One and eight SNPs were co-detected in two environments and by at least two methods, respectively, and they explained 5.87–9.59% of the phenotypic variation. Comparing the transcriptomes of two inbred lines with contrasting seed size, three and eight genes identified in the association panel showed significantly differential expression between the two genotypes at 15 and 39 days after pollination, respectively. Ten and 17 genes identified by a genome-wide association study were significantly differentially expressed between the two development stages in the two genotypes. Combining environment−/method-stable SNPs and differential expression analysis, ribosomal protein L7, jasmonate-regulated gene 21, serine/threonine-protein kinase RUNKEL, AP2-EREBP-transcription factor 16, and Zm00001d035222  were important candidate genes for maize kernel size and development.Kernel length, kernel width, and kernel thickness are important traits affecting grain yield and product quality. Here, the genetic architecture of the three kernel size traits was dissected in an association panel of 309 maize inbred lines using four statistical methods. Forty-two significant single nucleotide polymorphisms (SNPs;  Maize is one of the most important crops and is widely used as staple food, animal feed, and raw materials. Grain yield improvement is a longstanding breeding goal in maize. Kernel size traits, including kernel length (KL), kernel width (KW), and kernel thickness (KT), largely affect yield component kernel weight and product quality. In addition, large kernels have a favorable seed vigor and, finally, promote yield. Therefore, it is important to elucidate the genetic architecture of kernel size.2:3 families  for kernel size traits have been identified in Ffamilies , doubledfamilies , recombifamilies , three-wfamilies , four-wafamilies , and immpulation  with difGenome-wide association study (GWAS) is an effective approach for analyzing the genetic basis of complex traits. GWAS results are easily influenced by population structure and rare variants in natural populations. Therefore, many statistical methods have been developed to improve the power for identifying phenotype-genotype associations such as single-locus mixed linear model MLM; , compresZmCKX10, encoding cytokinin oxidase, was cloned by fine mapping of a major QTL (qKL1.07) for KL  for KL . Althoug) for KL , the app) for KL .Arabidopsis. Transcriptome analysis not only reveals a large number of genes associated with kernel size and development but also some biological processes and signaling pathways including DNA methylation, ovule development, cell cycle, cell division, ubiquitin, phytohormone signaling pathways, and transcriptional regulatory factors during seed, endosperm, and embryo development in maize , and quantitative trait transcript analyses, in maize . These sTo date, the dissection of kernel size traits by integrating GWAS and transcriptome analysis is rare. In the present study, we used an association panel including 309 inbred lines to identify significant SNPs and candidate genes for KL, KW, and KT in multi-environments using four GWAS methods. We also identified differentially expressed genes of two inbred lines with contrasting seed size that were selected from the association panel during two seed development stages. We finally determined consistent genes associated with kernel size by combined GWAS and transcriptome analysis.A total of 309 maize inbred lines were assembled into a panel. The panel was comprised of 128 China core germplasms, 16 new selected inbred lines, and 165 US public inbred lines whose plant variety protection had expired . They were planted in Yuanyang , Dancheng , and Sanya  experimental stations of Henan Academy of Agricultural Sciences in 2017. In 2019, the association panel was planted in YY station. The field experiment was arranged in a randomized complete block design with three replicates. Each inbred line was grown in two rows with 15 plants, 0.60 m in row spacing, and 0.25 m in plant spacing. Best linear unbiased estimate (BLUE) values of each trait in the four environments were calculated by the software QTL IciMapping v4.0  and werePearson correlation was calculated by R software . For sinK). K was set from 1 to 8 with three-time iterations. Length of burnin period was 5,000 and the number of MCMC replicates after burnin was 50,000. The results were visualized by Structure Harvester  were incorporated in the four methods. All parameters were set by default. Stringent Bonferroni correction is usually adopted for multiple testing correction in a single-locus model, whereas no multiple testing correction is needed in multi-locus methods . The reads were aligned against the maize B73 genomearvester , and delhe panel . One subhe panel . The kin methods . Therefo methods . Phenoty methods , and tha methods .To validate the candidate loci identified by GWAS, we selected two inbred lines from the association panel mainly according to the KL and KW value in SY2017 where the ear pollination is not easily influenced by the climate. AJ525 represented large kernel size because its KL and KW values both ranked second, whereas A350 represented small kernel size due to the two-trait values of it belonging to the bottom 10% in the association panel. The three-trait value of AJ525 was consistently higher than that of A350 across all environments. The population structure analysis revealed that AJ525 and A350 were assigned to one subgroup.Maize kernel filling includes three stages: a lag phase of minimal gain in dry weight (10–18 DAP), a linear phase of dry weight accumulation (18–40 DAP), and a period of diminishing dry weight accumulation approaching physiological maturity  enrichment analysis of the differentially expressed genes (DEGs) was performed by the GOseq R package-based Wallenius noncentral hypergeometric distribution (Fragments per kilobase of transcript per million fragments mapped (FPKM) was used as the gene expression level. The differential expression analysis was implemented by DESeq2 . Genes wribution . The KOBribution . The GATribution  was usedr = 0.72) and DC2017 (r = 0.85) and moderate in SY2017 (r = 0.40) and YY2019  and SY2017 (r = −0.12), indicating the trade-offs between them. The correlation between KL and KT was significantly positive in DC2017 . KW significantly and positively correlated with KT in DC2017  and SY2017 . For KL and KW, a significantly positive correlation  was observed between YY2017, SY2017, and YY2019. DC2017 showed no or weak correlations with other environments for the three traits , followed by KW (0.62–0.94), and KT was the lowest  for three traits were identified under five environments, of which five were detected by CMLM, 25 by SUPER, 14 by FarmCPU, and 11 by MLMM , whereas S6_22359567 was significantly associated with KW in YY2017 . KT-associated SNP S8_5131540 was detected by CMLM (p = 1.53E-05), FarmCPU (p = 9.68E-06), and MLMM (p = 8.37E-06) in BLUE. Five SNPs were co-detected by two methods (p = 1.58E-05) and MLMM  in terms of KL. It was also significantly associated with KW  by MLMM, indicating the SNP had a pleiotropic effect on KL and KW. S8_159308804 explained 7.18–8.58 and 8.38% of the variation of KL and KW, respectively.S2_138681134 was the only one environment-stable SNP, which was co-detected in BLUE and SY2017 environment in the SUPER model . It coul methods . Among tWe used these 42 significant SNPs to identify candidate genes for kernel-related traits, and 70 candidate genes were found . Four TFFrom the association panel, we selected two inbred lines with contrasting kernel size from field trials. In large seed AJ525, the mean value of KL, KW, and KT was 1.11, 0.85, and 0.54 cm, respectively, across different environments . For A352fold change| ≥ 1. There were 5,156 and 8,280 DEGs between the two genotypes at DAP15 and DAP39, respectively, indicating that the major difference between the two genotypes was shown at DAP39  in at least three samples, and 26 genes were expressed in all 12 samples. Twenty-two genes were differentially expressed between the two inbred lines or between the two development stages , S9. ThrTen and 17 genes identified by GWAS were significantly differentially expressed between the two stages in the two genotypes , S9. In The allele variations detected by GWAS and transcriptome analysis were identified through 22 common genes. The positions of SNPs from 22 common genes did not match exactly in both omics levels. With a distance of 0.20–71 kb, 23 SNPs detected in the transcriptome showed consistent allele variations as five significant SNPs identified by GWAS. Of these, 20 transcriptome SNPs corresponding to three significant SNPs by GWAS showed different alleles between the two genotypes at DAP39, whereas three transcriptome SNPs from two GWAS SNPs exhibited allele variations between the two development stages in A350 .Kernel size is a complex quantitative trait and is coordinately regulated by kernel length, width, and thickness. Elucidation of the variation of kernel size will facilitate to reveal the regulatory mechanisms of maize kernel development. GWAS and transcriptome analysis are effective methods to identify key loci and genes for kernel size in different omics levels. The combination of GWAS and transcriptome analysis is useful to improve the efficiency of gene identification.To control false associations, two single-locus methods (CMLM and SUPER) and two multi-locus methods (MLMM and FarmCPU) were used to identify significant SNPs for kernel size. Forty-two significant SNPs for the three kernel traits were identified. Eight SNPs were co-detected by at least two methods, of which two were detected by four methods, one by three methods, and five by two single-locus and multi-locus methods. However, most of the significant SNPs were specific to each GWAS method. Similar results were also found in previous studies. Due to a significant GEI effect, only one SNP was co-detected in two environments. Although KL and KW showed a significant and positive correlation in four environments, only one SNP (S8_159308804) was detected with a pleiotropic effect on KL and KW. Except KL and KW, the pleiotropism between KW and KT or KL and KT was also found in other studies , 2020a. Twelve SNPs were located within QTL regions for kernel size in previous studies. S5_201017684 detected by four methods and S8_159308804 detected by three methods were located within QTL regions for KT, KL, and KW that were identified by Three SNPs were located closely to the identified SNPs from previous studies . S2_2341Seventy candidate genes were identified from 42 significant SNPs, and 22 GWAS genes showed significantly differential expression between genotypes or stages , S9. In Dek44 encodes mitochondrial RPL9 and regulates cell growth and kernel development via cyclin/cyclin-dependent kinase-mediated activities -KD (kinase domain) interacting protein 130 were candidate genes for KW, and they showed significantly differential expression between the two genotypes. Ethylene receptor genes CM-ERT1 and Cm-ERS1 play a role in the early development of melon fruit . BR-defiOsGW2 encodes a RING-type E3 ubiquitin ligase and negatively regulates cell division, resulting in a decrease of grain width and weight , was highly expressed in AJ525 and showed significantly two-fold differential expression compared with A350. In soybean, GmPIP2-9-overexpressing plants had significantly more pod numbers and larger seed sizes than wild-type plants  for KW. Only RHY1A showed significantly differential expression between two development stages in the two inbred lines. d weight . DA2, thng seeds . E3 ubiqe plants .In summary, 42 significant SNPs for KW, KL, and KT were identified. In particular, one and eight SNPs were co-detected in two environments and by at least two methods, respectively. GWAS combined with transcriptome data revealed that RPL7, JRG21, serine/threonine-protein kinase RUNKEL, EREB16, and Zm00001d035222  were important candidate genes for kernel size and development.The original contributions presented in the study are publicly available. This data can be found at: SRA database of NCBI (accession no. PRJNA681326).JM constructed the phenotypic measurement and data analyses including phenotypic analysis, kinship analysis, and GWAS analysis and wrote the manuscript. JM, LW, and YC collected the seeds of 12 samples for transcriptome sequencing. HL, LW, and HW provided the 309 inbred lines. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P<0.05) SNPs associated with tail-type traits were detected. For significant SNPs' loci, we determined their physical location and performed a screening of candidate genes within each region. By combining information from previously reported and annotated biological functional genes, we identified SPAG17, Tbx15, VRTN, NPC2, BMP2 and PDGFD as the most promising candidate genes for tail-type traits. Based on the above identified candidate genes for tail-type traits, BMP2 and PDGFD genes were selected to investigate the relationship between SNPs within the tails in the Altay and Tibetan populations. rs119 T>C in exon1 of the BMP2 gene and one SNP in exon4 (rs69 C>A) of the PDGFD gene were detected. rs119 was of the TT genotype in Altay sheep, while it was of the CC genotype in Tibetan sheep. On rs69 of the PDGFD gene, Altay sheep presented with the CC genotype; however, Tibetan sheep presented with the AA genotype.Chinese indigenous sheep can be classified into three types based on tail morphology: fat-tailed, fat-rumped, and thin-tailed sheep, of which the typical breeds are large-tailed Han sheep, Altay sheep, and Tibetan sheep, respectively. To unravel the molecular genetic basis underlying the phenotypic differences among Chinese indigenous sheep with these three different tail types, we used ovine high-density 600K single nucleotide polymorphism (SNP) arrays to detect genome-wide associations, and performed general linear model analysis to identify candidate genes, using genotyping technology to validate the candidate genes. Tail type is an important economic trait in sheep. However, the candidate genes associated with tail type are not known. The objective of this study was to identify SNP markers, genes, and chromosomal regions related to tail traits. We performed a genome-wide association study (GWAS) using data from 40 large-tailed Han sheep, 40 Altay sheep (cases) and 40 Tibetan sheep (controls). A total of 31 significant ( Summary: This is a study on candidate genotyping of sheep with different tail types in China. According to the shape of their tails, Chinese indigenous sheep can be divided into three types, namely, fat-tailed, fat-rumped and thin-tailed sheep, of which the typical breeds are large-tailed Han sheep, Altay sheep and Tibetan sheep, respectively.Large-tailed Han sheep are mainly distributed in the hinterland of the North China Plain, which has a typical temperate continental monsoon climate with obvious changes in the four seasons; cold and dry in winter and hot and rainy in summer. The large-tailed Han belongs to the long fat-tail group, where the fat tail is large, fan-shaped, and hangs down to the hocks. The peach-shaped tail tip is close to the tail groove and is upturned. Altay sheep are mainly distributed in Fuhai and Fuyun counties in the Altay region of Xinjiang Uygur Autonomous Region. This area is dominated by a typical continental climate, with an annual average temperature of 4.0°C, an extreme minimum temperature of −42.7°C, an annual snow cover of 200–250 days, and a snow thickness of 15–20 cm. The fat deposited on the buttocks forms a rounded fat hip, which is wide, straight and rich. There is a shallow groove in the middle of the lower edge of the fat hip, which is divided into two symmetrical halves. Tibetan sheep are native to the Qinghai-Tibet Plateau and are mainly distributed in the Tibet Autonomous Region and Qinghai. The central production area is located at 26°50′∼36°53′ north latitude and 78°25′∼99°06′ east longitude. It is located in the southwestern part of the Qinghai-Tibet Plateau with an average elevation of over 4000 m. The climate is characterized by long periods of sunshine, strong radiation, low temperature, large temperature difference, clear and wet, long night rain, dry winter and spring, high wind pressure, low air pressure and low oxygen content.Large-tailed Han sheep and Altay sheep store large amounts of fat in their tails. Worldwide, more than 25% of sheep breeds are fat-tailed or fat-rumped . Fat-taiMEF2B, RFXANK, CAMKMT, TRHDE, RIPK2, GRM1, POL, MBD5, UBR2, RPL7 and SMC2 genes were involved in growth and meat production traits in sheep. The mutation of the TMEM154 gene was identified to be associated with Ovine lentivirus  is one in which a group of genetic markers that are representative of a phenotype are analyzed for variation within a set of DNA samples . This mentivirus . A novel rickets . Two novin sheep .Mass spectrometry can be used as a single nucleotide polymorphism (SNP) typing technique based on primer extension . The extBMP2 and PDGFD genes were taken to investigate the relationship between SNPs within the tails of Altay and Tibetan sheep.The primary aim of this study was to perform GWAS analysis using an Illumina Ovine SNP600 BeadChip to identify SNPs associated with Chinese indigenous sheep breeds with different tail types at the genome level, and to forecast and explore the major candidate genes associated with fat deposition in sheep tail. The filtered SNP may be used for molecular marker-assisted selection and will guide studies aimed at elucidating some complex traits. As secondary goal was to validate the GWAS results and screen casual genetic variants as genetic markers that are beneficial for the identification of sheep tail type in an independent sheep population, the After quality control, 119 individuals remained , of whichttp://pngu.mgh.harvard.edu/purcell/plink). The PCA results showed that the samples from the three breeds were clustered by two principal components: PC1 and PC2. According to PC1, Chinese sheep with different tail types could be divided into two groups consistent with their fat deposition: fat-tailed sheep  and thin-tailed sheep , as shown in Principal components analysis (PCA) of genome-wide SNPs was performed using R snpStats Software package  and PlinA case-control design GWAS was performed to research the genetic basis of fat deposition with different tail types. Tibetan sheep were coded as controls and large-tailed Han sheep and Altay sheep as cases. The general linear model (GLM) in Tassel 3.1 was used for whole genome-wide association analysis. We defined the whole-genome significance cut-off as the top 5% .In the results as shown in http://www.ncbi.nlm.nih.gov/assembly/457978) to compare significant SNP loci to confirm their chromosomes and physical locations, the significant SNP was extended approximately 100 kb in the upstream and downstream directions. The results showed that 31 SNPs were significantly (P<0.05) associated with tail type at the genome level, distributed on seven different chromosomes, as shown in supplemental (Table S2).By comparing genomics and bioinformatics, making full use of the USCS and NCBI databases and the latest sheep Ovis_aries_3.1 genomic information (DAVID v2.1 was used to conduct the gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses for a more in-depth understanding of the function of these identified genes.As can be seen from the BMP2 gene and one SNP in the fourth exon of the PDGFD gene. MALDI-TOF assay process used to extend the single site in the, rs119 T>C . Each locus has two genotypes . rs119, which was located on exon1 of the BMP2 gene, was of the TT genotype in Altay sheep, while it was of the CC genotype in Tibetan sheep. On rs69 of the PDGFD gene, Altay sheep presented with the CC genotype; however, Tibetan sheep presented with the AA genotype.Single SNP association analysis showed that the two genotypes at the rs119 and rs69 loci were significantly different in Altay sheep and Tibetan sheep  may cause bone stagnation in mice, such as shortened hind-limb length, sternal segment fusion, and bone mineralization defects. Gesta et al. (Tbx15 (T-Box Transcription Factor 15) can regulate adipocyte differentiation and mitochondrial respiration, and the Tbx15 gene plays an important role in limb bone development  is anti-lipogenic and inhibits pre-adipocyte differentiation, but the PDGF gene is highly expressed in adipose tissue , approval no. GSAU-AEW-2017-0003.In our study, 120 animals, were from Liaocheng in Shandong Province, including 40 large-tailed Han (20 rams and 20 ewes), Altay in Xinjiang Province, including 40 Altay sheep (20 rams and 20 ewes), and Tianzhu in Gansu Province, including 40 Tibetan sheep (20 rams and 20 ewes), respectively. These samples were collected from research station, the flock size is greater than 1000. All of the experimental animals were randomly selected, then blood was collected from the jugular vein, 8 ml blood was collected from each sheep, and stored at −20°C after slight shaking.A TIANamp Blood DNA Kit  was used to extract Genomic DNA from samples of whole blood. A NanoVue Spectrophotometer producer was used to assess the purity and concentration of genomic DNA.The Illumina Ovine SNP 600 BeadChip , which contained 604,715 SNPs spanning the ovine genome, was used to genotype the genomic DNA of each animal, with an average distance of 4.28 Kb between two SNPs.http://pngu.mgh.harvard.edu/purcell/plink/) was used to control the quality of the genotype data. Stringent quality control criteria were applied to increase the accuracy of GWAS inference: (1) sample call rate >95% and SNP call frequency >90%, (2) minor allele frequency (MAF) >3%, (3) Hardy–Weinberg equilibrium test, P<10–6. After quality control, 58762 SNP sites remain.PLINK software   softwareThe phenotypic traits determined by this experiment included: tail length, tail width and tail circumference. Tail length refers to the distance in the fat-tailed sheep from the leading edge of the first caudal vertebra to the tail tip. Tail width refers to the straight line distance at the widest point of the tail. The tail circumference is the length of one circle around the tail tip. The tail length, width and circumference of Lanzhou big-tailed sheep are 38, 32, and 106 cm, respectively. The tail length, width and circumference of Altay sheep are 22, 36, and 98 cm, respectively. The tail length, width and circumference of Tibetan sheep are 15, 4, and 7 cm, respectively.http://cran.r-project.org), a more general analysis model – the GLM  at each marker locus in the genome. In order to correct the possible false-positive effects of genome-wide association analysis by genetic relationship and population stratification, PCA was conducted using snpStats (v.1.4.0) in R (v.4.0)  and NCBI databases and the latest sheep Ovis_aries_3.1 genomic information (http://www.ncbi.nlm.n.gov/assemblv/457978/), significant SNP loci were aligned to confirm their chromosomes and physical locations, and gene function annotations were performed in the 100 Kb region upstream and downstream of the SNP. The Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/) was used to perform the GO enrichment analysis and KEGG pathway analysis. To better understand the functions of the genes within the 100 Kb region upstream and downstream of the SNP, the Ovis aries Ensembl gene IDs were converted into human orthologue Ensembl gene IDs using BioMart (http://www.biomart.org/), since the annotation of the sheep genome is limited.By comparing genomics and bioinformatics, making full use of the UCSC ( (DAVID)   were collected from Altay sheep in Altay, Xinjiang, and from Tibetan sheep. Genomic DNA extraction of the collected blood samples was performed using the TIANamp Blood DNA Kit, the detection of DNA sample concentrations using NanoDrop (Thermo NanoDrop 2000) and the detection of DNA quality by 0.8% agarose gel electrophoresis. 30 DNA samples were randomly selected and evenly diluted to 50 ng/μl, and then 2 μl of each sample was taken and evenly mixed to construct the DNA pool.http://bioinfo.ut.ee/primer3-0.4.0/) online design software was used to design primers for all exons of the gene. Primer information is shown in the Supplementary file (Table S1). Primers were synthesized by Cybrex Biotech Co., Ltd. The total PCR reaction system was 20 μl:1 μl of 5′ primers, (10 pmol/μL), 1 μl of 3′ primers (10 pmol/μL), 10 μl for the 2×Taq PCR Master Mix (Taq DNA polymerase (0.05 U/µl), reaction buffer, 4 mM MgCl2, 0.4 mM of each dNTP and 4×1.25 ml Nuclease-free water. Producer of Taq pol and dNTPs Tiangen Biotech Co. Ltd., Beijing, China), 2 μl for the DNA template, and 6 μl for the ddH2O. PCR reaction program: 94°C pre-denaturation 5 min; 94°C denaturation 30 s, annealing temperature 30 s, 72°C extension 30 s, 35 cycles; 72°C extension 10 min; 4°C preservation. The PCR product was detected by 0.8% agarose gel electrophoresis, and the target band was sent to Bio Miao Biological Technology (Beijing) Co., Ltd. for sequencing. According to the sequencing results, sequence comparisons using DNAMAN 5.2.10 software  and Chromas (v.2.0) software were performed to screen for SNP sites.PRIMER 3 (v.0.4.0.) , two SNPs were then genotyped in 200 Altay and 185 Tibetan sheep for genotyping. According to the sequence information of the SNP site, use the primer design software Assaydesign of Sequenom Company 3.1 to design the PCR reaction and single base extension primers and synthesize them. After the sample resin is purified, use Mass ARAY Nanod Ispenser points the purified product and transfers it to the Spectro CHIP (Sequen-om) chip, and uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for analysis. The detection results are typed using TYPER 4.0 software and the results are output.BMP2 and PDGFD genotype effect, p is the field effect, m is the gender effect, and e is the random residual.SPSS 22.0 software and a case-control analysis method were used to compare the frequency of genotypes of large-tailed and thin-tailed individuals , at each marker locus in the genome. The statistical model is as follows:BMP2 gene, Altay sheep presented with the TT genotype, while Tibetan sheep presented with the CC genotype. On exon4 of the PDGFD gene, Altay sheep presented with the CC genotype; however, Tibetan sheep presented with the AA genotype. These genes can be molecular markers for the selection of sheep tail type.Chinese indigenous sheep can be classified into three types based on tail morphology. Some candidate genes were identified by genome-wide association analysis and genotyping technology, and it was discovered that on exon1 of the


The genetic parameters of the rib eye area were estimated using a mixed linear mixed model. The rib eye area showed medium heritability (0.32 ± 0.13). Whole-genome sequencing of 40 large rib eye sheep  and 40 small rib eye sheep  was performed. Case-control genome-wide association studies and the fixation index identified candidate rib eye-associated genes. Seven single nucleotide polymorphisms (SNPs) in six genes  were identified as candidates. The study population was expanded to 1050 lambs to perform KASPar genotyping on five SNPs, which demonstrated that SNPs in LOC105611989, DPP6, and COL12A1 correlated significantly with the rib eye area, which could be used as genetic markers for molecular breeding of the rib eye area. The results provided genetic parameters estimated on the rib eye area and information for breeding based on carcass traits in Hu sheep.In sheep meat production, the rib eye area is an important index to evaluate carcass traits. However, conventional breeding programs have led to slow genetic progression in rib eye muscle area. Operationalizing molecular marker assisted breeding is an optimized breeding method that might improve this situation. Therefore, the present study used whole genome sequencing data to excavate candidate genes associated with the rib eye muscle. Male Hu lambs ( Improvement of carcass traits is very important for farmers and the sheep industry, because the commercial value of a sheep carcass is determined by a range of different aspects, notably the rib eye area. Studies suggest that the rib eye area is significantly associated with the carcass lean meat yield . Among mIn the last 20 years, the heritability of sheep carcass traits has been estimated in many sheep breeds from different regions and countries using different models and approaches . SeveralReducing the generation interval while maintaining a good level of selection accuracy would improve breeding efficiency . Early sDesigning effective breeding programs based on accurate estimates of genetic parameters and molecular markers is one way to solve the problem of slow genetic progression of the rib eye area of sheep. In the present study, the genetic parameters of the rib eye area were estimated using different models, and some genetic correlations between the rib eye area and economic traits were estimated. WGS of High and Low rib eye area groups from a reference population of Hu sheep was performed. The aim was to estimate the heritability of the rib eye area. In addition, we aimed to establish the feasibility of adding the rib eye area trait to breeding goal, and to identify candidate genes that could be used in molecular marker assisted breeding, which will increase the breeding value of Hu sheep.In the present study, a total of 1050 male Hu lambs, born between 2018 and 2019, were included. Among them, 776 individuals possessed a complete pedigree record. The full pedigree of the experiment population comprised 1500 individuals, including 70 male parents and 600 female parents, which were from three National Core Breeding Farms of Sheep and Goats (NCBFSG)  and a large scale Hu sheep farm (Gansu Sanyang Sheep Industry Co. Ltd.). The population with pedigree information could be used to estimate genetic parameters. Each lamb was weaned at 56 days old and transferred to Minqin Defu Agricultural Technology Co., Ltd. . There were two batches in 2018  and two batches in 2019 .From 64 days old, all male Hu lambs were raised indoors in individual 0.8 × 1 m pens until the lambs were 180 days old, and each column of pens had a separate trough and drinking bowl for the sheep to feed freely. Before the study, a 14-day transition period was implemented, during which the proportion of pellets in the diet was gradually increased by 7.1% per day, until the diet became totally granulated. The formula, raw materials, and manufacturing plant of the feed were consistent. The experiment began with lambs at 80 days of age. They were weighed at 80 days to obtain their initial weight (BW80) and raised for up to 180 days. The body weight of each lamb at 180 days old was measured as the final weight (BW180). The feed intake of the lambs during the experimental period was recorded every 20 days. The feed intake for 80–180 days was calculated from five records. Then, we calculated the average daily gain (ADG) and average daily feed intake (ADFI). In addition, individual feed efficiency [residual feed intake (RFI) and feed conversion ratio (FCR)] were estimated. RFI was the residual of the multiple linear regression of ADFI on ADG and the medium metabolic weight [MBW = ))0.75] . The FCRAt the 180 days old, blood was collected and the lambs were slaughtered in accordance with established national standards by Ministry of Agriculture and Rural Affairs of the People’s Republic of China (NY/T 3471-2019). The blood was stored at −20°C until DNA isolation. Twenty-four hours after slaughter, the rib eye muscle area was covered with oleic acid paper from between the fifth and sixth thoracic vertebrae, and used to sketch out the muscle area in a one-to-one ratio. These sketches were scanned using an EPSON Scanner  and analyzed using ImageJ  from which we obtained the rib eye area [REA , according to the manufacturer’s product description. The quality and integrity of the DNA was measured using the A260/280 ratio and by agarose gel electrophoresis. Then, 1.5 µg of high quality genomic DNA from the high and low groups  were used to generate 80 paired-end sequencing libraries with an inset size of 500 bp. The libraries were sequenced as 150 bp paired end reads using the Illumina NovaSeq PE 150 platform .Ovis aries reference genome (Oar_rambouillet_v1.0) using BWA (Burrows-Wheeler Aligner) (Version 0.7.8) software with default parameters . Reads with joint sequences and sequences for which the number of undetected bases in single-end sequencing exceeded 10% of the total length of the sequence were eliminated. Sequences with low-quality bases with a mass value of Q ≤ 5 in the single-end sequence were removed. The clean reads were mapped onto the rameters . To redurameters . SNPs werameters .p-value < 0.05), as determined using the ASReml Wald program, were incorporated into the (co)variance estimation. Three models that accounted for the genetic effects and bivariate genetic (co)covariance were fitted and were as follows:To accurately estimate (co)variance components, we constructed an animal mixed model with univariate and bivariate analysis between each pair of feature pairs, using the restricted maximum likelihood (REML) method implemented in the ASReml software . First, 1 and y2 are the phenotypic measurement vectors of traits 1 and 2, b1 and b2 are the fixed effect vectors previously described; 1 and e2 are the vectors of random residuals. X1 and X2 are design matrices of the fixed effects and Z1 and Z2 are design matrices relating traits to random animal genetic effects. The bivariate model was as follows:p-values using PLINK (version 1.9)  was used to calculate the FDR. (Three methods (fixation index (FST), Fisher’s exact test, and the Chi-squared test) were used to screen mutation sites with significant differences in their allele frequency between the High and Low groups. FST quantified the allele frequency differences between the High and Low groups using Vcftools software (version 0.1.14) . We usedion 1.9) . In addithe FDR. .We selected SNPs that met the requirements in all three screening methods . PCR extension primers for the SNPs were designed using the genomic DNA sequences. DNA samples from 1050 lambs with phenotypic records were genetyped using KAspar genotyping technology   to furthp < 0.05).In this model, In the present study, 776 animals with their REA phenotypes were included to estimate the genetic parameters. The average REA was 12.64 ± 2.46 Heritability and variance components for the REA are shown in Estimates of genetic and phenotype correlations between the REA and important economic traits  are shown in To identify variation in the REA in sheep genomes, we identified the candidate genes associated with an increased REA in Hu sheep. We performed WGS of Hu lambs in the high and low groups. We obtained a total of 700 Gb of raw data, with an average depth of 5-fold for each individual. The average number of reads was 58785931 and the average %GC was 43.6. . The effp-value of 0.0014. This verified that there were no subgroups in the population (p < 0.05). To detect strongly selected signals, we searched the sheep genome for single SNPs with an increased genetic distance (FST). We set a threshold to select the top 20 SNPs and we used the PLINK software for PCA. The first five principal components were included in Fisher’s exact test. According to the p-value of every SNP, eleven significant SNPs . Subsequently, seven target SNPs were selected that overlapped among the three methods and were annotated to the closest gene in the Oar_rambouillet_v1.0 genome. Six genes containing these seven SNPs were defined as candidate genes .ST6GAL2  SNP g.65927208 T > C, LOC105611989 SNP g.65927208A > T, DPP6 (encoding dipeptidyl peptidase like 6) SNP g.126636893A > G, COL12A1  SNP g.2261361 T > A, and COL12A1 SNP g.2261369 T > A) were subjected to genotyping, which generated three genotypes ; however, ST6GAL2 SNP g.65927208 T > C did not have a significant impact on the REA. For LOC105611989, the male Hu sheep with the TT genotype had largest REA and those with the AA genotype had the smallest REA among the three genotypes (p < 0.05). The REA of male Hu sheep carrying the AA and AG genotypes of the DPP6 SNP g.126636893A > G was increased compared with that in male Hu sheep carrying the GG genotype (p < 0.05). The effect of the COL12A1 g.2261361 T > A and COL12A1 g.2261369 T > A SNPs s were significant on the REA. For COL12A1 SNP g.2261361 T > A, male Hu sheep with the TT genotype had a larger REA than those with the AA genotype. By contrast, the REA in the experimental population with the TT genotype of the COL12A1 g.2261369 T > A SNP was significantly lower compared with that in the sheep with the AT genotype.To determine whether the selected candidate genes had a significant effect in the REA, in the enlarged experimental population (n = 1050), five SNPs . However, ST6GAL2 SNP g.65927208 T > C and DPP6 SNP g.126636893A > G did not conform to the Hardy–Weinberg equilibrium (p < 0.05).Genetic improvement has played an important role in productivity gains in animal farming. The REA is a valuable economic trait that could affect production traits in animals . The REAIn our study, we estimated the genetic parameters of the REA in Hu breeding, and candidate gene mining was carried out. The heritability of the REA-based genetic parameters with different models was moderate. Roden et al. reported lower estimates of for rib eye muscle depth and width (0.11 and 0.08) in Scottish Blackface breed than those in the present study . In our  to 0.26 . In 2007 to 0.26 . Our res to 0.26 .In addition, we found interesting correlations between the REA and certain important economic traits. Positive genetic correlations were estimated between the REA and production traits . As expected, the size of an individual has a decisive impact on the size of the REA. Mortimer et al. reported high genetic correlations (from 0.37 to 0.70) between the REA and body weight at different stages . PhenotyWe identified negligible genetic correlations between REA and feed efficiency traits (−0.05 and −0.01), and ADFI and BW correlated positively with the REA, suggesting that animals with a larger REA grow faster and consume more feed, so it is expected that their feed efficiency would be the same. A negative genetic correlation between the measured muscle depth and feed efficiency traits (−0.3 ± 0.15) with RFI and −0.15 ± 0.18 with FCR) has been observed in other breeds . This coALS2 (encoding Alsin Rho guanine nucleotide exchange factor 2) SNP g.218145714 T > C, ST6GAL2 SNP g.65927208 T > C, LOC105611989 SNP g.163720238A > T, PLXNA4 (encoding plexin A4) SNP g.104628299 TC > T, DPP6 SNP g.126636893A > G, COL12A1 SNP g.2261361 T > A, and COL12A1 SNP g.2261369 T > A.The REA is commonly used to reflect the muscular development of the carcass. To further investigate the mechanisms affecting the REA, we identified genes that regulate the REA in the Hu sheep genome using three methods. SNPs were identified in six genes: ALS2 gene may case Familial amyotrophic lateral sclerosis type 2, which is a juvenile autosomal recessive motor neuron disease. The ALS2 gene product, ALS2/alsin, forms a homophilic oligomer and acts as a guanine nucleotide–exchange factor (GEF) for the small GTPase Rab5. This oligomerization is crucial for both Rab5 activation and ALS2-mediated endosome fusion and maturation in cells , and RhoD functions. Rho GTPases are known to regulate actin dynamics  in different human populations of European ancestry  channels, which is known to enhance channel surface expression and potently accelerate their kinetics. Many studies suggest that ancestry . This suCOL12A gene is related to the size of the REA of Hu sheep. COL12A is mainly involved in the degradation of the extracellular matrix pathway and collagen chain trimerization. In 2014, a myopathy/anhydrotic ectodermal dysplasia (EDA)-like disease caused by heterozygous COL12A1 variants was reported (Col12A1 knockout showed muscle weakness with decreased grip strength, combined with bone fragility, short stature, and kyphoscoliosis (COL12A1 mutation. We believe that this disease also occurs in Hu sheep.After fitting multiple factors in the association analysis in a large population, we found that the reported . Mice wicoliosis . DelbaerIn addition, we performed genotyping and association analysis on the seven SNPs in the enlarged experimental population. Five loci were typed successfully and three loci were significantly associated with the REA. These three SNPs could be used for marker-assisted selection. Therefore, we hypothesized that these candidate genes might affect muscle development in Hu sheep. We cannot perform whole genome sequencing in a large Hu sheep population because of the cost; however, further studies are needed to confirm the biological effects of the identified SNPs in the rib eye area.ST6GAL2 SNP g. 65927208 T > C has no association with the REA. The results showed that the SNP has skewness in this population, which led to a lack of association with the REA. Although this research verified the association between candidate SNPs and the REA in a large population, verification at the cell and protein levels is still needed.According to the association analysis of candidate genes, we found that in male Hu sheep LOC105611989 SNP g.65927208A > T, DPP6 SNP g.126636893A > G, COL12A1 SNP g.2261361 T > A, and COL12A1 SNP g.2261369 T > A, whose protein products have metabolic functions in muscle cells, as candidate genetic markers of the rib eye area. Our results could be applied to marker assisted breeding of sheep and are expected to improve for the slow genetic progress of the rib eye area in sheep. Moreover, this study provides important information for estimating the heritability parameters of the rib eye area and for molecular breeding of carcass traits in Hu sheep.The results obtained in the present study showed that the rib eye area was heritable and there is genetic variability, which could theoretically be exploited for the genetic improvement of sheep. We identified


Amphioxus is a model organism for vertebrate evolutionary research. The significant contrast between morphological phenotypic similarity and high-level genetic polymorphism among amphioxus populations has aroused scientists' attention. Here we resequenced 21 amphioxus genomes to over 100X depth and mapped them to a haploid reference.More than 11.5 million common SNPs were detected in the amphioxus population, which mainly affect genes enriched in ion transport, signal transduction and cell adhesion, while protein structure analysis via AlphaFold2 revealed that these SNPs fail to bring effective structural variants.Our work provides explanation for “amphioxus polymorphism paradox” in a micro view, and generates an enhanced genomic dataset for amphioxus research.The online version contains supplementary material available at 10.1186/s12863-022-01038-w. Cephalochordata, providing evolutionary insight into the origin of vertebrates [The amphioxus, also known as lancelet, is the modern representative of the subphylum tebrates , 2. It itebrates –6.Branchiostoma belcheri inhabiting Xiamen waters as an example, most adult individuals among these amphioxi present no apparent morphological differences, even females and males only differ in the reproductive organs [Previous studies have revealed a contradictory phenomenon that extreme phenotypic similarity and high-level genetic diversity , 3 7–11–117–11 ce organs , howevere organs . The “poe organs .B. belcheri (20 chromosomes), B. japonicum (18 chromosomes), and B. floridae (19 chromosomes), got completely resolved [Variant calling based on resequencing of main amphioxus species is an approach to seek the reasons behind “amphioxus polymorphism paradox”, for it helps researchers to get whole-genome variant distribution, where affected genes and regulatory elements can be identified for functional analysis. High-quality reference genome is the prerequisite of variant calling, while it is certain that representative amphioxus genomes have been successfully assembled , 3 15, , 15, 16,resolved , 11, yetresolved  might maIn addition, the booming protein structure prediction algorithms have paved the way for exploring detailed variant effect on the protein level. As evidenced by the results of the biennial Critical Assessment of protein Structure Prediction (CASP), structure prediction has seen substantial progress in recent years . In CASPB. belcheri) genomes to over 100X depth using the Illumina HiSeq X Ten platform, selected the new haploid genome [Here we resequenced 21 amphioxus  turns out to be larger than that of previous researches using low-depth sequencing data [B. belcheri and 4% for B. floridae of the same genus). The improvement of reference genome and sequencing method can exert positive influence on SNP calling, for it helps researchers to capture more mutation sites, thus reducing false negative rate and laying foundation for deeper studies on a larger sample size. In fact, genomic polymorphism rates of various marine lives are identified as high, for example, 1–1.5% in sea squirts (urochordate) [The polymorphism rate of ing data , 11, thohordate) , 4–5% inhordate) , 2–2.5% hordate) , etc. Gehordate) . The actAs for amphioxus, it indeed owns widely-distributed SNPs, while two factors limit their functional effect. For one thing, the majority of SNPs (more than 90%) hit non-coding regions, and those located in protein-coding regions mainly trigger to synonymous variants, in that pN/pS ratios of most genes (more than 75%) are below 1 Fig S, the symWhile it’s still discovered that in contrast to the overall variant distribution, genes identified as highly missense-mutated turn out to outnumber those synonymous-mutated in several pathways, mainly involved in signal transduction, ion transport and cell adhesion  and eggNOG-mapper (http://eggnog-mapper.embl.de) [HMMER 3.1 package was employed for Pfam  annotatiembl.de) –47.B. belcheri individuals, 10 male, 10 female and 1 hermaphrodite, were obtained from shoals (within 1km2) in Zhanjiang, Guangdong province during the summer breeding season, and kept at 28–32 °C at the Beihai Marine Station of Nanjing University, Guangxi, China.Twenty-one Twenty-one samples were anesthetized and fixed by ethanol  before muscles were dissected. Genomic DNA was extracted separately using a QIAamp® DNA mini kit  following the standard manufacturer's protocol . The purRemoval of reads with more than 10% unidentified nucleotides (N);Removal of reads containing more than 50% of bases with a Phred score ≤ 5;Removal of putative PCR duplicate reads generated by PCR amplification.Quality control criteria  were app-q 20 -c).Then sequences got further filtering via fastp  (-q 20 --M -k 19), then secondary or supplementary alignments were filtered by sambamba [- “ "not " -p -l 9).After removing low-quality reads, the clean paired-end reads were mapped to the haploid reference genome using BWA  ;BaseRecalibrator and ApplyBQSR modules were applied to generate recalibrated bam files for each individual;–emit-ref-confidence GVCF).HaplotypeCaller module was applied again to detect variants , and got annotation by SnpEff [All GVCF files were processed by CombineGVCFs and GenotypeGVCFs modules to generate the population genotype file, then SNPs were selected using SelectVariants and VariantFiltration modules . Common SNPs in the population were selected by PLINK   genes with largest number of intron variant , synonymous variant , and missense variant  using clusterProfiler [pvalueCutoff = 0.05, pAdjustMethod = 'BH', qvalueCutoff = 0.2).GO enrichment analysis was performed on top-1000  was run to get the highest-score structures (ranked_0.pdb) to make comparison.Sequences of missense-variant-enriched genes were extracted based on enrichment analysis and SNP statistics above, then 100 crucial protein domains according to Pfam annotation were chosen for structure analysis. For each raw-mutated sequence pair, AlphaFold2  , missense variants (b), and intron variants (c). A: RNA processing and modification; B: Chromatin structure and dynamics; C: Energy production and conversion; D: Cell cycle control, cell division, chromosome partitioning; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism; G: Carbohydrate transport and metabolism; H: Coenzyme transport and metabolism; I: Lipid transport and metabolism; J: Translation, ribosomal structure and biogenesis; K: Transcription; L: Replication, recombination and repair; M: Cell wall/membrane/envelope biogenesis; N: Cell motility; O: Posttranslational modification, protein turnover, chaperones; P: Inorganic ion transport and metabolism; Q: Secondary metabolites biosynthesis, transport and catabolism; R: General function prediction only; S: Function unknown; T: Signal transduction mechanisms; U: Intracellular trafficking, secretion, and vesicular transport; V: Defense mechanisms; W: Extracellular structures; X: Unnamed protein; Y: Nuclear structure; Z: Cytoskeleton. Fig S3. Box plot of total pN/pS ratio distribution. Table S1. Number of common SNPs on each chromosome. Table S2. Number of variants affecting each gene calculated via SnpEff. Table S3. Statistics of SNPs detected in each type of genomic region in each amphioxus sample. Table S4. Full tables of GO enrichment results.


Condensed tannins (CTs) are secondary plant metabolites known for their antinutritional properties but also for their beneficial attributes for animal health and food quality, including antioxidant activity. Condensed tannins sources, such as plants and agro-industrial by-products or extracts prepared from these vegetal materials, have been used in ruminant diets to improve the animal antioxidant status and the oxidative stability of their products. However, this nutritional strategy has shown inconsistent results. Furthermore, unlike other phenolic compounds with low molecular weight, CTs are high molecular weight oligomers and polymers with poor bioavailability, which limit their absorption into circulation and direct antioxidant effect in living animals and post-mortem. Therefore, the action mechanism by which dietary CT exerts an antioxidant effect on ruminants is poorly understood. So, this review briefly presents the chemical structure of tannins, with particular emphasis on CT chemical structure, summarizes several studies focused on the effect of dietary CT sources on ruminants’ antioxidant status and oxidative stability of their products, and discusses the possible action mechanisms by which CT can exert such effects.Condensed tannins (CTs) are widely distributed in plants, and due to their recognized antioxidant activity are considered as possible natural antioxidants for application in ruminant diets. A wide range of CT-rich sources has been tested in ruminant diets, and their effects on animal antioxidant status and oxidative stability of their products are reviewed in the present work. Possible mechanisms underlying the CT antioxidant effects in ruminants are also discussed, and the CT chemical structure is briefly presented. Utilization of CT-rich sources in ruminant feeding can improve the animals’ antioxidant status and oxidative stability of their products. However, the results are still inconsistent. Although poorly understood, the evidence suggests that CTs can induce an antioxidant effect in living animals and in their products through direct and indirect mechanisms, which can occur by an integrated and synergic way involving: (i) absorption of CTs with low molecular weight or metabolites, despite CTs’ poor bioavailability; (ii) antioxidant action on the gastrointestinal tract; and (iii) interaction with other antioxidant agents. Condensed tannins are alternative dietary antioxidants for ruminants, but further studies should be carried out to elucidate the mechanism underlying the antioxidant activity of each CT source to design effective antioxidant strategies based on the use of CTs in ruminant diets.  Livestock species are frequently exposed to oxidative stress, generated by overproduction of free radicals that cannot be handled by the body’s antioxidant defense capacity, resulting in oxidative damage of several vital cell components and deterioration of many physiological functions, such as growth, reproduction, and immunity ,2. Oxidative damage in cells and tissues results from the action of several reactive molecular species, generally designated as reactive oxygen (ROS) and nitrogen species (RNS), although some non-radical reactive derivatives of oxygen and nitrogen are also included  that areSeveral factors can create favorable conditions for the development of oxidative stress in farm animals. Lipid supplementation of ruminants’ diets has been used for a long time to increase their energy density, and more recently to improve the fatty acid profile of ruminant products by inclusion of lipid sources rich in polyunsaturated fatty acids (PUFA) in diets ,7,8. Hown-3 PUFA, highly susceptible to lipid oxidation [The animals’ antioxidant status is also reflected in the quality of their products, determining a higher susceptibility or resistance to oxidative deterioration. Animal-derived foods are subjected to oxidative reactions, resulting in deterioration of color and development of undesirable odor and flavor, loss of nutrients such as essential fatty acids, vitamins, and other bioactive compounds, and production of compounds harmful to human health, a major cause of the quality loss in food ,14. Thesxidation ,17. Foodxidation .When oxidative stress occurs, an adaptive stress response is triggered to minimize the damaging effects of free radicals, increasing the capability of antioxidant systems to eliminate the free radicals . The endAs plant secondary metabolites, condensed tannins (CTs) are part of the plant chemical defense system against biotic and abiotic stressors ,21. Morepost-mortem antioxidant effects when applied in ruminant diets have been contradictory, with doubts remaining as to the effectiveness of this approach, as well as the action mechanisms. So, the main objective of the current review is to critically analyze the available knowledge on the effect of CT sources’ incorporation in ruminant diets on animal antioxidant status and oxidative stability of their products and discuss the CT antioxidant mechanisms in ruminants. Tannins’ chemical structure will also be briefly reviewed.Probably due to the association of various diseases with oxidative stress, the antioxidant activity has been one of the most studied biological effects of CTs, in both in vitro and in vivo models. The strong antioxidant capacity of CTs is demonstrated in several in vitro assays ,31,32, aTannins are a heterogeneous group of phenolic compounds that can be classified into 3 major groups based on their chemical structures: hydrolysable tannins (HTs), condensed tannins (CTs) and phlorotannins (PTs) ,35. The cis or trans-configuration) [cis orientation, whereas catechin and gallocatechin show trans orientation [For the purposes of this review, our focus will be on the CTs. Known also as proanthocyanidins, CTs are oligomers and polymers composed of flavan-3-ols linked through C4-C8 and/or C4-C6 bonds, both called B-type CTs, and doubly linked with an additional ether bond at C2 → O → C7 or C2→ O → C5, designed as an A-type CT ,37. The uration) ,40. The uration) . The steentation . Moreoveentation . Accordientation . The CTsentation . Taking into account the different pattern of hydroxylation, types of interflavan-3-ol linkages, stereochemistry of the C2 and C3 and degree of polymerization, a great structural diversity of CTs is found in plants . Their sCistus ladanifer, quebracho and mimosa), and widespread legumes forages (sainfoin and sorghum). Plants or by-products rich in CTs may represent a greater proportion of the diet (50–750 g/kg dry matter (DM), A wide range of CT sources has been extensively tested in ruminant diets, as modulators of the ruminal biohydrogenation in order to improve the fatty acid profile of meat and milk fat ,45, and 2 and 20% of CO2) after 10 days of storage in retail conditions [2:CO2 atmosphere and stored at 2 °C during 14 days, there was no improvement of the lipid and myoglobin stability when 50 g/kg DM of pomace grape was included in the concentrate supplied to the lambs [Grape pomace, consisting of grape seeds, skin and pulp, is one of the most studied CT sources in terms of its efficacy to improve ruminants’ antioxidant status and the oxidative stability of their products. Inclusion of 50 and 100 g/kg DM of red wine grape pomace in lamb diets resulted in the improvement of several muscle antioxidant status parameters . A reducnditions . Convershe lambs . Higher levels of grape pomace in lamb diets than those tested by other authors  were used in experiments by Chikwanha et al. . The musIn previous studies, the grape pomace was previously dried after incorporation into the diets. Ensiling the grape pomace is an alternative way to conserve this by-product. Inclusion of grape pomace silage  in the soybean oil-supplemented diet (40 g/kg DM of soybean oil) of dairy cows increased the reducing power of milk to reduce the ferric ions . HoweverCarob pulp is an agro-industrial by-product widely available in the Mediterranean area and commonly used in animal feed , which iOther agro-industrial by-products rich in CTs were also tested in ruminant diets, although the results on their antioxidant effect are still limited. Supplementation of goat diets with pine bark, a by-product of the timber industry containing up to 130 g/kg DM of CTs, also did not affect the meat MDA levels . Meat liCistus ladanifer, a perennial shrub very abundant in the Mediterranean area rich in CTs (32–161 g/kg DM of CTs [Cistus ladanifer (leaves and soft stems) in a high-forage diet, supplemented or not with 6% of a blend of soybean and linseed oils , reduced the MDA levels in meat after lipid oxidation induction [Cistus ladanifer, showing the effectiveness of the dietary Cistus ladanifer to enhance the meat’s resistance against lipid oxidation, even in PUFA-enriched meat [Cistus ladanifer  were incorporated in a forage and concentrate diet (1:1) supplemented with 0, 40 and 80 g/kg DM of soybean and linseed oils  [Cistus ladanifer to reduce the lipid oxidation in lamb meat without oxidative induction has also been proven [Cistus ladanifer  in diets gradually reduced the MDA levels in meat samples stored for 7 days at 2 °C [Cistus ladanifer in the diet [Cistus ladanifer resulted in a significant protection of the meat against lipid oxidation. In a more recent work, the partial replacement of dehydrated lucerne with 150 g/kg DM of Cistus ladanifer in oil supplemented diets (50–60 g/kg DM of soybean oil) composed by 1:1 forage and concentrate did not reduced the MDA levels in lamb meat [Cistus ladanifer on lipid stability. Differences in basal diets, levels and type of lipid supplement and inclusion of other antioxidant compounds in diets might have created a different antioxidant–pro-oxidant balance among experiments, leading to the inconsistent results on the antioxidant effect of the dietary Cistus ladanifer [The utilization of endemic species due to their richness in CTs has been another approach to supply CTs in ruminant diets. The aerial part of M of CTs ), has benduction . As expehed meat . Increas:2, v/v) , not alln proven ,61. Thesamb meat . In thisadanifer . Larrea divaricata (125 g/kg DM of leaves) and Acacia aroma (125 g/kg DM of leaves)—in goat diets composed by forage and concentrate did not change the antioxidant activity (DPPH assay) and total phenols content in meat, but reduced the MDA levels in meat stored at 4 °C for 6 days, at −18 °C for 30 days and at 26 °C for 6 h [Acacia farnesiana pods . On the other hand, when the FRAP and oxygen radical absorbance capacity (ORAC) assays were used, an increase of the antioxidant activity of milk was reported, reaching similar or higher values than those presented in the milk of goats fed exclusively on pasture. Increasing levels of total phenols in goats’ milk with increasing amounts of Acacia farnesiana pods in diets was also observed; however, the catechin levels remained unchanged. The inclusion of two different CT-rich woody species— for 6 h . Delgadi for 6 h  also didFicus bengalensis (119 g/kg in concentrate), a tanniferous native tree from the Indian Subcontinent [Ficus infectoria (evergreen tree abundant in northern parts of India) were included in their diets  [Ficus infectoria and Psidium guajava leaf mixture (70:30) in concentrate feed  supplied to lambs enhanced the erythrocytic antioxidant status, increasing the intracellular activity of SOD and CAT and the levels of GST, GSH and total and protein thiol (P-SH) [Quercus electrophori) containing 3.35% of CT and 3.10% of HT, suggesting an improvement in the antioxidant status of those animals [The impact of some native species on animal antioxidant status was evaluated by analyses of several antioxidant parameters in the erythrocytes, including enzymatic and non-enzymatic antioxidants and lipid peroxidation parameters. In cows, the dietary replacement of rice bran by dried and ground leaves of ontinent , seems tontinent . Similar12 g/kg) . Inclusil (P-SH) . Increas animals . Forage legumes with high levels of CTs, such as sainfoin and high-tannin sorghum, were also evaluated in terms of their effectiveness to limit meat lipid oxidation when incorporated in ruminant diets. Feeding lambs with silages containing sainfoin improved the lipid oxidative stability of meat subjected to pro-oxidant conditions  compared to grass silages . ConsistDifferences in CT chemical structure and concentration in the diet, as well as the diversity of basal diets used and the presence of other bioactive compounds with antioxidant activity in the diet, can help to explain the inconsistent results on the antioxidant effect of CT-rich plants and by-products, either between different CT sources, or with the same CT source. It is also important to note that within each CT source type, the CT levels can be variable among experiments, since the biosynthesis of phenolic compounds, such as CTs, depends on several factors, including variety, plant/fruit development stage, environmental temperature and water availability ,77,78,79Utilization of CT extracts allows a better understanding of the specific effect of dietary CTs on ruminant antioxidant status and oxidative stability of their products. Quebracho, mimosa and grape seed have been the main CT extracts applied in ruminant diets, as possible sources of antioxidants . The protective effect of dietary quebracho CTs against lipid oxidation was verified by Lobón et al.  in meat v/v)) reduced the MDA levels in meat stored for 3 and 7 days at 4 °C and subjected to lipid oxidation induction [2 atmosphere  and stored at 4 °C for 14 days [n-3 enriched meat stored in a modified atmosphere  over 12 days at 4 °C was also not affected by supplementation of lamb diets with 900 mg of red wine extract per kg of concentrate [In lambs, the incorporation of 25 g/kg DM of grape seed extract in a high-forage diet supplemented or not with a blend of vegetable oils  CT extract to protect lamb meat against lipid oxidation was tested in two studies, where 40 g/kg of mimosa extract was included in concentrate [Acacia mearnsii CT extract  and found no differences in the MDA milk levels. However, they found increasing diene conjugates concentration, which indicates a greater lipid peroxidation, with increasing CT levels.The efficacy of dietary supplementation with mimosa  of CTs Choerospondias axillaris peels after incubation. Gültekin-Ozgüven et al. [The degradation of CTs into compounds with a lower degree of polymerization or even to monomers throughout the GIT would allow greater availability of absorbable compounds, but the CT metabolization in the GIT is still poorly understood. Despite the conflicting results, several reports have shown that CTs are not inert during transit through the GIT, instead they are subject to extensive biotransformation processes ,100,101.n et al.  reportedn et al. . So, duen et al. ,102,108.n et al. ,102,108.n et al. ,110,111 14C-labeled CTs, a substantial disappearance of CTs from sheep and goat gastrointestinal tracts was also identified [The studies on the fate of CTs in the GIT have been performed mainly using in vitro models and in humans and animals such as rats and pigs ,108, whientified ,114. Glaentified  reportedentified  associatentified ,116 coulentified , and furCondensed tannins are known for their ability to form complexes with various types of molecules, primarily with proteins and to a lesser extension with polysaccharides, nucleic acids and metal ions . These cDespite the conflicting results on CT metabolization and absorption, increasing evidence supports that CTs undergo transformation along the GIT. The discrepancy in these results can be attributed to several factors, such as type of CT , possible interactions with food/feed matrix , and aniAcacia farnesiana were added to goats’ diets [Several studies reported an increase of the total phenolic content, determined using Folin–Ciocalteau reagent, in plasma, muscle and milk of ruminants fed diets contain CTs shown in , which cs’ diets .Although the CTs in intact form or their metabolites can be absorbed and transported to organs by blood inducing beneficial effects, the great proportion of CTs and their metabolites that are excreted in feces is an indicator of their poor bioavailability . So, theCistus ladanifer in lamb diets also resulted in increased levels of α-tocopherol in muscle [Cistus ladanifer can be a source of vitamin E, other mechanisms that may be responsible for these higher levels of α-tocopherol in muscle cannot be excluded. Various mechanisms by which phenolic compounds might induce such an increase have been hypothesized, such as by protecting vitamin E against oxidation, restoring the vitamin from oxidation, and inhibition of metabolism and enhancement of absorption [The gastrointestinal tract has been proposed as the main site of CTs’ action, where they and/or their metabolites can directly exert several biological activities, as antioxidant activity. In the intestine, CTs can scavenge free radicals, chelate metals, and reduce lipid peroxidation and the production of lipid oxidation derivatives and toxic compounds, which could result in improvements of the animal’s overall antioxidant status ,122. Morn muscle . Althougsorption . Moreover, CTs are also able to protect other antioxidant compounds in addition to vitamin E, such as ascorbic acid . Iglesialongissimus dorsi muscle from lambs fed on grape seed CT. Higher antioxidant enzymes gene expression was also observed in human hepatoblastoma cells incubated with grape seed procyanidins [Moreover, it has also been shown that CTs’ beneficial effects may be related to an ability to modulate the cell signaling pathways, thus affecting the expression of specific genes . Supplemyanidins . Consistyanidins  or grapeyanidins . Nrf2 isyanidins . The Nrfyanidins ,131. It yanidins ,133,134.The results showed that inclusion of CT-plants and plant extracts in ruminant diets can improve the animal antioxidant status and produce edible products with better oxidative stability. However, the antioxidant effect of dietary CT sources in ruminants has not been proven in all works, which can be due to several factors, such as CT chemical structure and concentration in the diets, composition of basal diet, presence of pro- and antioxidant compounds, or other uncontrolled factors that might create different balance between antioxidant and pro-oxidant agents.post-mortem by preventing oxidative damage in products, may be due to multiple mechanisms of action, which could occur in an integrated and synergic way. The direct antioxidant effect, which implies the absorption of CTs or their metabolites and presence in circulation and tissues, probably is not the most relevant CT antioxidant mechanism considering the poor bioavailability of CTs. However, this mechanism cannot be excluded and can contribute to overall antioxidant status when CT sources contain or originate higher levels of absorbable CTs. The antioxidant action of CTs in the GIT is the most consensual method, which can also occur in different ways, reducing oxidation reactions and protecting other antioxidant compounds. Interaction with other enzymatic and non-enzymatic components of the antioxidant system seems to be another possibility by which CTs can exert antioxidant activity. In addition to other factors, the chemical structure of CTs seems to be an important factor in determining the antioxidant action mechanisms of CTs in vivo, so the way CTs exert antioxidant activity and its effectiveness probably depend on the CT’s origin. However, the chemical structure of several CT sources used in animal nutrition is poorly characterized, and their characterization should be improved. In addition to better knowledge of the chemistry of each CT source, it is important to carry out further studies in ruminants to understand the action mechanism by which each CT source exerts antioxidant activity in living animals and in their products, which will allow design of effective antioxidant strategies based on the application of CT sources in diets.Evidence supports that the antioxidant effects of CTs, which are observed both in living animals and


Fusarium culmorum is an important pathogen causing head blight of cereals in Europe. This disease is of worldwide importance leading to reduced yield, grain quality, and contamination by mycotoxins. These mycotoxins are harmful for livestock and humans; therefore, many countries have strict regulatory limits for raw materials and processed food. Extensive genetic diversity is described among field populations of F. culmorum isolates for aggressiveness and production of the trichothecene mycotoxin deoxynivalenol (DON). However, the causes for this quantitative variation are not clear, yet. We analyzed 92 isolates sampled from different field populations in Germany, Russia, and Syria together with an international collection for aggressiveness and DON production in replicated field experiments at two locations in two years with two hosts, wheat and rye. The 30x coverage whole-genome resequencing of all isolates resulted in the identification of 130,389 high quality single nucleotide polymorphisms (SNPs) that were used for the first genome-wide association study in this phytopathogenic fungus.pG), however, four SNPs were associated with major quantitative trait loci (QTLs) with pG ranging from 12 to 48%. The QTL with the highest pG was involved in DON production and associated with a SNP most probably located within the Tri4 gene.In wheat, 20 and 27 SNPs were detected for aggressiveness and DON content, respectively, of which 10 overlapped. Additionally, two different SNPs were significantly associated with aggressiveness in rye that were among those SNPs being associated with DON production in wheat. Most of the SNPs explained only a small proportion of genotypic variance (F. culmorum were captured using a heuristic approach. Key phenotypic traits, SNPs, and candidate genes underlying aggressiveness and DON production were identified. Clearly, many QTLs are responsible for aggressiveness and DON content in wheat, both traits following a quantitative inheritance. Several SNPs involved in DON metabolism, among them the Tri4 gene of the trichothecene pathway, were inferred as important source of variation in fungal aggressiveness. Using this information underlying the phenotypic variation will be of paramount importance in evaluating strategies for successful resistance breeding.The diversity of 92 isolates of The online version contains supplementary material available at 10.1186/s12864-021-07931-5. Fusarium belongs to one of the most diverse and widely distributed fungal genera producing a broad variety of secondary metabolites [Fusarium species [F. graminearum and F. culmorum are frequently isolated members [F. culmorum infection can be even more problematic since the fungus produces an array of mycotoxins, including type B trichothecenes, which are inhibitors of protein synthesis in plants and animals. The main trichothecenes reported in F. culmorum-infected cereals include deoxynivalenol (DON), nivalenol (NIV), and their acetylated derivatives [F. culmorum isolate was described that produced nine additional mycotoxins on wheat, one of which was zearalenone [Fusarium spp. is a worldwide problem and therefore strict regulatory limits are established in many countries regarding the amount of mycotoxins legally permitted in grains for food/feed usage and in processed food in the EU, USA, and Canada [Fusarium infection of cereals results in decreased market value or even the rejection of the grain.abolites . It caus species , where F members –5. They  members , 7 and s members . A commo members . In addiivatives , but in ralenone . Crop myd Canada . ConsequF. culmorum has a wide host range, it offers an ideal opportunity to unravel the hidden genetic basis of quantitative pathogenicity. Previous studies consistently reported a large variation in aggressiveness of F. culmorum [FgFGL1 [, other hydrolyzing enzymes [F. culmorum and F. graminearum [F. graminearum, many other potential genes and genomic regions have been identified [F. graminearum aggressiveness, in particular the Tri5 gene encoding the first pathway enzyme was reported in multiple studies to be linked with aggressiveness [F. culmorum and F. graminearum cause reduced symptoms compared with wild-type isolates [F. graminearum populations [Pathogenicity is the ability of the pathogen to infect a susceptible host . If infeculmorum –21. Olde [FgFGL1 , other h enzymes  and ABC minearum , 25. Howentified , 26–29. entified . Accordisiveness –34. DON-isolates , 33, 35.F. graminearum are clustered within a 25-kb region on chromosome II that is inherited as one core TRI cluster [Tri101 and the two-gene Tri1-Tri16 locus, both located in unlinked chromosomal regions [Tri7 and Tri13 genes is required for NIV-producing chemotypes in both F. graminearum [F. culmorum [F. culmorum genome is structured similarly to that of F. graminearum, as shown by Schmidt et al. [F. culmorum isolate. In the meantime, however, NIV-producing isolates are increasing in frequency in several geographic regions, as has been reported for different Fusarium species [F. culmorum population with a frequency of 75.1% compared to 15.0% in F. graminearum at least in maize [Fusarium spp. Indeed, several other genes have been described as aggressiveness factors in candidate-gene association studies in F. culmorum [F. graminearum [Twelve of the trichothecene genes in  cluster , 37. Twominearum  and F. cculmorum . The tri species , 41. Forculmorum , 42 and minearum .F. graminearum was the first member of cereal-infecting Fusaria whose complete genome assembly was provided [F. culmorum, there are two genome sequences available, one from the Australian strain CS7071 [(PRJEB1738) [https://downloads.bioplatforms.com/wheat_pathogens/) and the other from a United Kingdom field strain, UK99 [F. graminearum strain PH-1 with 36.6 Mb [F. culmorum revealed four chromosomes using alignments to F. graminearum PH-1, but also yielded two small pseudomolecules concatenated by unplaced contigs > 2 kb (“chromosome 5”) and < 2 kb (“chromosome 6”) [The understanding of the physiological function of genes, chromosomal organization and related gene products or metabolites has been boosted by the arrival of next generation sequencing technologies, assisting to elucidate the genetic and molecular features that facilitate success of some fungal species as plant pathogens , 44, 45.provided , which hprovided –48. For JEB1738) , GenBankin, UK99 . The lat 36.6 Mb . The drasome 6”) .F. culmorum is available, to date only two genes have been associated with pathogenicity or aggressiveness according to the Pathogen-Host-Interaction database (http://www.phi-base.org/searchFacet.htm?queryTerm=Fusarium+culmorum): an ABC transporter and a ROGDI like leucine zipper domain regulating Trichothecene type B biosynthesis [Fusarium species [F. graminearum with the fast sub genome relating to host-pathogen interaction. High-throughput gene deletion studies have also proven useful in identifying genes essential for pathogenicity, e.g., [F. culmorum [Although the full genome sequence of ynthesis , 51. Dif species , 45, 52.y, e.g., , 54. Neiy, e.g., . Candidaculmorum , 42, butF. culmorum and used 134,789 high quality single-nucleotide polymorphisms (SNPs) to conduct a genome-wide association study (GWAS). We thereby took advantage of the natural diversity produced by historical mutation events among field populations of F. culmorum sampled from different countries and phenotyped them in multi-environment trials. This study is unique since, until now, no resequencing study, e.g., [To obtain a broader picture of the genetics of aggressiveness and DON production of this important fungal pathogen in its natural environment, we re-sequenced the genome of 92 isolates of y, e.g., , 45 compF. culmorum chromosomes was 3.66 per kb on average , the mean value was 15.5% with a minimum of 6.9% and a maximum of 19.5% for isolates S129 and S109, respectively. Values of aggressiveness in rye (AGG-RYE) ranged from 3.4% for isolate S129 to 27.9% for isolate FC95 with a mean of 15.7%. The entry-mean heritabilities were high for all traits, ranging from 0.85 to 0.89, thus providing a good starting point for association analysis.For DON content in wheat (DON-WH), the lowest concentration detected was 0.44 mg kg22 Table  Table. TWhen color-coding the subpopulations in a scatterplot for aggressiveness on wheat and rye Fig. , no clusP < 0.001) different from zero for all traits. The correlation between aggressiveness in wheat and DON production was significant  .The genotypic variance (< 0.001)  Fig. FurClassical multidimensional scaling (MDS) of the distance matrix was performed and the resulting principal components revealed some degree of population structure  Fig. ResThe association analysis resulted in the identification of 27 SNPs that were associated with DON production  that is significantly associated with DON production and explains 48.4% of the genotypic variance. Although this SNP explains just 1.33% of the genotypic variance of aggressiveness in wheat, it is the only SNP with a significance value above the Bonferroni threshold  was significantly associated with aggressiveness towards rye but not towards wheat. This was attributed by the authors to the different composition of the cuticles of wheat and rye. Moreover, rye and wheat were overlapping only in 1 year (2015), in 2014 only wheat and in 2016 only rye was analyzed. This might have caused differences due to the highly significant genotype × environment interaction observed also in this study that is typical for the pathosystem. The complex genetic control underlying aggressiveness is corroborated by the finding of this study, that no QTL for aggressiveness were common between wheat and rye.Because aggressiveness is inherited quantitatively, each gene contributing to this complex trait is expected to explain only a small proportion of the genotypic variance. This has been recently shown by candidate-gene based association mapping , 42 wherWhen the allele frequency of the SNPs was assessed in the whole population, we found that most of the isolates carry the high aggressiveness allele Fig. . This miF. culmorum genome. A blast of the genomic and the peptide sequences of both genes against the NCBI database and the UniProt database did not reveal for FCUL_06122 any more specific functions. The best hits, all on Fusarium species, also resulted in “uncharacterized protein”. For FCUL_10689 the best hits on UniProts were also unknown proteins, but in Fusarium longipes, Fusarium sporotrichioides, and Fusarium vanettenii (syn. Fusarium solani f. sp. pisi) peptide sequences were annotated as Transaldolase. Transaldolase (EC 2.2.1.2) was associated with the sugar and energy metabolism and one of the most abundant protein spots identified in B. cinerea mycelium [Cochliobolus heterostrophus was identified as a Hog1-dependent gene playing an important role in protection against oxidative stress [Hog-1 was significantly associated with aggressiveness and DON production in our candidate-gene association mapping of 100 F. culmorum isolates [Two SNPs significantly associated with aggressiveness in wheat were found to be the proteins FCUL_06122 and FCUL_10689 with no predicted function in the mycelium . Interese stress . And Hogisolates  of whichThe correlation between aggressiveness and DON production in wheat was significant, corroborating previous studies , 67, 68.Tri4 gene of F. graminearum. This gene encodes a multifunctional cytochrome P450 monooxygenase for different oxygenation and hydroxylation steps in the trichothecene pathway leading to DON [Tri4, but it is nonetheless an important factor in DON production having a high impact on aggressiveness. However, our study also shows that the large differences in aggressiveness among F. culmorum isolates most probably involve a multitude of quantitative loci of which Tri4 is only one, although an important one. Accordingly, Talas et al. [F. graminearum, each explaining 8 to 24% of the genotypic variance. As in our study, most genes detected did not have a known function.Results of this association study strongly promote an important role of DON content for aggressiveness. The SNP FC2_5593616, explaining 48.4% of the total genotypic variation in DON content and as the only SNP exceeding the conservative Bonferroni threshold, is located in a gene with a very high identity (95.7%) with the published sequence of the g to DON  and is, g to DON . Alternas et al.  found 26F. culmorum is most likely caused by a multitude of genes of whom those of the DON pathway have an important function. Among them, we could show here that the Tri4 gene or a gene in close linkage to Tri4 explained nearly half of the total genotypic variation. More functional studies will be needed in future to further decipher the molecular mechanisms underlying their associations with fungal aggressiveness.The high phenotypic variation of aggressiveness and DON production of F. culmorum collected from three different geographic origins, were used  cultivar that is moderately susceptible  and a highly susceptible, homogeneous rye single cross . Inoculations and phenotyping were done at two locations: Hohenheim  and Oberer Lindenhof  in Germany in 2 years (2014 and 2015 for wheat as host and 2015 and 2016 for rye), which is equivalent to four environments (=location-year combinations).Aggressiveness of o et al. , 42. BriTriticosecale) in the wheat experiment. Plots were sown with 220 kernels m− 2 resulting in a homogeneous dense plant stand. Experiments were designed following an incomplete block design  with two replications per environment and an incomplete block size of 10.Seeds were grown in two-row plots (rye) or three-row plots (wheat), respectively, with 1 m length and a row distance of 0.21 m. To decrease the drifting or secondary spore dispersal and avoid possible interference among plots, a chessboard-like design was used to arrange the entry plots that were bordered by long-strawed rye in the rye experiment and triticale  and the percentage of infected ears per plot (incidence) in a single value from 0 to 100% , 42. For2) between traits was calculated based on the BLUEs. The software package ASReml-R 3.0 [Statistical analyses of phenotypic data were done using the software R , outliernt hosts . Insteadnt hosts . Pearsonml-R 3.0  was usedml-R 3.0 .Plugs from each isolate were cultivated on PDA plates during 5 days at 20 °C. From each isolate, 200 mg of mycelia was scraped off from several plates with a sterile spatula and dried out with silica gel for 1 week. DNA extraction was performed following the cetyltrimethylammonium bromide (CTAB) method. DNA samples were run on a 1% agarose gel to check DNA stability and integrity with a O’GeneRuler 1 kb DNA ladder (Thermo Scientific) and the 23 kb DNA Molecular Weight Marker II (Sigma–Aldrich). DNA quality was measured with nanodrop, and quantity was measured with QuantiFluor® dsDNA System (Promega). Finally, the DNA was concentrated to 50 ng/μl. DNA samples were sent to Novogene Corporation  where Illumina libraries were prepared for whole-genome sequencing using a Illumina HiSeq X with 150 bp paired-end sequencing. The resulting sequencing libraries had an average insert size of 300 bp.F. culmorum strain UK99 that has almost 42 Mbp and consists of the 4 F. culmorum chromosomes plus two scaffolds of concatenated sequences that could not be assigned to the chromosomes [F. culmorum chromosomes were calculated using the SnpEff tool [F. culmorum strain UK99 covering an average of 87.7% of it, with a minimum of 81% and a maximum of 91.6%. The average sequencing depth was 33X and the mean insert length was 287.8 bp and ranged from 2 up to 636.3 kb. During the filtering process, SNPs that were mapped to repetitive sequences and copy number variations were excluded. Genotypic quality control of the final SNPs was performed using the R package GenABEL [Pair-end reads from all isolates were mapped to the reference genome with the Burrows-Wheeler Aligner software package   using thomosomes . The NGSomosomes  was usedEff tool  and vcftEff tool  and mappEff tool . UpstreaGiven that isolates coming from different geographic regions were used for this study, stratification was assessed by principal component analysis with the R package GenABEL . For thip < 0.0001 were used. All significantly associated markers of a trait were fitted into a linear model in the order of the strength of their association, i.e. with the most strongly associated marker fitted first [2 of this linear model to the heritability of the trait pG-combined). In addition, we also calculated the proportion of explained genotypic variance of each marker when fitted singly in a linear model (pG-single). The combination of the two values allows to draw conclusions whether significantly associated markers from one genomic region identify the same QTL, as the joint fit of all significantly associated markers corrects for collinearity among them. The additive effect (α-effect) of each individual significant SNP was derived from a linear model in which just the respective SNP was considered. The local GWAS plot was created using the R package LDheatmap [Association analysis between SNP markers and traits was done with GenABEL , using aed first . The pro=Radj2h2 . The proDheatmap .F. culmorum genome. Translated sequences were compared with the protein sequences of closely related species. For the identification of secondary metabolite biosynthesis gene clusters in the whole genome of F. culmorum the web server antiSMASH 3.0 was used [To analyze the sequences that harbor the significantly associated SNPs, sequences of 100 bp around the SNPs were extracted with BEDTools  and genowas used .Additional file 1: Table S1.Fusarium culmorum isolates used in this study.Additional file 2: Table S2. Number of SNPs identified and SNP density in each chromosome.Additional file 3: Table S3. Best linear unbiased estimates (BLUES) for aggressiveness in wheat (AGG.WHEAT), aggressiveness in rye (AGG.RYE), and deoxynivalenol content in wheat (DON-WHEAT) for all tested isolates across locations and years.Additional file 4: Figure S1. SNP density along the four chromosomes of F. culmorum. Histogram of variants per 100 Kb.Additional file 5: Figure S2. SNP count across all the samples of F. culmorum classified by the mutation type.Additional file 6: Figure S3. Frequency distritbution, phenotypic correlation coefficients and scatter plots for and between the evaluated traits for 92 isolates: Aggressiveness in wheat (AGG-WH) and in rye (AGG-RYE) and deoxynivalenol production in wheat (DON-WH). *** Significantly different from zero at 0.001 level of probability.Additional file 7: Figure S4. Scatterplot of the first two principal components (PC) of 92 isolates of F. culmorum originating from Germany, Syria, Russia and isolates from the international collection. The proportion of explained variance is shown in brackets at the corresponding axes.Additional file 8: Figure S5. Quantile-quantile plots (QQ-plots) for the aggressiveness in wheat (AGG-WH) and in rye (AGG-RYE) and deoxynivalenol production in wheat (DON-WH) showing the relation between expected and observed P values (−log10) for all SNPs after GWAS analysis adjusted for population stratification; Genomic inflation factor λ is given. The straight black diagonal lines represent the values under the null hypothesis of no association.