponded to the soft-state dermis not only because it was softer but also because it showed softening by repetitive straining , Table 1H-tensilin, and softenin are involved. H-tensilin is hypothesized to form crossbridges between collagen fibrils and softenin is supposed to compete for the binding sites of H-tensilin to collagen (Softenin had no effect on TFT dermis, which is a reasonable result if softenin was a softener of the standard dermis but not of the already soft dermis. TFT dermis stiffened by tensilin was softened by softenin only to the stiffness value before the application of tensilin, which implied that softenin cancelled the stiffening action of tensilin . The aggcollagen .Apostichopus japonicusH-tensilin S. chloronotus, which will be used for RT-PCR to obtain the objective cDNA fragments. We will analyze its total base alignment to clone coding DNA of softenin. Another strategy we are going to adopt after having succeeded in sequencing several tryptic fragments is to find a softenin-like protein in the genome of the S. purpuratus. Catch connective tissue is found also in sea urchins and thus we can expect the presence of the sequence of softenin-like protein in the genome. Although we could not find the sequence with high similarity scores in the already characterized proteins, more sequence data than the present short N-terminal sequence might pick up the whole sequence of sea-urchin softenin from the characterized and uncharacterized sea-urchin proteins.Some sea cucumbers have been known to show extensive softening of body walls in which they become a viscous mass in response to variety of environmental and mechanical factors


Solanum tuberosum), invertases are involved in 'cold-induced sweetening' of tubers, an adaptive response to cold stress, which negatively affects the quality of potato chips and French fries. Linkage and association studies have identified quantitative trait loci (QTL) for tuber sugar content and chip quality that colocalize with three independent potato invertase loci, which together encode five invertase genes. The role of natural allelic variation of these genes in controlling the variation of tuber sugar content in different genotypes is unknown.Invertases are ubiquitous enzymes that irreversibly cleave sucrose into fructose and glucose. Plant invertases play important roles in carbohydrate metabolism, plant development, and biotic and abiotic stress responses. In potato  analysis in an association-mapping population of 219 tetraploid individuals. Haplotype modeling revealed two to three major haplotypes besides a larger number of minor frequency haplotypes. cDNA alleles associated with chip quality, tuber starch content and starch yield were identified.For functional studies on natural variants of five potato invertase genes we cloned and sequenced 193 full-length cDNAs from six heterozygous individuals (three tetraploid and three diploid). Eleven, thirteen, ten, twelve and nine different cDNA alleles were obtained for the genes Very high natural allelic variation was uncovered in a set of five potato invertase genes. This variability is a consequence of the cultivated potato's reproductive biology. Some of the structural variation found might underlie functional variation that influences important agronomic traits such as tuber sugar content. The associations found between specific invertase alleles and chip quality, tuber starch content and starch yield will facilitate the selection of superior potato genotypes in breeding programs. Invertases are ubiquitous enzymes that irreversibly cleave sucrose into the reducing sugars fructose and glucose. Plant invertases not only play an important role in the partitioning of carbon between source tissue (photosynthetic leaves) and heterotrophic sink tissues such as seeds, tubers and fruits, they also function in plant development and in responses to biotic and abiotic stress. Three types of invertase isoenzymes, which are encoded by small gene families, are regularly found in plants. Cell wall-bound acidic invertases cleave sucrose in the apoplastic space (apoplastic invertases). Soluble acid invertases are located in the vacuole (vacuolar invertases), whereas soluble neutral invertases are located in the cytoplasm ,2.Solanum tuberosum), carbon is stored as starch polymers in tubers. Besides starch, tubers also contain small amounts of sucrose, glucose and fructose. The amounts of starch and sugars present in tubers depend on the genotype and on environmental factors. Storage at low temperature (e.g. 4°C) for several weeks leads to conversion of a small fraction of starch into sugars in tubers, with consequent accumulation of glucose and fructose, in particular [In the potato  for tuber starch and sugar content on the one hand ,16 and lvertases . Two tan of 9 kb . InvGE amosome 9 . The Invd tomato , InvGE/I'pCD141'  as Invapand LIN8 . GenomicPain-1 gene on chromosome III [Association mapping in populations of tetraploid potato varieties and breeding clones has revealed 'single-strand conformation polymorphisms' (SSCPs)  in inversome III . These msome III . Unfortusome III  nor highS. tuberosum lines H82.337/49 (P18), H80.696/4 (P40) and H81.839/1 (P54) [Invertase alleles were cloned from the tetraploid cultivars Satina, Diana and Theresa, and from the diploid /1 (P54) . The tet/1 (P54) , because/1 (P54) . Plants /1 (P54) .free™ Kit (Ambion). RNA concentration and quality were analyzed by measuring the A260 nm/A280 nm (1.8 - 2.0) and A260 nm/A230 nm (2 - 3) ratios using a Nanodrop ND-1000 spectrophotometer . RNA integrity was tested on 1% agarose gels loaded with 300-500 ng of total RNA. Total RNA was stored at -80°C. First-strand cDNA was synthesized according to the supplier's protocol from 2.0 μg of total RNA, using 200 U of Superscript™ II Reverse Transcriptase (Invitrogen) per reaction and 500 ng of oligo(dT)16-18  as primers. First-strand cDNA was treated with RNase H  for 20 min at 37°C. First-strand cDNA (1 μl per reaction) was then used for allele amplification and cloning.Total RNA was extracted from leaves and flowers using the ToTally RNA Isolation Kit  following the supplier's protocol. Total RNA was extracted from tuber tissue powdered in liquid nitrogen, using the Plant RNA Isolation Kit from Invitrogen  following the supplier's protocol. Tuber RNA was further purified by high-salt precipitation to remove polysaccharides and by lithium chloride precipitation to remove low-molecular-weight RNA. The RNA solution was adjusted to 1 mL by adding RNase-free water, mixed with 250 μl isopropanol and 250 μl high salt solution  and incubated on ice for 2 h. RNA was recovered by centrifugation at 13,000 rpm for 30 min at 4°C. The pellet was rinsed with 70% ethanol, and centrifuged at 13000 rpm for 5 min at 4°C. After removing the ethanol, the pellet was air-dried and dissolved in RNase-free water at a minimum concentration of 200 ng total RNA per μl. High-molecular-weight RNA was precipitated by mixing with 0.5 volumes of 5 M LiCl and incubating on ice overnight at 4°C. RNA was collected by centrifugation as above, rinsed with 70% ethanol, dried and dissolved in 20-50 μl RNase-free water depending on pellet size. All RNA samples were further purified using the DNA-L29099, X70368 (Pain-1), AJ133765 (InvGE and InvGF), Z21486 (InvCD111) and Z22645 (InvCD141). Pain-1 alleles were amplified using as template first-strand cDNA from tubers stored for 25 days at 4°C. InvGE and InvGF alleles were amplified from first-strand cDNA templates obtained from leaves and flowers. InvCD111 and InvCD141 alleles were amplified from leaf cDNA templates. Oligonucleotides were purchased from Invitrogen , Sigma-Aldrich Chemie  and Operon Biotechnologies . Polymerase chain reactions (PCR)  were performed using the Fast Start High Fidelity PCR System  or KOD Hot Start DNA Polymerase  according to the supplier's protocols. PCR products were purified with the High Pure PCR Purification Kit  and ligated into the pGEM®-T/T Easy vector  following the supplier's protocols. Competent cells of E. coli strains DH5α and DH10B  were transformed with recombinant plasmids [http://multalin.toulouse.inra.fr/multalin/multalin.html). Due to the large number of cDNAs sequenced, most variants were represented at least two times in independent PCRs primed with first-strand cDNA from the same genotype. cDNA alleles were then defined based on the consensus sequences of all clones obtained from an individual genotype. In some cases, the number of full-length cDNA sequences per genotype was low  by primer walking on the BACs using the dideoxy chain-termination method [Pain-1 gene by primer walking was performed on the BAC selected for complete sequencing, whereas the gene InvCD141 was sequenced using BAC BC37c23. BAC sequences were annotated using the Apollo Genome Annotation and Curation Tool, version 1.9.8 [The BAC  libraries BA and BC, both constructed from high-molecular-weight genomic DNA of the diploid, heterozygous genotype P6/210 and arrayed on high density filters, were screened by filter hybridization with labeled probes for cDNAs  [pCD141  as descr [pCD141 ,33. Posi [pCD141 . In addin method . Sequencon 1.9.8 .Phylogenetic trees were generated using the maximum parsimony method based on a Clustal W amino acid alignment  of all i®  and custom sequenced at the Core Facility for DNA Analysis of the Max-Planck Institute for Plant Breeding Research. The dideoxy chain-termination sequencing method was employed using an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and an ABI PRISM 3730 automated DNA Sequencer . SNPs were identified by sequence alignment and visual examination of the sequence trace files for overlapping base-calling peaks. In each tetraploid individual bi-allelic SNPs were assigned to one of five allelic states (two homozygous and three heterozygous). The SNP allele dosage in heterozygous individuals  was estimated from the relative heights of the overlapping base-calling peaks, both manually and with the Data Acquisition and Analysis Software DAx . Pyrosequencing [Pain1_SNP1544 alleles, the following primers were used to generate an amplicon of 252 bp: Forward : 5'-GGACCATTTGGTGTCGTTGT-3', reverse: 5'-(biotin)TCTTCCTCCTTGAGCAAAGC-3'. The sequencing primer was 5'-CGTTGTAATTGCTGATCA-3'.Amplicons were generated from genomic DNAs of the heterozygous individuals of the ALL population with locus-specific primers Table . The ampquencing  was carrWithin the SATlotyper (v.1.0.5) software  the SAT SNPs were tested for association with the phenotypic values using the general linear model (GLM) procedure in SPSS 15.0 . The model used wasOrigin is a factor (fixed) with four classes to identify the origin of each genotype in the ALL population from one of three breeding companies or from the standard varieties [Marker is a factor (fixed) with five levels, corresponding to the five SNP allele dosages: 0 for allele absent, 1, 2, 3 and 4 for allele present in simplex, duplex, triplex or quadruplex dosage. Population structure has been evaluated and described in [where y* stands for the adjusted phenotypic means . Origin arieties . Marker ribed in .InvGE and InvGF at the Invap-b locus on chromosome IX is already known [Pain-1 and Invap-a have been reported. We therefore isolated, sequenced and annotated the BAC clones BC149o15 (HQ197978) and BC163l15 (HQ197979), which were selected from BAC libraries based on cross-hybridization with Pain-1 and InvCD141 probes. In addition to 454 sequencing of whole BACs, the genes Pain-1 and InvCD141 were also sequenced by the dideoxy chain-termination method and primer walking. BC149o15 contained one full-length copy of the Pain-1 gene. The Pain-1 sequences obtained from the same BAC by two different sequencing techniques (454 and Sanger sequencing) differed by a six-nucleotide insertion in intron 2. The Pain-1 gene consists of seven exons and six introns and is around 4 kbp long  revealed a gap of around 1 kbp in the assembly of the 454 sequences in intron 2. Besides that, the two sequences differed by five nucleotides. The full annotation of BACs BC149o15 and BC163l15 is shown in Table S1 in additional file Pain-1, InvCD141 and InvCD111 are available as GenBank accessions HQ110080, HQ110081 and HQ197977, respectively.Whereas the genomic organization of the tandemly duplicated genes dy known , no genod pCD141 . The twoStpain1_a from cv Russet Burbank [Stpain1_c from cv Kufri Chipsona and AAQ17074 = Stpain1_b from an unknown genotype) in the alignment uncovered sixteen additional SNPs. Sequencing of exons 1, 3 and 7 of Pain-1 in the 34 standard varieties included in the association mapping population ALL identified four further SNPs. The total of 78 SNPs included one tri-allelic SNP and resulted in amino acid changes at 35 positions, corresponding to 5.5% of the deduced Pain-1 protein sequence . Conversely, four additional SNPs absent in the cDNA alleles were detected and scored in the amplicon sequences of the ALL population . The best correspondence between presence/absence of SNP alleles and the associated SSCP markers in the ALL population was found for the SNP alleles in group a , b (SNP528), c (SNPs 612 and 1068) and d (SNPs 612 and 637), a novel allele x not found among the cDNA clones (SNP 825), and the individual alleles Sa (SNP741), P18b (SNP1050) and Stpain1-a (SNP639 from cv Russet Burbank). Haplotypes were modeled using SATlotyper [A, B and C with frequencies higher than 10% accounted for 60% of all chromosomes in the population (4 × 189 = 756), whereas 35% were accounted for by 12 haplotypes with frequencies between 1% and 10%. Among the latter were five haplotypes that included the associated SNP alleles C552, A718, A1544 and T741. Five haplotype models were verified by corresponding cDNA clones, whereas the remaining ten haplotypes were novel .Fifty-nine es Table , and suble InvGF , we founInvGE cDNAs identified 13 different cDNA alleles encoding 12 amino acid sequences  [InvGE protein sequence  with TSC and TSY . Fourteen haplotypes with frequencies between 1% and 10% accounted for 60% of the chromosomes, including the associated alleles Sa and Da. Six haplotype models were compatible with cDNA sequences, whereas the remaining eleven haplotypes were new.Comparative sequence analysis of the invGE-c)  identifieviously  that HisInvGE-6f . One newInvGF, ten cDNA alleles were identified that coded for eight different amino acid sequences  [InvGF protein. Five similarity groups were distinguished. As in the case of InvGE, group a was the most divergent and group d was the most heterogeneous. The a and d alleles of InvGE and InvGF might be part of the same haplotype block. The InvGF group a alleles are therefore likely to correspond to the marker InvGF-4d.For es Table . AlignmeinvGF-b)  revealedInvap-a locus were cloned from leaf tissue. Fewer clones were sequenced than in the case of the loci Pain-1 and Invap-b. Twelve InvCD141 cDNA alleles (11 amino acid sequences) were represented among 28 sequences from six genotypes, and 9 InvCD111 cDNA alleles (8 amino acid sequences) were obtained from 14 sequences of five genotypes (Table StinvCD111-a) [StinvCD141-d) [InvCD141) including three tri-allelic SNPs, and 71 SNPs (InvCD111) caused 32 and 36 amino acid changes, respectively, equivalent to 5-6% protein diversity  were all in high linkage disequilibrium with each other. The presence/absence of this Sa-specific haplotype , indicating that the cDNA allele Sa corresponds to pCD141-3c. Association analysis of the SNPs confirmed Sa as an allele that is negatively associated with chip quality and tuber starch content. In addition, one novel, positive association of InvCD141-G765 with CQS, TSC and TSY was detected (Table InvCD141 haplotype models (Table Sa (haplotype E), whereas the remaining 14 haplotypes were new.Invertase cDNA alleles at the es Table . Two addCD111-a)  and Z226CD141-d) . One hunty Table  and 11. Pain-1, InvGE, InvGF, InvCD141 and InvCD111 (S. tuberosum) and seven tomato invertase genes from S. lycopersicum and S. pennellii , which formed a sixth branch, the tomato genes Slpain1-a (AAB30874), SpLIN5-a (CAB85898), SlLIN5-a (CAB85897), SlLIN7-a (AAM22410), SlLIN6-a (BAA33150) and SlLIN8-a (AAM28822) clustered with the respective orthologous potato genes. The interspecific distances between potato and tomato invertases were larger than the intraspecific distances between potato invertase alleles. Pain-1 was more closely related to the gene pair InvCD111/InvCD141 than to InvGE/InvGF.A phylogenetic tree was constructed based on the amino acid sequences deduced from 46 full-length cDNA sequences of i Figure . The trePain-1 and InvCD111 fell within this range with one SNP per 24 and 25 bp, respectively. The highest variability, with one SNP per 13 bp, was observed in the InvGE gene. InvGF and InvCD141, both with one SNP per 17 bp, also had higher than average variability. A total of 479 SNPs were detected, and nine (1.6%) were tri-allelic. The 55 identified sequence variants represent a minimum estimate of the number of invertase alleles present in the six genotypes. Other alleles may have been missed due to template bias during PCR amplification [InvCD141 and InvCD111 in some genotypes. The sequence variants encode 46 distinct invertase proteins that differ from each other by between 4 and 9%. We took various precautions to eliminate sequence variants resulting from mistakes in reverse transcription or PCR. High-fidelity Taq polymerases were used to minimize PCR-derived errors, and cDNAs were cloned from different PCR reactions. Three quarters of the cDNA alleles were supported by at least two cDNA sequences, and alleles were defined based on consensus sequences of all cDNA clones derived from an individual genotype, eliminating singletons . Although we cannot prove that all of the 479 SNPs are authentic, it is highly unlikely that more than a tiny minority of the SNPs observed were generated in the test tube. The generally high level of DNA polymorphism in the genome of Solanum tuberosum is well documented [StAOS2) have been cloned from three diploid genotypes and functionally characterized [Solanum lycopersicum). The two genomes are highly colinear, but tomato shows very little intraspecific variation [Analysis of 193 cDNA sequences obtained from three tetraploid and three diploid potato genotypes revealed a high level of natural allelic variation in five potato invertase genes. Fifty-five different full-length cDNA sequences were identified, none of which were previously represented in the databases. Most were genotype specific: only nine were isolated from more than one of the cultivars examined. The average SNP density in cultivated potato is one SNP per 21-24 bp ,42. The fication  or becaucumented ,33,41,42cterized . High secterized . The reacterized . In suchcterized . The bufariation . DifferePain1-9a, Pain1-8c, InvGE-6f/InvGF-4d and pCD141-3c, but not for Pain1-5b and Pain1-5c, perhaps because an insufficient number of cDNA clones was analyzed. Alternatively, these SSCP markers might result from intron polymorphism that is not detectable in cDNA sequences. The full range of invertase alleles present in a population of 219 tetraploid individuals can be captured by genotyping 15, 11 and 38 SNPs, respectively, at the three invertase loci. Association analysis of the SNPs identified two new associations, one between the SNP allele InvGE-A85 and TSC and TSY, and the other between InvCD141-G765 and CQS, TSC and TSY  differed in their associations. These SNPs are in strong but not absolute linkage disequilibrium with each other. Pain1-C552 and Pain1-A718 were more strongly associated with chip quality after cold storage, whereas Pain1-A1544 was mainly associated with tuber starch content. Whereas the cDNA alleles Pain1-Da, Sa and P18a contained all three SNPs, the allele StPain1-a (AAA50305) contained only A1544. This and the four different haplotype models obtained for these three SNPs  and a large number of minor haplotypes with low frequencies. This haplotype distribution might be typical for nuclear genes of the European potato and a signature of its domestication, introduction and breeding history [Solanum species during 20th-century breeding programs [The direct inference of haplotypes from amplicon sequences requires that loci be homozygous, which is rarely the case in diploid and tetraploid potato genotypes. In amplicon sequences derived from heterozygous loci, the phase of the SNPs is unknown. The SATlotyper software was developed to model haplotypes in polyploid species based on unphased SNP data . Haploty history -50. MoreHaplotype modeling correctly predicted some, but not all cDNA alleles that were obtained experimentally from the cvs Satina, Diana and Theresa and could have been detected based on the chosen SNP set. This is because, based on the analysis of sequence trace files, the SNP allele dosage cannot be scored with complete accuracy in all individuals used for haplotype modeling. A small percentage of scoring errors leads to haplotype artefacts with low frequency. To eliminate such artefacts, we considered only haplotype models with a frequency above 1%. As this is an arbitrary threshold, we cannot exclude the possibility that some of the remaining low-frequency haplotypes are also artefacts. Nevertheless, our results demonstrate that SATlotyper is a valuable tool for the fast identification of the major haplotypes present in populations of tetraploid potatoes. The determination of the exact haplotype composition of a tetraploid individual, including rare haplotypes, calls for an allele cloning approach such as that performed in this study.SlLIN9 located on chromosome 8 . This gene might encode a soluble neutral invertase not characterized so far in potato. The five characterized potato invertase genes are all located within segmental chromosome duplications of unknown size, which show structural conservation with other, distantly related plant species [As in tomato , the fou species ,52. FuncVery high natural allelic variation in five potato invertase genes was uncovered by sequence analysis of full length cDNA clones from six different genotypes and SNP analysis in a larger association mapping population. This variability is explained by the potato's reproductive biology. Some of the structural variation found might be causal for functional variation, which influences important agronomic traits of the potato such as tuber starch and sugar content. The invertase cDNA clones described here are the basis for further functional studies. The associations found between specific invertase alleles and tuber starch content, starch yield and chip quality facilitate the selection of superior potato genotypes in breeding programs. Finally, our results point out that natural variation should be taken into account when conducting molecular and functional characterization of potato genes.ASA: allele specific amplification; BAC: Bacterial artificial chromosome; CQA: Chip quality in autumn; CQS: Chip quality after storage at 4°C; LD: linkage disequilibrium; PCR: Polymerase chain reaction; QTL: Quantitative trait locus; SCAR: sequence characterized amplified region; SSCP: single strand conformation polymorphism; TSC: tuber starch content; TSY: tuber starch yield; TY: tuber yield.AD carried out the cDNA cloning, the sequence analyses and the haplotype modeling, and participated in the SNP analysis. SM performed the SNP analysis and contributed to the association statistics and haplotype modeling. LL carried out the pyrosequencing. CG conceived the study, participated in its design and coordination, carried out statistical analyses and drafted the manuscript. All authors read and approved the final manuscript.InvGE cDNA allelesTable S3: SNPs differentiating .Click here for fileInvGF cDNA allelesTable S4: SNPs differentiating .Click here for fileInvCD141 cDNA allelesTable S5: SNPs differentiating .Click here for fileInvCD111 cDNA allelesTable S6: SNPs differentiating .Click here for fileTable S1: Annotation of BAC clones BC149o15 and BC163l15.Click here for filePain-1 cDNA allelesTable S2: SNPs differentiating .Click here for filePain-1 cDNA allelesFigure S1: Amino acid alignment of .Click here for fileInvGE cDNA allelesFigure S2: Amino acid alignment of .Click here for fileInvGF cDNA allelesFigure S3: Amino acid alignment of .Click here for fileInvCD141 cDNA allelesFigure S4: Amino acid alignment of .Click here for fileInvCD111 cDNA allelesFigure S5: Amino acid alignment of .Click here for file


KDELC2, PHOX2A, and TMEM40. Findings from the uni-SNP models were used to develop highly accurate predictive multi-SNP models in the training data set. Despite the substantially smaller size of the validation data set, the training multi-SNP models had slightly lower predictive ability when applied to the validation data set. Six Gene Ontology molecular functions related to ion transport activity were enriched  among the genes associated with the detected SNPs. The findings from this study demonstrate the complementary value of the uni- and multi-SNP models, and univariate and bivariate GWAS analyses. The identified SNPs can be used for genome-enabled improvement of feed efficiency in feedlot beef cattle, and can aid in the design of empirical studies to further confirm the associations.Single nucleotide polymorphisms (SNPs) associated with average daily gain (ADG) and dry matter intake (DMI), two major components of feed efficiency in cattle, were identified in a genome-wide association study (GWAS). Uni- and multi-SNP models were used to describe feed efficiency in a training data set and the results were confirmed in a validation data set. Results from the univariate and bivariate analyses of ADG and DMI, adjusted by the feedlot beef steer maintenance requirements, were compared. The bivariate uni-SNP analysis identified  11 SNPs, meanwhile the univariate analyses of ADG and DMI identified 8 and 9 SNPs, respectively. Among the six SNPs confirmed in the validation data set, five SNPs were mapped to  In feedlot beef cattle enterprises, feed consumption dominates the input (and costs) and weight gain dominates the output (and return). Average daily gain (ADG) per animal, computed as the difference between final and initial trial weight divided by the number of days in the trial, is a frequently used indicator of weight gain. The cost of feed represents 62% to 84% of the total costs in a beef cattle production unit Genomic improvement of feed efficiency in beef cattle relies on the identification of genomic variants (single nucleotide polymorphisms or SNPs) associated with feed efficiency components. A genome-wide association study (GWAS) can be used to identify SNPs to be included in genome-enabled selection decisions. The study of feed efficiency requires the consideration of output (ADG) and input (DMI) indicators, adjusted for maintenance requirements (MBW).The majority of the SNPs reported to be associated with feed efficiency were identified from the analysis of each component (ADG or DMI) separately, or functions thereof such as residual feed intake and residual average daily gain All procedures were conducted following the guidelines recommended in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching Measurements were collected from 1,321 feedlot steers from five ranches in Montana between 2005 and 2008. The combination of ranch, harvest group, and harvest year resulted on 27 contemporary groups (CGs). Pedigree and breed information from 3,331 animals 0.73, 366.40±40.12 kg, and 332.58±29.32 days, respectively.Two feed efficiency components, ADG and DMI, were analyzed. Individual steer ADG (kg) was the difference between adjusted final weight (FW) and initial weight (IW) in the trial divided by days in the trial. The FW was estimated by dividing the individual hot carcass weight by the average dressing percentage of the harvest group. Individual daily DMI (kg) was measured using the GrowSafe automated feeding system . Individual MBW was calculated using the estimated BW mid-trial. The age of the steer at mid-trial  was also recorded. The average (± standard deviation) ADG, daily DMI, IW, FW, MBW, and mA were: 1.61±0.24 kg, 10.48±1.42 kg, 310.10±40.08 kg, 597.50±48.43 kgThe DNA was extracted from blood samples using the salting out method ijklmnY denoted the observed ADG or DMI, μ denoted the overall mean, iSNP denoted the fixed effect of an individual SNP genotype, jB denoted the fixed effect of breed (5 levels), kD denoted the fixed effect of diet  that has a Normal distribution  and bivariate (ADG and DMI) analyses of uni-SNP mixed-effect models were used to detect SNPs associated with feed efficiency. The uni-SNP model for the univariate analysis ; Eq.1 waThe GWAS was implemented using Qxpak v.5.05 A multi-SNP model was developed for the univariate and bivariate analyses. Starting with the SNPs detected at P-value <0.001 in the uni-SNP models and all other explanatory variables equal, a stepwise feature selection approach was used. The final multi-SNP model included the SNPs that entered (were added to the model) and stayed  in the multi-SNP model at P-value <0.0001.The SNPs were first identified using complementary models and analyses on a training data sets. Subsequently, the findings were evaluated on a validation data set that included a separate group of steers. Training and validation data sets were generated from the records that passed the quality control based on sire family VRMSE is the RMSE from the validation data set and TRSME is the RMSE from the training data set. For the bivariate analysis, model inadequacy was the average of three root mean (co)variance terms: RMSE for ADG, RMSE for DMI, and the root means square covariance (RMSC) between ADG and DMI. The change in MA was computed as for the univariate analysis.The SNPs detected  in the training data set using the uni-SNP model and univariate and bivariate analyses were validated at P-value <0.05 2r statistic Some of the SNPs detected could be an artifact of the dependency between SNPs that exhibit high linkage disequilibrium (LD). This situation is the result of the average probe spacing (49.4 kb) of the platform The genetic parameters of ADG and DMI were estimated to assess the potential amount of genetic variability that could be associated to SNPs. Heritability and genetic and phenotypic correlations between ADG and DMI were estimated using an animal model and univariate and bivariate analyses implemented in WOMBAT The detected SNPs were mapped to harboring or proximal (within 2 kb of the 5′ untranslated region or 0.5 kb of the 3′ untranslated region of a gene) genes in the Bos_taurus_UMD_3.1 assembly. The SNP mapping and gene information was obtained from the National Center for Biotechnology Information, SNP and Gene databases Bos Taurus genome as background.Functional analysis of the genes corresponding to the SNPs detected  in the bivariate analysis offered insights into the categories enriched among the genes. The consideration of genes from the bivariate analysis was motivated by the goal of identifying functional categories among genes that could have pleiotropic effects on both feed efficiency components. Genes farther upstream and downstream from the detected SNPs were not included in the functional analysis because the number of spurious  genes added to the functional analysis could have overwhelmed the fewer real (true positive) loci, potentially biasing the results. The enrichment of Gene Ontology (GO) FAT categories and KEGG pathways among the genes was investigated using Fisher's exact test in DAVID Gene networks associated with feed efficiency were visualized using the genes affiliated to the enriched functional categories. The network was visualized using the BisoGenet plug-in Cytoscape The heritability estimates of ADG and DMI were 0.14 and 0.25, respectively. These heritability estimates confirmed the opportunity for genome-based improvement of these feed efficiency components. The phenotypic and genetic correlations between ADG and DMI were 0.52 and 0.18, respectively. These estimates were consistent or slightly lower than in previous reports A summary of the number of significant SNPs (and corresponding genes) detected by the univariate and bivariate analyses is presented in The potential relationship between feed efficiency and the genes harboring or in the proximity of the detected SNPs was investigated. The connection between the feed efficiency components and genes was based on gene annotation information available at the National Center for Biotechnology Information, Gene database KLF6) and is within the 500 kb LD span reported in cattle KLF6 regulates genes in the transforming growth factors β signaling pathway ZHX2). This QTL region has been linked to ADG in Japanese Black (Wagyu) cattle Previous studies reported genomic regions associated with ADG on BTA 2, 4–7, 9, 11, 14–20, 22, 23, 26, and 28 NCKAP5) and dipeptidyl-peptidase 6 (DPP6), respectively. The three SNPs on BTA15 associated with ADG were: rs41620774 mapped to engulfment and cell motility/CED-12 domain containing 1 (ELMOD1), rs108964818 mapped to Lys-Asp-Glu-Leu containing 2 (KDELC2), and rs41768978 mapped to paired-like homeobox 2a (PHOX2A). Both rs41620774 and rs108964818 were mapped less than 1 Mb apart, and for both SNPs, steers homozygous for the minor allele  had the lowest ADG. The protein encoded by ELMOD1 has a GTPase-activator function on small G proteins of the arf family PHOX2A, a gene associated with respiratory rhythm  and autonomic nervous system development PAK1IP1) on BTA23, was associated with ADG. The p21-activated protein kinase-interacting protein 1 encoded by this gene has been associated with cell proliferation and signal transduction The remaining six SNPs associated with ADG were mapped to known genes. Steers heterozygous for rs109934193 on BTA2and rs110787048 on BTA4, had higher ADG than the average homozygous steer. These SNPs were mapped to NCK-associated protein 5 (ATAD1), lipase, family member J (LIPJ), 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2), phosphatase and tensin homolog (PTEN), and renalase, FAD-dependent amine oxidase (RNLS). These genes play roles in mechanisms related to energy expenditure, including ATAD1 role on ATP catabolism LIPJ role on lipid catabolism PTEN role on inositol phosphate metabolism The SNPs associated with DMI are presented in ADAMTS3), group-specific component vitamin D binding protein (DBP), and neuropeptide FF receptor 2 (NPFFR2). These genes have been associated with protein processing KIF16B), N-acetylneuraminic acid phosphatase (NANP), otoraplin (OTOR), phosphoribosylaminoimidazole carboxylase pseudogene (PAICSP), and small nuclear ribonucleoprotein polypeptide B (SNRPB2). In particular, NANP muschlparticipates on the amino sugar and nucleotide sugar metabolism Four additional SNPs associated with DMI were not mapped to genes. Every additional C allele on rs41663978 was associated with lower daily DMI (−0.22±0.05 kg) relative to the A allele. This SNP was mapped on BTA6, approximately 5 Mb from a QTL previously associated with DMI 2r = 0.052). These two SNPs are in the intronic region of the coxsackie virus and adenovirus receptor-like membrane protein (CLMP). This gene encodes for a type I transmembrane protein of the CTX family and transmemrane proteins have activating or suppressing roles on cell growth Although rs42128656 was associated with DMI, this SNP was not mapped to a known gene. This SNP was 600 kb from other two SNPs (rs43291568 and rs43291603) on BTA15 also associated with DMI. This pair of SNPs were mapped within 27 kb of each other, however the pairwise LD among these SNPs was low (TMEM40). Similarly, rs41588990 was mapped to the intronic region of CCR4-NOT transcription complex, subunit 6-like (CNOT6) on BTA6. This gene plays a role in the deadenylation of mRNAs in the cytoplasm and deadenylation has been associated with cell growth Among the SNPs associated with DMI and mapped to known genes, rs108942504 was found on a gene on BTA22 that encodes a structural protein, the transmembrane protein 40  were presented in 2r = 0.046). Furthermore, rs41565199 was mapped to pseudo metalloendopeptidase (OMA1) that inhibits growth and approximately 400 kb upstream from hyaluronan synthase 2 (HAS2) that mediates cellular growth Among the SNPs uncovered by the uni-SNP bivariate analysis, rs41722387 was mapped approximately 450 kb from rs41565199, a SNP associated with ADG on BTA14 . DespiteKDELC2) had positive associations with ADG and DMI albeit a much higher additive estimate for ADG than DMI. The SNPs detected by the one univariate and the bivariate analyses  identified by the uni-SNP univariate ADG, DMI and bivariate analyses. The final multi-SNP ADG, DMI, and the bivariate models included nine, eight, and seven SNPs, respectively , UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), and Sin3A-associated protein 130 kDa (SAP130). These genes are known for their roles on glycosaminoglycan biosynthesis pathway and cell growth ZNF608) on BTA7, and rs109945988 was mapped 4 Mb from a QTL on BTA11 previously associated with ADG BIRC6), RAS guanyl releasing protein 3 , tetratricopeptide repeat domain 27 (TTC27), and latent transforming growth factor beta binding protein 1 (LTBP1) that is associated with cell growth The additional SNPs uncovered by the multi-SNP approach were mapped on BTA 2, 7, 11, 13, 20, 22, and 29. On BTA2, rs108939474 was associated with ADG and was within 500 kb of heparan sulfate 6-O-sulfotransferase 1 (CELF2). This gene modulates the cellular apoptosis program ECHDC3), and USP6 N-terminal like (USP6NL). Two additional SNPs in the multi-SNP univariate DMI analysis were rs41577655 and rs110911295. Mapped to BTA15, rs41577655 was located less than 250 kb upstream of apoptosis inhibitor 5 (API5) and tetratricopeptide repeat domain 17 (TTC17) that was associated with growth. Mapped to BTA20, rs110911295 was located 500 kb upstream from the PAP associated domain containing 7 (PAPD7) and steroid-5-alpha-reductase, alpha polypeptide 1 , and downstream the mediator complex subunit 10 (MED10) and NOP2/Sun domain family, member 2 (NSUN2). Both, SRD5A1 and NSUN2 have been associated with abdominal subcutaneous and visceral fat Among the SNPs included in the multi-SNP univariate DMI analysis, rs41629972 was detected by the uni-SNP DMI analysis. Also on BTA13, rs41577108 was proximal to CUGBP Elav-like family member 2 (CHL1) on BTA22. The cell–cell adhesion function of this gene enables cells to assemble into organized tissues. The remaining SNP, rs42459305, was mapped to a gene dense region on BTA29 with several predicted loci and located less than 3 kb downstream from the olfactory receptor, family 8, subfamily G, member 5 (OR8G5) and could impact feed consumption.The multi-SNP model selection for the bivariate analysis of ADG and DMI resulted in three SNPs not detected by the uni-SNP bivariate model. Among these, rs109945988 was associated in the multi-SNP ADG model, and rs41600811 and rs42459305 represent new associations. In particular, rs41600811 was mapped 100 kb downstream the cell adhesion molecule with homology to L1CAM  validated were: rs108942504 (univariate DMI); rs41629972 and rs108964818 (univariate ADG); and rs108964818, rs41768978, and rs110522962 (bivariate).For the multi-SNP models, validation was assessed by the change in the MA between the validation and training data sets, relative to the validation set . OverallThe 236 genes corresponding to the SNPs detected at P-value <0.01 by the uni-SNP bivariate analysis were considered for functional analysis. The P-value threshold was selected because of the high number of SNPs detected by the univariate analyses at P-value <0.01 that were also detected in the bivariate analysis.TRPC2). This gene was reported to be associated with several behavioral responses Seven functional categories were enriched  among the genes corresponding to the detected SNPs . The mosIon channel activity was associated with maintenance of normal gradient on plasma membranes, participation in cellular de- and re-polarization, neurotransmitter release, immune function, insulin secretion, and active transport mechanisms required for the digestion and absorption of nutrients FGF2 and the intermediate gene ubiquitin C (UBC). These genes could have driver or hub role on feed efficiency components. In addition, FGF2 was implicated on smooth muscle cell differentiation and signaling A comprehensive network of the genes affiliated to the enriched molecular functions is reconstructed . In thisSingle nucleotide polymorphisms associated with the feed efficiency components ADG and DMI in feedlot beef steers were identified using uni-SNP and multi-SNP models and univariate and bivariate analyses. The complementary set of SNPs detected by the univariate and bivariate analyses confirmed the value of considering both GWAS approaches. For certain SNPs, the bivariate analyses could gain precision through the consideration of covariation between ADG and DMI relative to univariate analyses. For other SNPs, univariate analyses could benefit from lower noise of each trait studied separately, relative to the bivariate analysis. Genomic loci that had favorable associations with ADG and DMI simultaneously, or favorable associations with either trait with minimum detrimental association with the other trait, while accounting for the body maintenance requirements, were identified. The validation of models and SNPs suggest that the findings could be replicable. Functional analysis and gene network visualization facilitated the interpretation of the association between SNPs mapping to genes that have ion channel-related molecular function and feed efficiency components. Results from this study can be used for genome-enabled improvement of feed efficiency in feedlot beef cattle, to support further empirical confirmation of the associations, and as proof of concept of the value of complementary association analyses.


The Illumina BovineLD BeadChip was designed to support imputation to higher density genotypes in dairy and beef breeds by including single-nucleotide polymorphisms (SNPs) that had a high minor allele frequency as well as uniform spacing across the genome except at the ends of the chromosome where densities were increased. The chip also includes SNPs on the Y chromosome and mitochondrial DNA loci that are useful for determining subspecies classification and certain paternal and maternal breed lineages. The total number of SNPs was 6,909. Accuracy of imputation to Illumina BovineSNP50 genotypes using the BovineLD chip was over 97% for most dairy and beef populations. The BovineLD imputations were about 3 percentage points more accurate than those from the Illumina GoldenGate Bovine3K BeadChip across multiple populations. The improvement was greatest when neither parent was genotyped. The minor allele frequencies were similar across taurine beef and dairy breeds as was the proportion of SNPs that were polymorphic. The new BovineLD chip should facilitate low-cost genomic selection in taurine beef and dairy cattle. Genetic improvement of several key agricultural species is accelerating with the adoption of genomic selection A major challenge in implementing genomic selection in most species is the cost of genotyping. The expected value of the information gained by genotyping must exceed the cost of obtaining the genotypes. During the early stages of genomic selection in the dairy industry, the cost of high-density genotyping could be justified. The primary application was to evaluate bulls that were potential candidates for production of commercial semen. Using SNP information for those evaluations resulted in more accurate selection of bulls to acquire and extensively market. Once increased accuracies of genome-enhanced breeding values had been demonstrated, breeders and buyers quickly adopted this technology to improve accuracy of selection Imputation is the process of predicting unknown genotypes for animals from observed genotypes and often uses information from a reference population with dense genotypes to predict missing genotypes for animals with lower density genotypes. It is also applied to merge genotypes of similar densities but different SNPs. Most imputation algorithms use information from relatives and population linkage disequilibrium. A number of software programs for imputation have been developed based originally on human genetics http://www.illumina.com/documents/products/datasheets/datasheet_bovine3K.pdf), was developed and made commercially available. That product offered a significant advance toward low-cost genomic selection in cattle; however, imputation accuracy was highly dependent on the relationship of the individual genotyped with the Bovine3K chip to the reference population genotyped at a higher density In 2010, a low-density bovine SNP chip, the Illumina GoldenGate Bovine3K Genotyping Beadchip (http://www.illumina.com/documents/products/datasheets/datasheet_bovineLD.pdf) was developed to provide high imputation accuracy for higher density SNP genotypes in taurine dairy and beef populations. The main objective was to provide a tool that would enable genomic estimated breeding values to be calculated from accurately imputed genotype data from an Infinium-based SNP array with very low rates of failed samples. The main features of the new BovineLD chip are presented along with its imputation performance in a range of breeds and reference populations.In this study, the Illumina Infinium BovineLD Genotyping Beadchip (http://www.illumina.com/documents/products/datasheets/datasheet_bovineHD.pdf) SNP arrays, with priority given to BovineSNP50 content. From the known and validated SNPs, selection priority was 1) high minor allele frequencies (MAFs) in targeted breeds, 2) uniform spacing at a minimum of 2 SNPs per Mbp, with increased SNP density within 500 kbp of chromosomal ends, 3) inclusion of SNPs for determination of sex, parentage, Y haplotypes, and subspecies and maternal lineages, 4) SNP quality and fidelity criteria for robust reproducibility , and 5) a target overlap of 2,000 SNPs with the Bovine3K chip to ensure backward compatibility. The anticipated SNP spacing (2 SNPs per Mbp) obviated the need to check for highly correlated SNPs.To provide highly accurate imputation to BovineSNP50 genotypes in global taurine breeds, SNPs were selected from validated assays from existing higher density chips and similar SNP detection technology, i.e. the Illumina BovineSNP50 and BovineHD (http://www.cbcb.umd.edu/research/bos_taurus_assembly.shtml) was used to define 500-kbp segments over the 29 autosomes. A lack of flanking information at the end of each chromosome had resulted in lower imputation efficiency in preliminary tests. To correct that problem, the SNP density was doubled in the first and last segments of each chromosome. Reflecting the diverse membership of the Bovine LD Consortium, initial SNP selection was made by one member and updated by the others. The initial SNP selection was based on two independent criteria. First, SNPs with the highest mean MAF in each 500-kbp segment were selected over a broad range of European breeds including European Holstein, Montbéliarde, Normande, Jersey, Brown Swiss, Norwegian Red, Swedish Red and White, Finnish Ayrshire, Charolais, Limousine, Blonde d'Aquitaine, and Maine Anjou, with Holstein receiving double weight; the top two SNPs were selected in the segment at each end of the chromosome. Second, SNPs with the highest mean minimum MAF for six major European dairy breeds  were selected for each 500-kbp segment, with again 2 SNPs selected at each end of the chromosome. Selecting those SNPs with the highest mean of the two selection criteria within each 500-kbp segment (with doubling at the chromosome ends) resulted in 8,000 SNPs. Those 8,000 SNPs were subjected to a similar selection process using MAFs from North America and Oceania along with the European populations. For Holstein and Jersey breeds, the MAF used was the mean across the 3 populations; for Brown Swiss, only North America and Europe were included. The mean MAF was computed from Holstein, Jersey, Brown Swiss, Angus, and Brahman. The minimum MAF was from Jersey, Brown Swiss, and Angus. Again, the SNPs with the highest mean of the two selection criteria were selected with doubling at the chromosome ends.The SNPs were selected to be highly informative with a high MAF over a large range of breeds from around the world . The refNext, some of the selected SNPs were replaced by Bovine3K SNPs that were in nearby locations to ensure backward compatibility. In addition, SNPs used for breed determination and parentage testing that had not already been selected were included, and some SNPs were added to fill gaps generated by map inconsistencies.For the X chromosome, Bovine3K SNPs with high MAFs were selected and supplemented with BovineSNP50 SNPs, with consideration given to spacing, MAF, and fidelity. Because large gaps remained after that initial selection, additional X- chromosome SNPs were chosen from the BovineHD assay.For the Y chromosome and mitochondrial DNA (mtDNA), 9 Y-specific and 13 mtDNA SNP markers were identified from the BovineHD chip based on assay fidelity and performance across 27 breeds, MAF across those breeds, and ability of a SNP to discern subspecies and geographic locations of breed origins.http://faculty.washington.edu/browning/beagle/beagle.html) http://aipl.arsusda.gov/software/findhap/) Imputation efficiency was assessed in 10 populations . Beagle software . There were 236 chromosome X SNP on the final Bovine LD chip. Flanking sequences and base calls for the 6,909 SNP are given in The SNP assays for 6,914 loci were validated using data from 290 samples that represented 26 global dairy and beef breeds  and inclThe BovineLD chip, consisting of 6,909 final loci, was validated for 290 individuals from 26 major dairy and beef breeds . The meaThe concordance rate for 2,088 SNPs in common between BovineLD and Bovine3K assays was 98.78% for 281 females genotyped with both chips. The most likely cause of the differential performance between the BovineLD and Bovine3K chips is the chemistry difference between the Infinium and GoldenGate assays.Bos primigenius indicus breed), mean MAF was lower (0.18). Overall, >89% of the SNPs were polymorphic in Brahman, which suggested that the BovineLD chip may be useful for imputation in this breed.Data for calculating mean MAF  were priFor the 6,909 SNPs selected for the BovineLD chip, median spacing was 0.348 Mbp, with only 82 (1.1%) of intervals greater than 1 Mbp . These gCGCCGCAAC (haplotype 1) was observed only in cattle with indicine lineage . The second haplotype (TCTCCTCAC) was associated with central European lineage, haplotype 3 (TCTCCTCAT) was 1 base different from haplotype 2 and probably appeared to be associated with breeds that came to the island of Jersey from France or Spain, and haplotype 4 (TCTTGTCGC) was associated with northern European lineage, including islands. Only a few breeds had more than one haplotype, e.g. Santa Gertrudis and Beefmaster, both of which are taurine–indicine hybrids. Common haplotypes across breeds appeared to reflect a common origin. Phylogenetic analysis separated the 26 breeds into four distinctive clades, which agrees with a previous report on the dual origins of dairy cattle breeds in Europe AAGAGCAAAAAAG) was at highest frequency in indicine cattle. Most taurine×indicine cattle were derived from taurine cows. Therefore, the lack of haplotype 7 for taurine breeds in most regions is not unexpected. While more research is required, these preliminary results suggest the BovineLD markers could be useful in determining lineage origin between taurine and indicine breeds or identifying potential admixture within a population of locally adapted animals.The sex-specific and lineage identification SNPs also appeared to perform well. The nine Y-chromosome SNPs had a 100% call rate across 230 males of different breeds and no genotype calls for the 55 females. We investigated the frequency of the haplotypes of the alleles from these 9 SNP both within and across breeds. Four unique haplotypes were observed, which differed dramatically in frequency across breeds, Imputation accuracy was assessed in Australian, French, and North American cattle populations. In all cases, the accuracy of imputation to BovineSNP50 genotypes was ≥95% . Most imFor Australian and North American Holsteins, accuracy of imputation to BovineSNP50 genotypes was better for BovineLD genotypes than for Bovine3K genotypes. For Australian Holsteins, imputation accuracies were up to almost 6 percentage points higher with the BovineLD chip than with the Bovine3K chip using the same data . Mean imThe greatest improvement in imputation for BovineLD genotypes compared with Bovine3K genotypes was for individuals with no genotyped parents. For Australian Holsteins, difference in mean imputation accuracy with and without a sire in the reference population was 2.9 percentage points for Bovine3K genotypes but only 1.3 percentage points for BovineLD genotypes. The improvement was smaller for North American Holsteins: a difference of 2.7 percentage points between both parents genotyped and no genotyped parents for Bovine LD genotypes  comparedBos primigenius taurus beef and dairy cattle.The Illumina BovineLD BeadChip includes 6,909 SNPs selected to provide optimized imputation to BovineSNP50 genotypes in dairy breeds. The SNPs have MAFs of >0.3 in most breeds, and nearly uniform spacing across the genome except at the ends of the chromosome where densities were increased. The chip also includes SNPs on the Y chromosome and mtDNA loci that are useful for gender checking, determining subspecies classification and identifying certain paternal and maternal breed lineages. Accuracy of imputation to BovineSNP50 genotypes using the BovineLD chip was >99% when both parents were genotyped in the North American BovineSNP50 reference population. That high accuracy suggests that the design criteria for the BovineLD chip would be useful to consider in other species for which an “imputation chip” could dramatically lower the cost of implementing genomic selection. BovineLD imputation was about 3 percentage points more accurate across multiple populations compared with Bovine3K imputation. The improvement was greatest when neither parent had been genotyped. The gain in imputation accuracy is attributed primarily to the increased overall density of the BovineLD chip compared with the Bovine3K chip and also to the even further increased density at the ends of chromosomes. The high MAFs also contribute to the improved imputation accuracy. The MAFs were similar across taurine beef and dairy breed as was the proportion of SNPs that were polymorphic. Although it would be expected that accuracies of imputation would be highest for those breeds which were included in the design of the chip, which was dominated by dairy breeds, the similar SNP characteristics (particularly the high MAF across many beef and dairy taurine breeds) suggest that the BovineLD chip will perform well in imputation of taurine beef cattle. Our results suggest that the imputation accuracy will also be quite dependent on the size of the population genotyped with a higher density SNP assay. Overall, the new BovineLD BeadChip should facilitate low cost genomic selection in Table S1Genomic locations, flanking sequences and base calls for the 6,909 SNP on the bovineLD array.(CSV)Click here for additional data file.


Fr-H1, Fr-H2 and Fr-H3) have been identified from bi-parental genetic mapping but it can be argued that those mapping populations only capture a portion of the genetic diversity of the species. A genetically broad dataset consisting of 184 genotypes, representative of the barley gene pool cultivated in the Mediterranean basin over an extended time period, was genotyped with 1536 SNP markers. Frost tolerance phenotype scores were collected from two trial sites, Foradada (Spain) and Fiorenzuola  and combined with the genotypic data in genome wide association analyses (GWAS) using Eigenstrat and kinship approaches to account for population structure.Frost tolerance is a key trait with economic and agronomic importance in barley because it is a major component of winter hardiness, and therefore limits the geographical distribution of the crop and the effective transfer of quality traits between spring and winter crop types. Three main frost tolerance QTL (Fr-H2 and HvBmy loci on chromosomes 5H and 4HL, respectively. The other significant kinship associations were located in genomic regions that have not previously been associated with cold stress.GWAS analyses identified twelve and seven positive SNP associations at Foradada and Fiorenzuola, respectively, using Eigenstrat and six and four, respectively, using kinship. Linkage disequilibrium analyses of the significant SNP associations showed they are genetically independent. In the kinship analysis, two of the significant SNP associations were tightly linked to the Haplotype analysis revealed that most of the significant SNP loci are fixed in the winter or facultative types, while they are freely segregating within the un-adapted spring barley genepool. Although there is a major interest in detecting new variation to improve frost tolerance of available winter and facultative types, from a GWAS perspective, working within the un-adapted spring germplasm pool is an attractive alternative strategy which would minimize statistical issues, simplify the interpretation of the data and identify phenology independent genetic determinants of frost tolerance. Frost tolerance also depends on the intrinsic capacity of the vegetative tissues to survive freeze-induced desiccation, and their efficient recovery from the stress . In t. In tVrnpd-2, where σ2e, is the residual variance component and σ2g is the genotypic variance component. The BLUPs for each accession were classified according to their geographic origin, growth habit and ear morphology  was set for identifying significant SNP associations. Significant SNPs mapping within 5 cM of each other were considered as being linked to the same QTL and the highest was chosen as representing the QTL.DARwin v.5.0  , ear morphology (two-rowed / six-rowed types) and germplasm type (landraces / old cultivars / modern cultivars).Click here for fileTable S2. Summary statistics for Foradada (Spain). Genotypic means and standard error for frost tolerance for the fixed terms in the model. Number of individuals sampled in each class in brackets.Click here for fileTable S3. Summary statistics for Fiorenzuola . Genotypic means and standard error for frost tolerance for the fixed terms in the model. Number of individuals sampled in each class in brackets.Click here for fileTable S4. Germplasm list of the 184 barley lines assembled into the GWAS population providing country and region of origin, growth habit, spike morphology and frost tolerance genotypic means used in this paper.Click here for file