SNPs are the most abundant polymorphism type, and have been explored in many crop genomic studies, including rice and maize. SNP discovery in allotetraploid cotton genomes has lagged behind that of other crops due to their complexity and polyploidy. In this study, genome-wide SNPs are detected systematically using next-generation sequencing and efficient SNP genotyping methods, and used to construct a linkage map and characterize the structural variations in polyploid cotton genomes.G. hirsutum acc. TM-1. Recombination rates and hotspots are identified across the cotton genome by comparing the assembled draft sequence and the genetic map. Using this map, genome rearrangements and centromeric regions are identified in tetraploid cotton by combining information from the publicly-available G. raimondii genome with fluorescent in situ hybridization analysis.We construct an ultra-dense inter-specific genetic map comprising 4,999,048 SNP loci distributed unevenly in 26 allotetraploid cotton linkage groups and covering 4,042 cM. The map is used to order tetraploid cotton genome scaffolds for accurate assembly of G. hirsutum and G. barbadense. We construct and use an ultra-dense SNP map to correct sequence mis-assemblies, merge scaffolds into pseudomolecules corresponding to chromosomes, detect genome rearrangements, and identify centromeric regions in allotetraploid cottons. We find that the centromeric retro-element sequence of tetraploid cotton derived from the D subgenome progenitor might have invaded the A subgenome centromeres after allotetrapolyploid formation. This study serves as a valuable genomic resource for genetic research and breeding of cotton.We report the genotype-by-sequencing method used to identify millions of SNPs between The online version of this article (doi:10.1186/s13059-015-0678-1) contains supplementary material, which is available to authorized users. Single nucleotide polymorphisms (SNPs) are the most abundant and universal sequence variation across whole genomes, making them attractive markers for the genetic analysis of plants. It is estimated that one SNP occurs every 100 to 300 bp in any genome . Thus SNArabidopsis [G. raimondii [In most eukaryotes, the centromere is an essential chromosomal region and usually contains high-copy-number retrotransposons and satellite repeats, which are highly diverse and difficult to map, clone, and sequence. The precise position of the centromere determines the relative positions and linkage relationship between markers in or near the centromere region. Even though the identification of centromeres on genetic maps has been difficult due to highly repetitive sequence and low recombination rate in centromeric region , 20, cenbidopsis , and G. aimondii .G. hirsutum L. and G. barbadense L., which have a genome size of 2.5 Gb and large numbers of repetitive sequences derived from an allopolyploidization event between G. herbaceum L. (A1) and G. raimondii (D5) Ulbrich approximately 1 to 2 million years ago (MYA) [G. hirsutum and G. barbadense have been widely used to develop inter-specific maps because of the lack of polymorphisms between the intra-species [Cotton is an important textile and oil crop throughout the world. The cotton species include two commercially important cultivated allotetraploid species, go (MYA) . Due to -species –33. Howe-species –36.Here, we constructed an ultra-dense genetic map of allotetraploid cotton consisting of nearly 5 million SNPs  were developed from a cross between two cultivated tetraploid cotton species; G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. For SNP discovery, two parents were sequenced at relatively high coverage using the Illumina sequencing approach. In total, 154.7 Gb of high quality short read sequences (61.9-fold genome coverage) were generated for TM-1, and 97.9 Gb (39.1-fold genome coverage) for Hai7124  were retained for genotyping. Accurate genotyping of allopolyploid genomes based on short reads with low genome coverage was difficult due to two major obstacles: First, sequencing error and heterozygous regions in the genome led to inaccurate genotyping at given SNP sites, and second, excessive missing genotypes existed due to low genome coverage. To overcome these challenges, we performed a two-step genome-wide genotyping process. First, SNP positions were identified in the initial genotype of 59 F2 individuals using a minimum read depth of four and minimum read per allele of one as thresholds. A similarity score was calculated between SNP sites in each scaffold based on the initial genotype, and the SNP sites with high similarity scores were classified into a block in order to avoid mis-assembly in further analysis. Then, a sliding window approach was used to evaluate a group of consecutive SNPs in the same block for genotyping. The minimum read depth in a window containing adjacent SNP sites was 40 to avoid genotyping errors with a single allele at low genome coverage. The genotype of each sliding window was determined based on Bayesian theory , while in the second step screening, the missing genotype rate of SNP sites was reduced to 5.21 % when the sliding window approach was used and the accuracy of genotype calling at SNP sites was improved to 99.28 %. It is obvious that the accuracy and efficiency of the sequence-based genotyping of the mapping population were greatly improved through the use of the two-step method. As a result, 4,999,048 of 5,054,468 simple SNPs involved in 4,049 recombination bins were genotyped successfully, where a recombination bin represents a class of SNP loci without a recombination node and missing genotype detected. In our study, hemi and complex SNPs detected between two parents were not used for map construction because of their intricate nature that likely represents a mixture of two homoeologous or paralogous sequences due to the sequencing depth of the F2 individuals. Modern breeding has enhanced gene flow and post-domestication introgressions through deliberate hybridization between these two species. For example, the reciprocal introgressions between G. hirsutum and G. barbadense cultivars have developed Acala and Pima cultivar families. Therefore, many of these interspecific SNPs can be used in such introgressions in future cotton breeding.A total genome sequence of 784 Gb was generated for 59 Feory see . First s2 individuals and oriented by integrating the reference framework SSRs using Joinmap 3.0 [To effectively order the linkage groups, 441 framework SSR markers that were able to amplify 519 discrete polymorphic loci and were distributed evenly on 26 chromosomes were selected, with 10 cM intervals between the markers based on our microsatellite-based, gene-rich linkage map . The ultnmap 3.0 . The mapG. thurberi and G. barbadense. Finally, the highest density intra-specific linkage map comprising 4,153 loci over 3500-cM has been developed to date in Upland cotton [The inter-specific map was further compared to a high density intra-specific map which was developed to tag QTLs in Upland cotton using RAD sequencing technology . We idend cotton . The SNPAlthough the emergence of NGS technologies provides a rapid and inexpensive method for genome-wide assembly, the assembly of complex and polyploid genomes such as cotton is still challenging. It is difficult to distinguish homoeologous genomic segments with a high degree of sequence similarity, large gene families, and extensive segmental duplications such as multiple ‘paralogs’. In the present research, the ultra-dense SNP map was further used to validate the initial assembly of the TM-1 genome (scaffold N50: 210 kb) based on the Illumina short reads. We detected 90 instances of mis-assemblies of the scaffolds, which covered 36.0 Mb and accounted for 1.6 % of the entire assembled genome  reads of 2, 5, and 10 kb libraries used to generate the assembly, the longer scaffolds were enhanced by assembling 174,454 pairs of Sanger-sequenced bacterial artificial chromosome (BAC)-end sequences comprising 116.5 megabases (Mb) . After tAfter these false mis-assemblies were broken, the final assembly of the TM-1 genome was produced  . Accordiin situ hybridization (FISH) [Triticum aestivum) genome has been produced by sequencing isolated chromosome arms through flow-sorting technology [Allopolyploid plants often undergo major changes in genome structure and function induced by the combination of divergent genomes from two or more related parental species . Many stn (FISH) , and flon (FISH)  technolochnology . Howeverchnology . In factG. herbaceum or G. arboreum, and the D genome species, G. raimondii, contributed the constituent A and D subgenomes of tetraploid cotton, respectively [G. arboreum (A2) [G. raimondii (D5) [G. arboreum is an important cultivated diploid species and may have undergone some of its own chromosomal rearrangements during its evolution and improvement. When the ultra-dense tetraploid genetic map was aligned with the G. raimondii genome [G. raimondii genome was used for comparative genomic analysis.Generally, ancestors of the A genome species, ectively . Using teum A2)  and G. rdii (D5) , two extdii (D5) , partly i genome , we foun  and G. G. raimondii (GrChr03) at 33736–28298512 bp , and A03/A02 (66.75-146.42 cM) corresponding to GrChr05 at 17656841–64067579 bp ; A04/A05 (0–54.66 cM) corresponding to GrChr09 at 43817852–70655127 bp , and A05/A04 (160.43-213.26 cM) corresponding to GrChr12 at 591184–15299554 bp   observed here need to be accounted for in the ‘translation’ of information from the D genome in G. raimondii to allotetraploid cotton genomes. Genome variations such as inversions and translocations are considered important factors in species evolution. In the process of evolution from diploid to tetraploid cotton, these chromosomal structural rearrangements may have caused a series of changes in phenotype. These structural variations in the genome may help to elucidate the relationships between the phenotype and genotype of important agronomic traits in tetraploid cotton.Two reciprocal translocations revealed in the present study are in agreement with the results from prior studies , 32. ThrG. hirsutum and G. barbadense. In chromosome A01, for example, some QTLs related to fiber quality from interspecific hybrid populations have been mapped to the SNP-poor region [G. hirsutum and G. barbadense cultivars have developed the Acala and Pima cultivar families [A total of 4,999,048 mapped SNP loci (1 SNP/0.5 kb) were unevenly distributed on 26 chromosomes, with more loci in the A subgenome  than the D subgenome  Fig. . On averr region –51. Sever region , 53 in wr region . Furtherfamilies . MoreoveTo facilitate genetic analysis, we converted the SNP linkage map into a skeleton bin map Fig. . The twoArabidopsis [G. hirsutum. Genome-wide patterns of recombination have been described previously for some species, including Arabidopsis [Recombination can generate genetic variation and induce genetic diversity. By comparing genetic and physical distances between adjacent markers, we further examined the relative changes in recombination rate along each chromosome. Consistently high recombination at chromosome ends and recombination suppression were clearly observed in different linkage groups. We found 26 recombination suppression regions in the 26 linkage groups Fig. , which mbidopsis , where 1bidopsis , 60 and bidopsis . This rabidopsis , and cucbidopsis , but is bidopsis .Gossypium (CRG), CRG1 and CRG2, respectively. CRG1 and CRG2 were recently identified by co-localization immunostaining with antiserum to the centromere-specific histone CENH3 in cotton [Little is known of the dynamics of centromeric DNA in polyploidy plants. Centromeres usually contain high-copy-number retrotransposons and satellite repeats, which are difficult to map, clone, and sequence. Few studies have focused on centromeres in cotton , 65. In G. hirsutum acc. TM-1 [T-test: P value = 6.81e-07) . To construct paired-end libraries, DNA was fragmented by sonication, and DNA ends were blunted before adding an A base to each 3′ end. DNA adaptors with a single T-base 3′ end overhang were ligated to the above products. Ligation products were purified on 2 % agarose gels that each targeted a specific range of insert sizes. Quantification and quality assessment were carried out by running 1 μL of library on an Agilent DNA 1000 LabChip analyzer . We constructed Base-calling files were obtained from raw fluorescent images from the Illumina HiSeq 2000 platform using CASAVA 1.8 software in FASTQ format . IlluminAlignment of the sequences to the TM-1 reference genome  was perfFor each parental sample, putative SNPs with respect to the TM-1 reference sequences were first called using samtools  and an iUsing the mapping population, the initial genotypes of individuals were detected and classified into blocks based on similarity scores. The similarity score was calculated at SNP sites independently according to the following formula:Gij and Giz is the genotype of individuals i at the j-th SNP and at the z-th SNP, respectively, and s(GizGij) / n is the score of individuals i between the genotype at the z-th SNP and the genotype at the j-th SNP. n represents the number of individuals without the missing genotype at the j and z-th SNP. The similarity score is the sum of the scores of n individuals at the z-th SNP except for those with missing genotypes. The score of individual i at the j and z-th SNPs are: s = 1 if the genotypes of Giz and GijGij are identical ; s = 0.5 if the genotypes of Giz and Gij are different and one is ‘H’ ; and s = 0 if the genotypes of Giz and Gij are different and neither is ‘H’ (A vs. B). The minimum similarity score used for classifying similar SNP sites into a block was 0.7. There were two reasons that a minimum similarity score of 0.7 was used: (1) Markers with a high proportion of matches  were classified into a block in a similar study [2 population was 80.07 %.where w window, assume there are n reads at the SNP sites in the window. The error rate of parent 1 and parent 2 are E1 (0.058) and E2 (0.02), respectively. Under the condition of the allele from parent 1 (P1P1) or parent 2 (P2P2) or two parents (P1P2), the probability of observing n1 reads of parent 1 at the SNP sites in the window would follow a binominal distribution:For SNP sites in the same scaffold and block, an approach combining the sliding window approach  and BayeP(P1P1 | n1), P(P2P2 | n1), P(P1P2 | n1), be the posterior probabilities that the allele comes from parent 1, parent 2, or two parents given the observed n1 reads of parent 1 at the SNP sites in the window. These probabilities were calculated as:Let The genotype of the allele was determined based on the highest posterior probability at the SNP sites in the window:P1P1, P2P2 and P1P2 used for the Bayesian probability calculation were estimated based on the theoretical probabilities for an F2 population, that is, P1P1 : P2P2 : P1P2 = 0.25 : 0.25 : 0.5.The prior probabilities for genotypes To evaluate the accuracy of the genotypes of the 59 individuals, the genotypes of 26 random SSR markers that can be uniquely mapped to scaffolds by the size of their PCR product were identified in each individual. We assumed that the SNP genotypes of the 59 individuals in 10 kb flank intervals of 26 SSR markers’ physical locations were consistent with the genotypes of the corresponding SSR markers.After the imputed genotype was visually scored by a sliding window, unambiguous SNP sites were used for the construction of a genetic linkage map. Using SNP sites, an ultra-dense genetic linkage map based on the imputed genotype of allotetraploid cotton was constructed by the following procedure. The co-segregating SNP sites were clustered as a recombination bin, defined as a class of SNP sites that had identical genotypes across the 59 individuals with no recombination existing between each pair of SNP sites. The resulting 4,049 recombination bins combining SSR genotypes were used for the construction of the genetic linkage map using JoinMap Version 3.0  with a rEach scaffold within the SNP linkage map was divided into 201 bp segments based on the SNP positions. SNP segments were mapped to the assembled genome using BWA software . The staG. raimondii genome using MUMmer software, version 3.23 [We aligned the scaffolds from the ultra-dense genetic linkage map based on the SNPs of allotetraploid cotton to the whole ion 3.23 , and theThe estimated recombination rate (cM/Mb) was calculated using in-house Perl scripts, which divided the genetic length of the segment in cM by the physical length of the segment in Mb. We defined recombination suppression regions as those where the recombination rate was less than 1.0 cM/Mb, and recombination hot regions as those where the recombination rate was greater than 1.0 cM/Mb .G. hirsutum acc TM-1. To search the centromeric regions of each chromosome in TM-1, a sequence alignment method was applied. According to the structure of the retrotransposons, relatively conserved 5’LTR regions, designated as GhCR1-5’LTR, GhCR2-5’LTR, GhCR3-5’LTR, and GhCR4-5’LTR, were selected as query sequences to align with the TM-1 genome database. All the alignments were obtained by BLASTn analysis with its parameters set to the default values. Subsequently, GhCRs-5’LTR were screened for sequence similarity and e-value, with ≥80 % and ≤1e-20 set as the threshold values, respectively. To identify the centromeric regions of the A subgenome, the screened alignments of 13 chromosomes from GhCRs-5’LTR were mingled. They were then used to calculate the median size of the 95 % confidence interval for the median, which was defined as the centromeric region for each chromosome. To identify the centromeric regions of the D subgenome, the screened alignments of 13 chromosomes from GhCR1-5’LTR and GhCR3-5’LTR were used to analyze the centromeric regions. For further analyses, we used a multiple alignment consensus sequence that contained the GhCRs LTR retrotransposon RT domain. Phylogenetic analysis was performed using the NJ method in the MEGA 5.10 program [Four LTR retrotransposons were identified from the centromere-specific BAC clone, 97G20 , which i program . StatistG. hirsutum acc TM-1 and G. barbadense cv. Hai7124 are available at http://mascotton.njau.edu.cn/. The F3 descended from F2 population will be available for mapping the QTL/genes related to important traits for predictive breeding in the near future.The sequences have been deposited in DDBJ/EMBL/GenBank under the accessions (PRJNA274882). The SNP data between


Ictalurus punctatus) with male F1 hybrid catfish .Columnaris causes severe mortalities among many different wild and cultured freshwater fish species, but understanding of host resistance is lacking. Catfish, the primary aquaculture species in the United States, serves as a great model for the analysis of host resistance against columnaris disease. Channel catfish in general is highly resistant to the disease while blue catfish is highly susceptible. F2 generation of hybrids can be produced where phenotypes and genotypes are segregating, providing a useful system for QTL analysis. To identify genes associated with columnaris resistance, we performed a genome-wide association study (GWAS) using the catfish 250 K SNP array with 340 backcross progenies derived from crossing female channel catfish (pik3r3b, cyld-like, adcyap1r1, adcyap1r1-like, and mast2. In addition, 3 additional suggestively associated QTL regions were identified on linkage groups 7, 12, and 14. The resistant genotypes on the QTLs of linkage groups 7 and 12 were found to be homozygous with both alleles being derived from channel catfish. The paralogs of the candidate genes in the suggestively associated QTL of linkage group 12 were found on the QTLs of linkage group 7. Many candidate genes on the four associated regions are involved in PI3K pathway that is known to be required by many bacteria for efficient entry into the host.A genomic region on linkage group 7 was found to be significantly associated with columnaris resistance. Within this region, five have known functions in immunity, including The GWAS revealed four QTLs associated with columnaris resistance in catfish. Strikingly, the candidate genes may be arranged as functional hubs; the candidate genes within the associated QTLs on linkage groups 7 and 12 are not only co-localized, but also functionally related, with many of them being involved in the PI3K signal transduction pathway, suggesting its importance for columnaris resistance. Ictalurus spp.) accounts for approximately 60% of US aquaculture production (www.ers.usda.gov). However, due to devastating diseases, the catfish industry has been hindered by unprecedented challenges. Diseases can cause losses of up to one third of the industry each year. Flavobacterium columnare, a Gram-negative bacterium, is the causative agent of columnaris disease, which is very common in wild and cultured freshwater fish worldwide . The average water temperature was 25°C. A total of 340 backcross progenies were selected from the extremes of the disease resistance distribution of the 1200 fish based on the selective genotyping method [F. columnare were provided by the Aquatic Microbiology Laboratory, Auburn University. To get a single F. columnare colony, several fish were experimentally infected with a virulent isolate  [F. columnare for the present study, to which channel catfish was more resistant than blue catfish [6 CFU/mL. Control fish were treated with identical procedures except that they were exposed to sterile modified Shieh broth.The bacteria challenge procedure was conducted as previously described . The bacovar II) , and bac catfish . A singlDNA was isolated using standard protocols. Briefly, the blood samples were incubated at 55°C overnight and DNA was extracted twice with phenol and once with chloroform. DNA was precipitated by isopropanol and collected by brief centrifugation, washed twice with 70% ethanol, air-dried, and resuspended in reduced EDTA TE buffer . DNA was quantified using spectroscopy by Nanodrop (Thermo Scientific). The integrity of DNA samples was checked by 1% agarose gel electrophoresis stained with ethidium bromide. DNA was diluted to 50 ng/uL and the quality of genomic DNA satisfied the requirement for the genotyping platform of SNP arrays.We have developed a catfish 250 K SNP array using Affymetrix Axiom genotyping technology . Genotyp2 value. LD pruning was conducted with a window size of 50 SNPs, a step of 5 SNPs, and r2 threshold of 0.5, resulting in 14,420 independent SNP markers. The population structure was assessed by principal component analysis with the independent SNP markers.Statistical analysis was carried out using the SVS software package . Pairwise linkage disequilibrium for the backcross progeny population was calculated according to rY is the vector of phenotype; b is the vector of fixed effects including weight and first two principal components; a is the vector of random effects with covariance kinship matrix G; e is the vector of random residuals; X and Z are coefficient matrices.EMMAX (Efficient Mixed-Model Association eXpedited) analyses using all SNPs were conducted with the first two principal components and the fish body weight as covariates . The modThe threshold P-value for genome-wide significance was calculated based on Bonferroni-correction with the estimated number of independent markers and LD blocks . A ManhaThe upstream and downstream genes of the significant SNPs were determined. GENSCAN program  and FGENNo new SNPs were discovered in this manuscript. The SNPs used in this manuscript are from the catfish 250 K SNP array [


Meanwhile, cattle with wild-type combined haplotype Hap1/Hap1 had significantly (p < 0.05) greater body length than those with Hap2/Hap2. Our results indicate that variations in the SMO gene could affect body size traits of Qinchuan cattle, and the wild-type haplotype Hap1 together with the wild-type alleles of these detected SNPs in the SMO gene could be used to breed cattle with superior body size traits. Therefore, our results could be helpful for marker-assisted selection in beef cattle breeding programs.Smoothened (Smo)-mediated Hedgehog (Hh) signaling pathway governs the patterning, morphogenesis and growth of many different regions within animal body plans. This study evaluated the effects of genetic variations of the bovine SMO gene on economically important body size traits in Chinese Qinchuan cattle. Altogether, eight single nucleotide polymorphisms (SNPs: 1–8) were identified and genotyped via direct sequencing covering most of the coding region and 3ʹUTR of the bovine SMO gene. Both the p.698Ser.>Ser. synonymous mutation resulted from SNP1 and the p.700Ser.>Pro. non-synonymous mutation caused by SNP2 mapped to the intracellular C-terminal tail of bovine Smo protein; the other six SNPs were non-coding variants located in the 3ʹUTR. The linkage disequilibrium was analyzed, and five haplotypes were discovered in 520 Qinchuan cattle. Association analyses showed that SNP2, SNP3/5, SNP4 and SNP6/7 were significantly associated with some body size traits ( Smoothened (Smo)-mediated Hedgehog (Hh) signaling pathway is a critical regulator of cell growth and patterning during embryonic development, and also is involved in stem cell renewal and tissue homeostasis in adult animals ,2,3. PerIn mice, the forced expression of a constitutively actived SMO allele promoted chondrocyte proliferation, while chondrocyte-specific SMO knockout mice showed a marked decrease in chondrocyte proliferation and developed shorter long bones than wild-type littermates . SimilarLittle study on the bovine SMO gene has been performed, since most research has focused on model animals and human. Based on its role in osteogenesis and myogenesis as demonstrated in mice and zebrafish, we proposed the hypothesis that variations of the SMO gene affected body size traits in cattle. Here, we do research on Qinchuan cattle, for as a famous indigenous cattle breed in China, its growth rate and underdeveloped hind hip need to be improved to be comparable to imported beef cattle breeds . MoleculSMO gene maps to bovine chromosome 4, consists of 12 exons divided by 11 introns and 3ʹUTR, and encodes 780 amino acids. BLAST analysis revealed that the amino acid sequence of the bovine SMO (NP_001179149.1) shared high identity with other vertebrates. The percent identity with human (NP_005622.1), rat (NP_036939.1), mouse (NP_795970.3), chicken (XP_414970.4), zebrafish (NP_571102.1) and xenopus (NP_001128704.1) were 94%, 93%, 92%, 79%, 71% and 70%, respectively. The relatively high identity among mammalians (92%–94%) suggested that the SMO gene was more conserved within this group. The phylogenetic tree  intuitivResearch on model animals and human has revealed that Smo protein consists of three domains: the extracellular N-terminal domain, a seven heptahelical transmembrane domain, and the intracellular C-terminal tail . Using tWe amplified and sequenced most of the coding region and 3ʹUTR of the SMO gene. Compared with the SMO gene reference sequence (GenBank accession No. AC_000161), a total of eight SNPs , includiAll eight SNPs were successfully genotyped by PCR-direct sequencing. The detailed SNP information for each cattle was provided in 2 test indicated that genotype distributions of the eight mutations were all in Hardy-Weinberg equilibrium (χ2 < χ20.05). The PIC value  manifesr2-value, a pairwise measure of LD, SNP1 and SNP8 loci were in perfect LD (r2 = 1), so were SNP3 and SNP5 (r2 = 1), and SNP6 and SNP7 (r2 = 1). r2 = 1 indicates that, during the history of the sample, two SNPs have not been separated by recombination, and observations at one SNP provide complete information about the other SNP [r2 > 0.33) with each other. This may be a result of selection which can result in nonrandom associations among SNPs and thus elevate levels of LD in a gene; especially selection, during domestication, aiming at alleles of a gene can significantly elevate levels of LD in a given region [ther SNP . Thus, Sn region ,21.8), but both the perfect and strong LDs between SNP marker pairs notably decreased the number. Obviously, Hap1 (-CTCCCTTC-), the combination of wild-type alleles, was the most frequent (0.558), followed by Hap2 (0.234), Hap3 (0.092), Hap4 (0.085), and lastly the combination of mutant-type alleles Hap5 (-TCCACGCA-) (0.031). The high-frequency haplotypes might have been in the population for a long time and more adaptive to the environment or selection [Subsequently, a total of five haplotypes were identified . Theoretelection ,23. Conselection .p > 0.05) (data not shown).Association analysis between the single SNP markers and body size traits were performed . At the p = 0.005), wither height (p = 0.011), hip height (p = 0.022), hip width (p = 0.004) and heart girth (p = 0.018) than those with mutant genotype CC; significant difference was also found between genotype TC and CC for hip width (p = 0.015). As mentioned above, SNP2 led to the p.700Ser.>Pro. non-synonymous mutation in the intracellular C-terminal tail of bovine Smo. Intriguingly, in common with other GPCRs, phosphorylation of Smo regulates the switch between on/off signaling states [p < 0.05) might be caused by the attenuation of Smo activation via the substitution of Ser. residue with un-phosphorylatable residue Pro. Subsequently, the attenuation might undergo cascade amplification, consequently influencing the transcription of downstream target genes.At the SNP2 locus, animals with wild-type genotype TT had significantly greater body length , wither height (p = 0.046), hip width (p = 0.022) and heart girth (p = 0.024) compared to those with mutant genotype TT/TT. At the SNP4 locus, individuals with genotype CC demonstrated higher mean values for body length (p = 0.010), wither height (p = 0.049) and hip width (p = 0.025) than those with genotype AA. At the SNP6/7 locus, the body length (p = 0.028), hip width (p = 0.007) and heart girth (p = 0.018) of animals with wild-type genotype TT/TT were significantly larger than those of animals with mutant genotype GG/CC. Although these SNPs identified in the 3ʹUTR were non-coding DNA variants, there is growing indication that 3ʹUTR variants actively participate in modifying gene expression patterns. A G > A transition in the 3ʹUTR of the GDF8 gene created microRNA target sites for mir1 and mir206, consequently promoting the muscular hypertrophy phenotype in Texel sheep [At the SNP3/5 locus, animals with wild-type genotype CC/CC showed significantly larger body length  appeared to be beneficial for improving body size traits in cattle breeding programs.p = 0.031) than those with Hap2/Hap2 (CC-CC-TT-AA-TT-GG-CC-CC) (Haplotype combinations may provide greater power than a single marker for genetic disease and trait associations . ConsequG-CC-CC) , which sMarker-assisted selection (MAS) based on genetic variation is more effective and powerful than traditional selection methodologies for genetically improving livestock economic traits, for example, growth traits, milk traits, reproduction traits, meat quality traits . It is wet al. [520 adult animals  were selected from the following farms: Fineness Breeding Center of Qinchuan Cattle , Reserved Farm of Qinchuan Cattle  and Qinchuan Cattle Farm . Seven body measurement traits (body length (BL),wither height (WH), hip height (HH), rump length (RL), hip width (HW), chest depth (CD), heart girth (HG)) were measured for statistical analysis as Gilbert et al.  describeet al. .Bos taurus, Homo sapiens, Rattus norvegicus, Mus musculus, Gallus gallus, Xenopus laevis and Danio rerio) were acquired using BLAST provided by NCBI. A phylogenetic tree for SMO was constructed using MEGA6.06 software. Multiple sequence alignment for orthologous Smo proteins was performed using Clustalx2.0 software.The amino acid sequences of the SMO gene for different species , and 0.3U Golden DNA polymerase . The PCR was performed in a thermal cycler  with the following procedure: initial denaturing at 95 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 30 s at the optimum annealing temperature (w/v) agarose gel (containing 200 ng/mL ethidium bromide) and purified by Axygen kits , and then sequenced in both directions in an ABI PRIZM377 DNA analyzer . Sequence maps were imported into SeqMan of DNASTAR software (version 7.1) and analyzed to search for variations.Primers  used to perature , 72 °C fPrimers  were redGenotype frequencies were calculated by direct counting. Allele frequencies, Hardy-Weinberg equilibrium (HWE), heterozygosity (He), and effective allele numbers (Ne) were analyzed based on genotype frequencies as Nei and Rychoudhury  describeijklm = trait measured on each individual; μ = overall mean; Gi = fixed effect of the ith genotype; Aj = fixed effect of the jth age; Fk = fixed effect of the kth farm; Sl = fixed effect of the lth sex; Sm = fixed effect of the mth sire; and εijklm = random error.The associations between single SNP marker and body measurement traits were analyzed with SPSS software (version .19.0) using the general linear models (GLM) procedure and the following model:For the association analysis between combined genotypes and the body size traits, the statistical model was similar to the model 1 with a slight modification, which was that Gi was the fixed effect associated with the ith combined genotypes. Moreover, to obtain more robust results, all the p values of the statistical results were corrected by Bonferroni correction which was used to account for multiple tests.In summary, we analyzed the molecular characterization of the bovine SMO together with the SMO of several other species, and identified eight SNPs and five haplotypes in the coding region and 3ʹUTR of SMO in 520 Qinchuan cattle. Genotyping and association analyses demonstrated that wild-type alleles of some detected SNPs appeared to be more beneficial for selecting cattle with superior body size traits, and could be used as molecular markers for the improvement of Qinchuan cattle and perhaps other breedtypes that would benefit from marker-assisted selection for increased body size. Nevertheless, these results should be considered as preliminary, and further studies should be conducted to validate observed associations in a broad variety of cattle breeds. Further studies also should explore the molecular mechanisms responsible for these SNPs in the SMO gene associated with the variations in body size traits.


Almost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and consequently what is published is a composite of the two. This means that the relationship between alternate alleles at two or more linked loci is lost. We have developed a simplified method of directly obtaining the haploid sequences of each genome copy from an individual organism.The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Samples were: 1) lymphocytes from a single Angus steer; 2) sperm cells from an Angus bull; 3) lymphocytes from East African Zebu (EAZ) cattle collected and processed in a field laboratory in Eastern Kenya. Haploid sequence from a fosmid library prepared from lymphocytes of an EAZ cow was used for comparison. Cells were serially diluted to a concentration of one cell per microlitre by counting with a haemocytometer at each dilution. One microlitre samples, each potentially containing a single cell, were lysed and divided into six aliquots . Each aliquot was amplified with phi29 polymerase and sequenced. Contigs were obtained by mapping to the bovine UMD3.1 reference genome assembly and scaffolds were assembled by joining adjacent contigs that were within a threshold distance of each other. Scaffolds that appeared to contain artefacts of CNV or repeats were filtered out leaving scaffolds with an N50 length of 27–133 kb and a 88–98 % genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97–201 kb but only ~27–68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data.We have developed a set of laboratory protocols and software tools that will enable any laboratory to obtain haplotype sequences at only modestly greater cost than traditional mixed diploid sequences. The low cost and high throughput of next generation sequencing has led to numerous reference genomes and resequenced genomes being published . HoweverThe phase relationship between common alleles can be recovered using population or pedigree data based on linkage disequilibrium or co-segregation, respectively. However, it is becoming increasingly apparent that a substantial amount of the phenotypic variation within populations is attributable to rare genomic variants  and the There have been multiple reports of sequencing from single cells –9 but feHaploid sequences have been obtained by sequencing DNA diluted to less than a haploid genome per aliquot –3. HowevWe tested the method with three sets of samples: 1) diploid sequence of an Angus steer using 75 bp fragment reads that was validated against Illumina BovineHD SNP (770 K) haplotypes imputed by Beagle 3.3.2  without Analysis pipelines were developed using the 75 bp fragment reads for the Angus steer. The following results are from those data unless otherwise stated.Forty aliquots each predicted to contain a single cell were prepared from the Angus steer 72711. Seven of the 40 test Genomiphi amplifications produced readily visible products on an agarose gel. No DNA was detectable by agarose gel electrophoresis in the remaining reactions . The observed mean of 6 positive loci is less than the expected mean of eight, which would represent 1/3rd of the haploid genome (1/6th of the diploid genome). This may be a consequence of the dropout of genomic regions that is frequently observed when using phi29 to amplify trace amounts of DNA but is consistent with the subcellular samples being derived from the DNA of less than a single cell. Thirty-one subcellular samples that produced between 3 and 14 positive PCR reactions were chosen for library preparation and sequencing to obtain 75 bp fragment reads on an ABI 5500. A bulk genomic DNA sample was also sequenced to obtain 2 × 100 bp paired end reads on an Illumina GAII instrument.A second Genomiphi reaction was performed on the Genomiphi product of all positive samples and this product was used to screen the genome by PCR at 24 loci to estimate the proportion of the genome that was represented in each sample. There was a mean of 6.0 PCR positive loci  for each subcellular sample. There was no correlation between the concentration of the Genomiphi product from a subcell and the number of PCR positive loci  suggesting that the remaining 19 % of the genome was in regions derived from both chromosomes, not sequenced due to low depth of coverage for each library or lost to drop-outs in the amplification or during the sample preparation process.Phi29 amplification from trace amounts of DNA is expected to give rise to very uneven coverage with some regions having very high coverage and others very low or none . The covContigs from diploid cells had an N50 length of 359 bp and a read coverage of 7.7 X. Given the low and skewed genome coverage Fig. , it was Gaps between contigs were assigned to 1000 bp bins and the mean gap length in each bin was obtained. For bins with gap lengths > 1000 bp the mean gap length was close to the midpoint of the bin size , however the mean length of gaps that were < 1000 bp was 144 bp (σ = 175 bp). Therefore it appeared that gaps greater than 1000 bp were approximately randomly distributed and those that were less than 1000 bp were highly skewed in length.If junctions of contigs and gaps were randomly distributed they would be expected to follow a Poisson Distribution . Block lThe sensitivity of N50 scaffold length and total length of all scaffolds to variation in the maximum gap length for joining adjacent contigs into scaffolds was tested using threshold gap lengths from 100 to 2000 bp in increments of 100 bp for chromosome 12 Fig. . The N50Contigs and scaffolds that fall within repeats could compromise the construction of SNP haplotypes since the reads could have been incorrectly mapped and repeat regions can give rise to false SNP calls. To discover whether contigs or scaffolds were affected by repeats, the numbers of contigs and scaffolds in, or overlapping, repeats were calculated for chromosome ten and compared with the number of contigs that would be within repeats if the same scaffolds were randomly placed Table . There wInput files for the Single Individual Haplotyper (SIH) programme were prepared using BuildRefHapInput.V1.1.jar. Scaffolds were checked for excess heterozygous SNP before inclusion in the SIH input. Only loci that were heterozygous in both the bulk genomic DNA sequence data and had both alleles represented in the subcellular libraries were taken forward for SNP haplotype assembly. Before assembly, loci were tested against two quality criteria and loci failing these tests were excluded from the haplotype construction.2 = 2.8 % of the genome will be heterozygous but this is ~9 % of a sample that had gone into the library). For each subcellular library ~8 % of loci that were heterozygous in the bulk DNA were also heterozygous in that library; about the expected number. Apparent heterozygotes can also be a consequence of Copy Number Variants (CNV), repeats and sequencing errors. The coverage of all loci that were heterozygous in the sequence obtained from bulk genomic DNA was obtained and plotted against the number of libraries for which there was a heterozygote call at that position . This would suggest that at least some of the heterozygous SNP calls were artefacts of CNV or expanded repeats in the tested animal or collapsed repeats in the reference genome assembly.If contigs were randomly placed across the genome and each subcellular library contained 1/6th of the cellular genome then it would be expected that about 9 % of the contigs would represent both orthologous chromosomes and might therefore contain true heterozygous SNP (1/6th of each chromosome is amplified therefore (1/6)ion Fig. . There wThere was a more complex relationship between the number of subcell libraries represented at a locus and the depth of coverage in the bulk genomic DNA sample. Coverage was between 24 and 28 fold up to about 13 libraries at a locus. Howevever at loci with more than 13 libraries represented the coverage in the bulk genomic sample rose steeply suggesting that these loci are associated with CNV Fig. .Fig. 6AsUp to six libraries were prepared from each cell, but only two contigs should be found covering any given genomic region in the sequence from a single cell. It is possible to obtain more than two copies if the region has a copy number greater than 1 per chromosome. Higher numbers of copies of a region would lead to more libraries containing contigs across the region and hence the observed association between numbers of libraries containing heterozygotes and coverage. Approximately 1.3 % of the Angus genome is associated with CNV > 20 kb and more is associated with smaller CNV . In totay = 1.5× + c, r2 = 0.84). This suggests that these loci are caused by CNV, repeats or reference genome assembly errors rather than sample contamination.For any pair of adjacent SNP loci with alleles A/a and B/b there are 4 possible haplotypes , only one or two of which should be present in any given diploid animal. The number of haplotypes was counted for all pairs of adjacent loci on chromosome 21  and N50 (27 kb) scaffold lengths Table . TherefoAfter filtering out scaffolds with > 20 % or > 3 heterozygous loci and pairs of SNP loci with > 2 two SNP haplotypes, the remaining SNP loci were formatted for haplotype construction by SIH with input from scaffolds constructed using either ContigMetrics with a 1000 bp maximum gap or Samtools TargetCut. The mean and N50 size of scaffolds are shown in Tables The N50 scaffold length for the Angus steer was 27.4 kb and the N50 haplotype length for SNP based haplotypes obtained by SIH was 97 kb. 89 % of the genome had scaffold coverage of at least 1 X but only 64 % of the genome had haplotypes assigned by SIH. This may at least in part be due to the distribution of gaps between informative SNP. Although 50 % of the genome had SNP less than 2446 bp apart, 22 % had SNP > 10 kb apart and 15 % > 20 kb apart. Haplotypes in these regions might not be captured by the scaffolds, which had mean and N50 lengths of 16 kb and 27 kb respectively. Although <90 % of the genome was covered by informative scaffolds or SNP haplotypes > 99.96 % of heterozygous SNP loci were phased on scaffolds and 88 % on the longer SNP Haplotypes, supporting the hypothesis that <90 % of the genome was covered by informative scaffolds or SNP haplotypes because of an absence of informative SNP in these regions.Libraries were prepared from four sets of samples. Genomiphi amplified single cells from: 1) an Angus steer prepared from freshly collected cells at the University of Missouri; 2) single sperm cells from an Angus bull prepared from semen shipped at ambient temperature from Missouri USA to Liverpool UK; 3) blood of EAZ cattle collected in Western Kenya and stored at ambient temperature (20–38 °C) for 24–48 h prior to amplification. 4) a fosmid library from bulk genomic DNA of a single EAZ cow without Genomiphi amplification.Furthermore two different sequencing technologies were used. The Angus samples were sequenced from 75 bp fragment libraries on Applied Biosystems SOLiD 5500 machines and the EAZ samples were sequenced from 2 × 100 bp paired-end libraries on an Illumina HiSeq.2)/0.24). Sequence from each library covered approximately 22 % (σ 4.5 %) of the haploid genome suggesting that the stated fosmid pool size was accurate.The fosmid library was supplied in pools of 6000 fosmids with 30–45 kb inserts and three pools were combined for each library preparation. Each library was expected to cover 24 % of the haploid genome and 6 % of the sequence data in a given library was expected to come from both orthologous chromosomes ((0.24/2)The contig and scaffold length parameters reflected the different sources of the libraries and the different sequence data types and analysis methods.Effect of sequence data type: The 2 × 100 bp paired-end libraries sequenced on the Illumina HiSeq generated contigs at least four times longer than the 75 bp fragment libraries sequenced on the ABI SOLiD. However, the scaffold lengths from the comparable Angus Steer (SOLiD) and EAZ Cells (HiSeq) only differed by two to three fold. Contig lengths of the two 75 bp fragment libraries from Angus animals had similar distributions . Given that the sperm were shipped from the USA to UK at ambient temperature (within a preservative buffer), it is also an indicator of the robustness of these cells.The fosmid library generated scaffolds with N50 scaffold sizes that approximated to the expected insert size ~40 kb) and these had the smallest variability in scaffold size (standard deviation) relative to mean scaffold length Fig. . The N500 kb and 2 = 0.002), supporting the hypothesis that scaffold lengths represent the lengths of the underlying fragments.The importance of the sample source is further emphasised by the large variation in the amount of available data, which varied between 1.2 X and 12.5 X scaffold coverage. The scaffold coverage of the genome from the EAZ subcellular libraries was particularly sparse at 1.2 X. But there was no correlation between scaffold coverage of the genome and N50 scaffold length  for the fosmid library. However, where the template does not conform to this expectation, TargetCut risks joining contigs from adjacent shorter scaffolds. The mean lengths of scaffolds generated by TargetCut on subcellular fractions were 2–3 times longer than those generated with ContigMetrics when using a maximum permitted gap between contigs of 1000 bp. ContigMetrics with a gap of 2000 bp generated similar SNP haplotype lengths to TargetCut  SNP that would be on the same scaffold or SIH SNP based haplotype. Over 99 % of pairs of missense SNP within a given gene shared at least one scaffold Table  suggestiThe mean length of cattle genes in Ensembl79 was 32 kb (N50 length 106 kb) which is substantially greater than the 10 kb mean haplotype length estimated by linkage disequilibrium . The verAnnotation: SNP in genes were annotated with their consequences for gene structure using the Ensembl API. Counts of each type of structural change are shown in Table The counts of non-synonymous (missense) and synonymous variants were obtained for each gene in the EAZ cattle. The 414 genes with a ratio of non-synonymous/synonymous > 4 were submitted to DAVID to identify pathways that might be under strong positive selection. The only cluster of genes with an enrichment score > 1 contained 29 olfactory receptor genes (Enrichment score 4.29).We have demonstrated a simple method for obtaining direct molecular haploid sequences by isolating and amplifying fractions of individual cells. Molecular haplotypes have been obtained before, by a range of methods , 11, 14.−5. If scaffolds from both chromosomes can reliably be identified and filtered out then an optimal sequencing strategy would minimise the combined cost of sequencing and library construction. By removing 2.2 % of scaffolds from the data set we eliminated 82 % of heterozygous SNP in scaffolds. The majority of the remaining heterozygotes were assumed to be due to CNV and repeats. A cost calculator for different strategies is included in the Supplementary files . The project cost varies only by about 10 % for dilutions between 0.5 and 0.1 genome equivalents per library, but using a very small proportion of the genome, as did Kuleshov et al. [Kaper et al.  used 96–v et al. , increasv et al.  or MALBAv et al.  4 X coveThere are no established quality criteria benchmarks for a diploid genome sequence and criteria will ultimately depend on the intended application. For understanding long-range interactions within the genome of a given individual haplotypes of 1 Mb or greater could be desirable, for example for resolving HLA (or BoLA) haplotypes. However for understanding the haplotype structure of populations, to increase the power of mapping studies, then much shorter haplotypes that capture most SNP in linkage disequilibrium within genomic regions harboring quantitative trait loci would be sufficient. We used simulation to obtain the percentage of haplotypes that would be completely covered by at least one scaffold given ratios of scaffold length to population haplotype length of between 1 and 7 and scaffold coverage of 2–20 X, assuming haplotype lengths are exponentially distributed and that the mean haplotype length in the cattle that we sequenced was 10 kb  Table 88. With tSIH uses the SNP data from all scaffolds to form a single pair of SNP haplotypes from all overlapping scaffolds. The SNP haplotypes are expected to be larger than the scaffolds and this was found, although mean SNP haplotype lengths were only marginally longer than mean scaffold lengths for the EAZ cells, presumably because these cells were drawn from a population not a single animal and therefore overlapping scaffolds would not necessarily be consistent. Although N50 lengths for SNP haplotypes were large (97–190 kb) for the Angus steer there was a substantial loss of data since genome coverage fell from ~ 91 % for the informative scaffolds to ~66 % for the SNP haplotypes. The ability of any method to reconstruct whole genome haplotypes will ultimately be determined by the length of scaffolds used and the lengths of homozygous regions across the genome. Homozygous regions might be quite short in highly outbred populations, such as many human groups, but correspondingly large in intensively selected livestock such as the Angus cattle used here. The mean and N50 gap between informative SNP used for haplotype reconstruction was 1820 bp and 9791 bp. The maximum gap between informative SNP was 1.08 Mb on BTA7.The N50 length of scaffolds in the Angus steer was 46 kb, which is substantially longer than the 14 kb N50 scaffolds obtained by Kaper et al.  using diMolecular haplotypes have also previously been obtained by fosmid cloning and TargetCut and SIH for haplotype reconstruction, which produced N50 haplotypes of 1 Mb and 81 % of genes completely within haplotypes. This isWe have demonstrated a simple sample preparation method for generating DNA derived from single chromosomes. This together with the suite of data processing tools we have written will enable staff in any laboratory to obtain direct molecular haplotypes. The N50 size of scaffolds of paired end Illumina reads assembled with targetcut on the EAZ cells were 133 kb and covered 89 % of the genome. Using SIH to infer SNP haplotypes extended the N50 size in the EAZ cells to 201 kb. These diploid sequences of the EAZ cells were obtained using a genome read coverage of 19.5 X, similar to a standard high quality resequencing project. The preparation of 21 libraries rather than a single library was the only significant source of increased costs, and this cost penalty is likely to decline with the maturation of laboratory automation and library preparation kits.This strategy therefore puts direct haplotype sequencing of diploid genomes within the reach of any laboratory. The method may be particularly useful for haplotype sequencing of large numbers of samples around loci of interest, since amplified aliquots of single cells can be screened by PCR to identify those containing the locus. This provides a route to sequencing megabase regions without complex cloning or primer walking or the discovery biases inherent in sequence capture.The project followed UK, US and Kenyan Institutional and Government Guidelines for Experiments involving animals. The only samples used were blood and semen samples. Blood collection from cattle is not a regulated procedure in the UK and a Home Office licence is not required for its collection. Blood samples collected in Kenya and the US were aliquots of larger samples that were collected for other purposes and also did not require ethical approval. The semen sample was from a US semen bank.A diagram of the sample processing workflow is shown in Fig. Samples were collected from two sets of animals. First, from a single Angus steer (number 72711) from the University of Missouri research farm and a semen sample from a US registered Angus bull (Lab Id 1736). Second, from 18 East African Zebu (EAZ) cattle sampled in Busia district near Lake Victoria in Western Kenya in January and February of 2012. Finally, a fosmid library was prepared from EAZ cow 121-147 collected from Busia at the same time.Ten milliliters blood samples were collected into vacutainers with acid citrate dextrose anticoagulant and processed within two hours in the case of the Angus steer. In the case of the EAZ, blood samples were stored at ambient temperature (22–37 °C) for up to 48 h. Lymphocytes were separated from whole blood using Lymphoprep™  following the manufacturer’s instructions. As well as eliminating red blood cells Lymphoprep also eliminates neutrophils, which are the leukocytes with the shortest half-life (~4 h). Residual red blood cells were removed by ammonium chloride lysis  and the remaining lymphocytes were resuspended in phosphate buffered saline (PBS).A semen sample from Angus bull 1736 was used in the dilution procedure below without prior preparation.TM (Invitrogen) sample buffer was added and mixed by pipetting to neutralise the sample. Two μl of the neutralised lysate was used in each 20 μl GenomiphiTM reaction following the manufacturer’s protocol. The resulting product was called a subcell sample. The remaining 10 μl of lysate was stored at −20 °C. Genomiphi products were visualized by electrophoresis in 1.5 % agarose gels. For samples that generated detectable Genomiphi amplification products, the remaining 10 μl of lysate was used in 5 further Genomiphi reactions to make a total of six Genomiphi subcell products per cell.Cells were counted on a Fuchs-Rosenthal haemocytometer using the entire area within the triple grid lines; the volume over this area is 3.2 μl. Cells were serially diluted in PBS and at each dilution an aliquot was taken and stained with 1 % Lugol’s iodine for visualisation. This procedure was repeated until an average of ~3 cells were observed in the whole area of the haemocytometer over five replicate counts of the same dilution. Whole genome amplification of single chromosomes was adapted from the detailed protocol of Spits et al.  for wholTM reaction was performed using 20 ng of the first GenomiphiTM product to generate material for validation purposes. Each of the six subcell products from a single cell was expected to contain sequences from approximately 1/3rd of the haploid genomes (1/6th of the diploid genome). Each subcell product was tested with 24 PCR primers spaced across the bovine genome. The perfect subcell product is expected to yield ~8 positive reactions (2 × 1/6th × 24). Subcell products that had less than 15 positive PCR reactions were judged to be from a single cell and were selected for short read library preparation. The semen sample was diluted in the same way and aliquots predicted to contain whole single sperm were amplified and processed as described above. Whole genomic DNA was prepared from bulk cells from the Angus animals using the DNeasy Blood & Tissue Kit . A fosmid library with 35–45 kb inserts was prepared by LGC Genomics  from bulk DNA prepared from cells from a single EAZ cow that were stored and shipped frozen in 70 % ethanol.A second GenomiphiA barcoded library was prepared from each sperm or subcell aliquot of the Angus animals using the ABI SOLiD4 fragment library kit following the manufacturer’s instructions . Fragment reads of 75 bp were obtained using ABI 5500 instruments and the exact call chemistry (ECC) module. ECC makes it possible to convert the color space data to nucleotide base sequences prior to mapping.A separate library was prepared from DNA extracted from 50 cells from the Angus steer, which was amplified with Genomiphi and sequenced on the ABI SOLiD 5500 . A library was also prepared from whole genomic DNA of the Angus steer using the Illumina PE-102–1001 paired end library kit and 2×100 bp reads were obtained with an Illumina GAIIx instrument .Libraries from the EAZ samples were prepared using the Illumina TrueSeq protocol and 2 × 100 bp reads were obtained on an Illumina HiSeq2000.The fosmid library was aliquoted into 192 pools each predicted to contain ~6000 unique clones. Three pools of 6000 clones were merged to make each of 47 libraries using the Illumina TruSeq Paired End library preparation kit and 2 × 100 bp paired end sequence reads were obtained on an Illumina HiSeq2000.A diagram of the data processing workflow is shown in Fig. Programmes were written in Java to build scaffolds, filter out poor quality scaffolds and sites, format the data for SIH and validate the data by internal consistency and consistency with Illumina SNP chip and Beagle 3.3.2 derived haplotypes. To avoid excess memory usage and to take advantage of the available cluster all data processing was performed in parallel on a chromosome by chromosome basis leading to > 3000 calls to each programme to analyse data from each of the ~100 libraries across each of the 30 bovine chromosomes. Perl scripts were written to manage the calls to established and new software and undertake minor housekeeping tasks. A complete workflow together with scripts and source code for Java programmes are available on GitHub , 23.ABI SOLiD reads were converted from color space to FASTQ base-space files using the ABI tool ConvertFromXSQ.sh with the -f switch, which removes reads which fail the ABI quality criteria. Illumina FASTQ files were directly processed. Reads were mapped to the UMD3.1 bovine reference genome  using BWA Picard Tools module “ContigMetrics” was written to take BAM files from each library and build contigs of overlapping reads and then merge adjacent contigs that are within a user defined distance into scaffolds and output gff3 files with the co-ordinates of read contigs and scaffolds for each chromosome and library and also to report basic statistics for read and contig coverage.It has been observed that phi29 amplification from trace amounts of template gives rise to a highly skewed distribution of read coverage with some genomic regions having very high coverage and others having little or none . TherefoTargetCut also generates scaffolds from BAM files; it is available within Samtools and was developed for building scaffolds from sequences from fosmid libraries: “inserts were identified by computing read depth genome-wide in 1 kb windows for each clone pool and selecting runs of 25 to 45 kb for which at least two-thirds of the constituent windows had read depth above the predicted background level” . We alsohttp://www.broadinstitute.org/gatk/). Duplicate reads were removed using Picard Tools and reads were twice realigned around indels, using GATK before calling SNP with the UnifiedGenotyper. GATK was run in DISCOVERY mode with EMIT_ALL_CONFIDENT_SITES set to ensure that reference bases were called where data were available to support them. Positions with read coverage greater than 50 were ignored. All other parameters were left at default, which meant that SNP required a minimum Phred scaled score of 30 to be called as high confidence, but low confidence SNP were also recorded.SNP were called using the Genome Analysis Tool Kit V2.4.7  and PicaA Java Programme BuildRefHapInput.V1.1.jar was written to process gff3 files of scaffold co-ordinates and Variant Call Format (VCF) files of SNP and output the frag and allvars files for processing by the SIH programme .BuildRefHapInput.V1.1.jar filtered out all positions in the VCF file that were not informative amongst the processed libraries since these disrupted the consistency calculations. It also screened all scaffolds for an excess of heterozygous SNP that could be indicative of CNV or multiple chromosomes being represented in the sample. Scaffolds that contained more than 3 heterozygous loci or that had a heterozygote frequency greater than 0.2 were removed. These parameters are user defined and were chosen after inspection of a small number of clearly heterozygous scaffolds. BuildRefHapInput also compared all overlapping scaffolds from different libraries and calculated two measures of consistency between independent libraries as previously described , 13. SIHScaffold haplotypes and SIH generated haplotypes from the Angus steer were compared against haplotypes inferred by Beagle 3.3.2  from theA Java programme SimulateScaffoldCoverageHaplotypes.jar was written to simulate the exponential distribution of haplotypes across a chromosome in order to estimate the percentage of haplotypes in cattle that were completely covered by sequence haplotypes. Population haplotype lengths were assumed to be exponentially distributed with a mean of 10 kb . ScaffolData analysis pipeline; describes programmes to process the sequence data and their usage.CostCalculator.xls: Can be used to explore the trade-off between numbers of libraries and proportion of genome in each library and depth of coverage.Fastq files for all reads are deposited at the European Nucleotide Archive under SUBMISSION_ID ERA303977; STUDY_ID ERP005648.http://www.ncbi.nlm.nih.gov/projects/SNP/snp_viewTable.cgi?handle=CGR_LIV).SNP for each project will available from dbSNP with submitter handle CGR_LIV (https://github.com/LiverpoolHarry/HapSeqScripts and programmes are available from Github together with the Java source code http://www.genomics.liv.ac.uk/tryps/HaploSeq.html together with links to all other data.Scaffolds and SNP haplotypes are available from our website


Sesamum indicum L.) is an important oilseed crop and has an indeterminate growth habit. Here we resequenced the genomes of the parents and 120 progeny of an F2 population derived from crossing Yuzhi 11  and Yuzhi DS899 , and constructed an ultra-dense SNP map for sesame comprised of 3,041 bins including 30,193 SNPs in 13 linkage groups (LGs) with an average marker density of 0.10 cM. Results indicated that the same recessive gene controls the determinacy trait in dt1 and a second determinate line, dt2 (08TP092). The QDt1 locus for the determinacy trait was located in the 18.0 cM–19.2 cM interval of LG8. The target SNP, SiDt27-1, and the determinacy gene, DS899s00170.023 (named here as SiDt), were identified in Scaffold 00170 of the Yuzhi 11 reference genome, based on genetic mapping and genomic association analysis. Unlike the G397A SNP change in the dt1 genotype, the SiDt allele in dt2 line was lost from the genome. This example of map-based gene cloning in sesame provides proof-of-concept of the utility of ultra-dense SNP maps for accurate genome research in sesame.Sesame ( Sesamum indicum L., 2n = 2x = 26) belongs to the Pedaliaceae genus and is an ancient oil seed cropSesame (dt1 line) was created from line 91–0 of the sesame cultivar Yuzhi 11 (selected as the reference genome for the Sesame Genome Project) using ethylmethane sulfonate (EMS) mutagenesis in 2009 (China patent application No.: ZL2015108760163). The dt1 mutant line has a shorter flowering period and develops 8–20 capsule nodes. The seed ripens concurrently, making it more suitable for mechanical harvesting. An additional determinacy genotype, 08TP092 (dt2 line), has been induced by gamma ray  mutagenesisdt2 type limits the number of capsule nodes and this number is not affected by environmental factors3In angiosperms, floral induction initiates the transition of vegetative shoot meristems to inflorescence meristems. In indeterminate annual plants, floral formation continues throughout the whole reproductive life, giving rise to indeterminate inflorescences. Determinate plants, on the other hand, can form a terminal flower cluster, as the shoot apical meristem is converted into a terminal floral meristem356A genetic linkage map is a prerequisite for better understanding the inheritance of traits on a genome-wide level10111216171518192015172 population of a cross between Yuzhi DS899 (dt1) and JS012 (Dt) and a whole genome re-sequencing approach. SiDt and alleles controlling the determinacy of the inflorescence meristem in the three genotypes were identified and validated based on the ultra-dense SNP map and genome re-sequencing data. The findings are valuable for genome assembly, genome-wide association analyses of key agronomic traits, target gene mapping and further studies of Dt genes in sesame.Of note, since 2010, NGS technology is increasingly being applied to sesame genome assembly1920212213dt1, P1) and JS012 , and 120 F2 progeny were sequenced using an Illumina sequencing approach   in the two parents, representing 94.45% of all detected variants, were high-quality.To construct an ultra-dense SNP genetic linkage map for sesame, two parents, cv. Yuzhi DS899 ( 315474)  (see Sup 315474) . In tota 315474) , of whicr sample . After a2 progeny. A matrix containing 40,034 skeleton bins  was created for the 120 F2 progeny. On the basis of Mendelian separation ratios , 3,101 bins containing 30,494 SNP markers were chosen from the above skeleton bins for linkage mapping  grows indeterminately throughout the whole reproductive period, more than 20 capsule nodes are formed, and plant height reaches 165.0 cm  develops 8–20 capsule nodes, and the number of capsule nodes varies with latitude. 08TP092 (dt2 type) forms only 2–3 capsule nodes on the main stem and each lateral branch, and capsule node number is independent of latitude.To date, three inflorescence meristem termination phenotypes have been documented in sesame  see Sup. The sho165.0 cm . In dt1  cluster . Yuzhi Ddt1 and dt2 genotypes in sesame, we constructed F1 hybrids and F2 populations of five cross combinations, namely ‘Yuzhi DS899× Yuzhi 11’, ‘08TP092× Yuzhi 11’, ‘Yuzhi DS899× 08TP092’, ‘Yuzhi DS899× Ningbohei’ and a mapping population of ‘Yuzhi DS899 × JS012’  is dominant. In dt1 backcrosses of ‘Yuzhi DS899× Yuzhi 11’ and ‘Yuzhi DS899× Ningbohei’, the segregation ratios of the indeterminate and determinate (Dt:dt1) phenotypes fitted the expected 1:1 ratio (2 population fitted the expected ratio of 3 (Dt):1 (dt). Chi-square tests (P > 0.05) validated that the segregation of the determinate growth trait in dt1 and dt2 lines fitted the Mendelian inheritance mode, as the x2 values for trait segregation in the F2 population ranged from 0.22 to 0.53. We also investigated the genetic relationship between dt1 and dt2 phenotypes. All progeny of direct and reciprocal crosses, back-crosses and the F2 population developed from the ‘Yuzhi DS899 × 08TP092’ cross were found to be determinate (dt2 (08TP092) is allelic with dt1 (Yuzhi DS899), and that the two phenotypes are controlled by a regressive gene locus. We annotated the determinacy alleles in Yuzhi DS899 and 08TP092 as Sidt1 and Sidt1-2, respectively.In order to systematically investigate the inheritance of × JS012’ . The phe:1 ratio . In addierminate . Results2 progeny from the ‘Yuzhi DS899 × JS012’ cross, we performed gene locus mapping for the determinate growth trait using winQTLcart and QTLNetwork, respectively  like protein. SiDt alleles are comprised of 4 exons and 3 introns and code for 531 amino acid residues (nd exon was detected between the Sidt1 and SiDt genes and which caused a change in amino acid from Ser to Asn (S79N) (GenBank accession No. KU240042). Protein homology modelling showed that there is no difference in 3D structure between the SiDt and Sidt1 proteins  and ormation , and namidt1 See . Non-redresidues . A SNP, eins See . The PROdt2 line had the same recessive gene locus for the determinacy trait as the dt1 mutant line. We attempted to amplify the gene sequence of the SiDt allele (named Sidt1-2 in this study) using the primer pair SiDt F and SiDt R  showed that a ~24.9 kb fragment containing the SiDt (DS899s00170.023), DS899s00170.022, and DS899s00170.021 loci and part of the DS899s00170.020 sequence .We screened for homologous genes of cted See . Four ofSiDt, we performed gene alignment and DNA polymorphism analysis of SiDt sequences in Yuzhi 11, Yuzhi DS899 and 30 indeterminate varieties from a worldwide germplasm bank (See SiDt sequence of 25 the 30 indeterminate varieties (M1-M30) had 100% identity to that of Yuzhi 11, while 18 SNP sites were present in the other 5 varieties, whose SiDt sequence identity ranged from 99.06% to 99.28%. All 18 SNP sites in the SiDt gene were different from the G397A mutation, and 16 of the 18 sites were located in introns of the SiDt gene. We performed DNA polymorphism analysis of the SiDt gene using genome re-sequencing data from 715 germplasm resources in public sesame datasets (PRJEB8078 in NCBI), retaining SNP sites detected in ≥7 (1%) samples Sdt1 in Indian mustard (Brassica juncea)SiDt, we aligned the predicted sequence of the SiDt and Sidt1 proteins with six homologues from different plant species  with CEN in A. majus   remained in the vegetative stage until 57 d  . During the root . In shootil 57 d . In the 1 mutant . Under l57 d See . Express7 d–57 d . These rSiDt and Sidt1 alleles control the transition to inflorescence meristem development in the Dt and dt1 sesame genotypes, respectively, and that the other allele of SiDt in the dt2 genotype is missing from the genome. This study is the first example of the application of an ultra-dense SNP genetic map in sesame for QTL mapping and map-based cloning of functional genes involved in key agronomic traits.In this study, we constructed an ultra-dense SNP genetic linkage map for sesame using whole genome re-sequencing. The 13 LGs matched the 13 chromosomes in the sesame karyotype and can be used for fine genome assemblyS. indicum L.17272814151616172 population to construct an ultra-dense SNP genetic map for sesame. The average marker density in our map is approximately one bin per 0.98 cM or one SNP per 0.1 cM .During construction of the SNP map, we found that 78 gaps larger than 5 cM were present in the SNP map , some ofSidt27-1 SNP marker supplied the necessary information for SiDt gene cloning.To this point, it has not been possible to perform map-based cloning of sesame genes due to the low marker density of unsaturated genetic maps and limited population size. To determine target SNP sites and clone candidate genes at target intervals, strategies such as increasing the quantity of markers and genome coverage, enlarging population size and using a Mutmap-like strategy are necessarydt1 and dt2) of sesame. Similar to other determinate mutants or crops26dt1 type) and 08TP092 (dt2 type) lines were observed to form a terminal flower cluster at the apex. Moreover, the determinate growth habit in the dt2 type is independent of latitude change (See dt1 and dt2 genotypes are controlled by a recessive gene locus (Arabidopsis (tfl1-1) was investigated by Shannon and Meeks-Wagner (1991)TFL1 (TERMINAL FLOWER 1) and its orthologs cause early flowering and control inflorescence architecture in Arabidopsis33FLOWERING LOCUS T) genes with high identity to TFL1 was found to control downstream photoperiod-dependent and photoperiod-independent pathways to promote the transition to flowering272829303133343536373839SiDt, controls the determinate trait in sesame. NR annotation indicated that SiDt gene is a TFL1 homolog. Previous reports have indicated that 15 amino acid residues in TFL homologs, such as His88Tyr in TFL1, are important residues for maintaining switch functionalitySiDt, but protein functionality is likely affected with this deleterious change.Determination of inflorescence meristems is an important developmental transition in crop plants. Here, we systematically investigated the genetic basis of two determinate lines  varieties had SNP variants in the SiDt gene. Screening of the sesame genome uncovered 7 genes with homology to SiDt, protein identity ranging from 29–78%. Three proteins, Sis00157-2, Sis00154-1 and Sis00141-1, whose identity ranged from 52–78%, grouped together with SiDt protein, , genes DS899s00170.022, DS899s00170.021 and part of the sequence of DS899s00170.020 were not detected due to the deletion of a 24.9 kb segment. The DS899s00170.022 gene (predicted to be a poly ADP-ribose polymerase 3 protein) is 4,298 bp in length and the DS899s00170.021 gene  is 1,177 bp, while DS899s00170.020, whose function is unknown, is 2,888 bp in length.We used a sequence amplification and genome re-sequencing strategy to identify the r genome . In addiSiDt/Sidt1 has highest identity with the centroradialis protein (CEN) of Antirrhinum , is expressed at a late inflorescence phase, and controls the morphological switch between shoot growth and inflorescence development43Arabidopsis, the expression of TFL1 is weak during early development to delay the commitment to flowering, while expression at later stages increases to maintain the inflorescence meristemGtTFL1 in Gentiana is mostly expressed in stems with SAMs  at the vegetative stage. As the plant develops into the reproductive stage, its expression level significantly decreases. Overexpression of GtTFL1 prevents and delays floral initiation and developmentDt1 soybean, the GmTFL1b gene is expressed mainly in the root and shoot apical meristems at the vegetative stage. During early vegetative growth, the GmTLFL1b transcript accumulates in the SAM and is abruptly lost in the determinate lineSiDt transcript is similarly detected mainly in the root and stem apical meristems at the early vegetative and reproductive stages , weak expression delays commitment to flowering, whereas increased expression at later stages (after 27 d) corresponds with the transition to inflorescence meristem development. In addition, we found that, under short days, the transcript peak of Sidt1 in dt1 occurred earlier than in SiDt (dt1. We have shown that the SiDt gene cloned here using genetic mapping is the target gene for the inflorescence meristem transition in sesame and plays an important role in the inflorescence meristem termination pathway25TFL and TFL-like homologues are believed to be negative regulators of flowering time41irrhinum . In Antie stages 35. Howev in SiDt . This exDt type), Yuzhi DS899  and 08TP092  were chosen to investigate the inheritance of the determinate habit trait in sesame (dt2) was a kind gift from Prof. Amram Ashri of Hebrew University of Jerusalem, Israel in 1984. All the above varieties are available from HSRC, HAAS , Pingyu (113°62′ E and 32°97′ N) and Yuanyang (113°97′ E and 35°05′ N) experiment stations in 2013–2014. The parents, direct crosses, reciprocal crosses, back-crosses and Fions see  to invesdt1, P1) and JS012  were chosen to construct a mapping F2 population , 29 sesame varieties with an indeterminate growth trait were randomly chosen from the core collection of worldwide sesame germplasm at the HRSC for gene diversity analysis See . Young lGenomic DNA was extracted from young leaf tissues of each sample using DNeasy Plant Mini Kits  and fragmented by sonication. Standard paired-end (PE) libraries were constructed according to Illumina guidelines. DNA ends were polished and an ‘A’ base was then added to each 3′ end. After ligating DNA adaptors with a single ‘T’ base overhang at the 3′ end to the above products, they were purified on 2% agarose gel. Target products with an insert size of 300 bp were excised and purified using Gel Extraction Kits . Libraries were subsequently sequenced on an Illumina HiSeq2500 platform  according to standard sequencing protocols.2 progeny were merged using VCFTools2 progeny were filtered according to the following high quality (high-confidence) criterion: SNPs presenting 20–100 times in the 120 F2 progeny and two parents were retained.Raw reads obtained from the Illumina HiSeq2500 platform were filtered using Trimmomatic 0.321- and P2- homozygous SNPs were then used to create a population matrix. The genotypes of the P1- or P2- unique SNP loci in the 120 F2 progeny were coded using home-made Perl scripts according to the following criteria: (1) for homozygous SNPs from P1 called in F2 progeny, when read depth was ≥5, the SNP was coded as ‘AA’; otherwise, the SNP was coded as ‘D’; (2) for homozygous SNPs from P2 called in F2 progeny, when the read depth was ≥5, the SNP was coded as ‘BB’; otherwise, the SNP was coded as ‘C’; (3) If SNPs were heterozygous in the F2 progeny, they were coded as ‘H’; and (4) if the SNP was not called in the F2 progeny, it was regarded as missing and coded as ‘-’. All coded loci were classified in terms of their positions on contigs, and the ‘C’ and ‘D’ genotypes of each F2 progeny were corrected using a hidden Markov model approach from the R package MPRTo collect unique and homozygous SNPs, home-made Perl scripts were used to filter the above high-quality SNPs to retain SNPs that were homozygous in either parent,and to delete SNPs present in both parents. Unique PLocating the gene locus for the determinate habit trait was performed using QTL Cartographer 2.5http://www.premierbiosoft.com/prierdesign/index.html) according to the method of Wei et al.The physical distance of the QDt1 interval locus was determined in the appropriate scaffold of the Yuzhi 11 reference genome (PRJNA315784) based on the bin marker sequences of HS0831 and HS0817. The genomes of Yuzhi DS899 and JS012 were compared to screen unique SNP/InDel sites in the physical distance of the QDt1 interval using home-made Perl scripts. 16 SNP/InDels located in the QDt1 interval and adjacent regions were transformed into PCR-based SNP/InDel markers See  using thSiDt alleles in Yuzhi 11, and the dt1 and dt2 genotypes, the SiDt F and SiDt R primer pair was designed using Primer Premier 5.0  according to the manufacturer’s instruction. Standard PCR reactions were carried out on a PTC-225 machine  under the following conditions: 94 °C for 1 min; 30 cycles of 30 s at 94 °C, 1 min at 55 °C and 1 min at 72 °C, with a final 5 min extension at 72 °C. All PCR products were individually gel purified for Sanger sequencing with three replications.In order to clone the entire gDNA and cDNA sequences of  5.0 see . DNA ampSiDt allele sequence in dt2, the 08TP092 genome was re-sequenced on the Ion ProtonTM Sequencer platform  according to the manufactures’ instructions. Raw reads were filtered and aligned with the reference genome of Yuzhi 11 using BWA 0.7.12SiDt in Yuzhi 11. PCR reactions were performed using gDNA from Yuzhi 11 and 08TP092 as described above. The cDNA sequence of SiDt was sequenced and deposited in the NCBI (GenBank accession No. KU240042).To identify the SiDt homologues in sesame, we scanned the genome of Yuzhi 11 using BLASTP, with a cutoff E value of ≤1E-20 and coverage of ≥50%. To explore DNA polymorphisms and to verify the target SNP allele, SiDt sequences from the 30 varieties discussed above (M1-M30) (see http://www.lynnon.com/pc/framepc.html). Genome re-sequencing data from the 715 varieties available in public sesame datasets (PRJEB8078 in NCBI) were used for SiDt gene diversity analysis using MEGA 5.2 according to the Muscle methodSiDt gene are listed in SiDt homologs and DNA polymorphism analysis were constructed using the maximum likelihood methodSiDt and Sidt1 proteins were performed using SWISS-MODELhttp://provean.jcvi.org/index.php). The predefined threshold was set at −2.5.To identify any M30) see  were ampSiDt and Sidt1 were aligned with the 6 homologs of TFL1 , CEN (Antirrhinum majus), LeSP (Lycopersicon Esculentum), Dt1 (Glycine max), PsTFL1a (Pisum sativum) and VuTFL1 (Vigna unguiculata) using DNAMAN (http://www.lynnon.com/pc/framepc.html). A phylogenetic tree was constructed based on 12 TFL1 orthologs in Arabidopsis and other 9 crops (see Predicted amino acid sequences of rops see  legend uDt), Yuzhi DS899 (dt1) and 08TP092 (dt2) were sterilized with 3% sodium hypochlorite and grown in plastic pots (20 cm × 20 cm) with sterilized vermiculite and peat (V:V = 3:1) in chambers under a day/night temperature of 28 °C/24 °C with 70 ± 1% relative humidity. Photoperiodic conditions of 15 h light/9 h dark (LD) and 12 h light/12 h dark (SD) were applied using cool intensity light of 100 μmol/m2/s. Twenty seeds were sown per pot and there were five pots per genotype per treatment. Each treatment was replicated three times. All treatments were watered regularly with 10-fold-diluted liquid MS medium. Three replicate samples of root, leaf, stem and shoot apical tissues of each genotype were collected at 7, 17, 27, 37, 47 and 57 d after planting. All samples were frozen in liquid nitrogen and stored at −70 °C before extraction of RNA.Uniform healthy seeds of Yuzhi 11  according to the procedures of Wei et al.SiDt and Sidt1 genes was normalized against the β-tubulin gene and analyzed using ΔΔCt methodTotal RNA was extracted from tissues using RNAiso Plus Reagent . Genomic DNA was removed from the RNA products by DNase I treatment. First-strand cDNA synthesis was performed using a RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. The primer pair 2 population; and PRJNA316751 for the genome of 08TP092. The cDNA sequence of Sidt1 gene has been submitted to NCBI under accession number KU240042.All genomic data generated in this study has been deposited at the National Center for Biotechnology Information (NCBI) under the following accession numbers: (1) PRJNA315784 for the genome of Yuzhi 11 (version 2); PRJNA315474 for the genomes of Yuzhi DS899, JS012 and the 120 FHow to cite this article: Zhang, H. et al. Ultra-dense SNP genetic map construction and identification of SiDt gene controlling the determinate growth habit in Sesamum indicum L. Sci. Rep.6, 31556; doi: 10.1038/srep31556 (2016).


B. napus inbred lines using double-digested restriction-site associated DNA (ddRAD) and genotyped 19,327 RAD tags. A total of 15,921 RAD tags were assigned to a published genetic linkage map and the majority (71.1%) of these tags was uniquely mapped to the draft reference genome “Darmor-bzh.” The distance of LD decay was 1,214 kb across the genome at the background level (r2 = 0.26), with the distances of LD decay being 405 kb and 2,111 kb in the A and C subgenomes, respectively. A total of 361 haplotype blocks with length > 100 kb were identified in the entire genome. The association panel could be classified into two groups, P1 and P2, which are essentially consistent with the geographical origins of varieties. A large number of group-specific haplotypes were identified, reflecting that varieties in the P1 and P2 groups experienced distinct selection in breeding programs to adapt their different growth habitats. GWAS repeatedly detected two loci significantly associated with oil content of seeds based on the developed SNPs, suggesting that the high-density SNPs were useful for understanding the genetic determinants of complex traits in GWAS.High-density genetic markers are the prerequisite for understanding linkage disequilibrium (LD) and genome-wide association studies (GWASs) of complex traits in crops. To evaluate the LD pattern in oilseed rape, we sequenced a previous association panel containing 189  Genome wide association studies (GWASs) utilize historical recombination events and linkage disequilibrium (LD) in diverse germplasm to detect sequence variants associated with phenotypic traits. The approach has proven to be a powerful tool for exploring the genetic architecture of agronomically important traits in crop species –7. A preB. napus have primarily been interrogated using several hundred conventional molecular markers including simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. The mean r2 value was estimated to be 0.023 for linked loci and 0.019 for unlinked loci in a collection of 509 B. napus inbred lines with 89 SSR markers [B. napus. Thus, the extent and LD decay in B. napus were re-evaluated recently using high-density SNP arrays. The distance of LD decay was about 0.6 cM (r2 < 0.2) for the A and C subgenomes in 313 inbred lines as revealed by Infinium array containing 4,329 SNPs [Brassica 60 K SNP array, the distances of LD decay in the A and C subgenomes were estimated to be 210 and 810 kb (r2 < 0.2) in a collection of 472 rapeseed accessions, respectively [r2 < 0.1) in 203 Chinese semi-winter rapeseed breeding lines, respectively [Brassica 60 K Infinium SNP array is a good tool for genotyping, it remains expensive for a regular laboratory when the large population needs to be genotyped.The extent and pattern of LD in  markers . A simil markers . The dis markers , or to b markers . However329 SNPs . With thectively , while tectively . AlthougWith the advent of next generation sequencing (NGS) technologies and significant reduction of sequencing cost, SNPs are being discovered and genotyped in high-throughput manners such as whole genome resequencing (WGR) and double-digested restriction-site associated DNA (ddRAD) using the Illumina NGS platform. The NGS technology coupled with reduced representation libraries (RRLs) is an economic way to efficiently discover SNPs in large scale . In diplB. napus inbred lines using ddRAD. A total of 19,327 polymorphic tags containing 31,833 SNPs were discovered and genotyped using the UNEAK pipeline with modifications. Of which, 15,921 tags were assigned to a published genetic linkage map through LD-based mapping. The genetic linkage showed a good collinearity to the reference genome of the B. napus cultivar ‘Darmor-bzh’ [B. napus, and test their usefulness for GWAS with seed oil content.In this study, we sequenced an association panel with 189 mor-bzh’ . Using tB. napus inbred lines was used for this study . The pooled libraries were amplified and PCR products were separated on a 2% agarose gel. Fragments in a size range of 270–550 bp were purified with a Qiagen gel purification kit and submitted for sequencing on a HiSeq2000 platform with paired-end (PE) reads of 90 bp.Genomic DNA was extracted from young leaves of a single plant for each inbred line. ddRAD libraries for all inbred lines were constructed as previously described . BrieflyThe 90 bp PE reads that passed quality filtering were parsed into different individuals, with the outermost barcodes and remnant restriction sites at both ends exactly matching the adaptors used. The clean sequence data was submitted to National Center for Biotechnology Information (NCBI) with the project SRP066134. After trimming the 5 bp barcode and the 5 bp on the 3’ end of sequences, the remaining 80 nucleotides of each PE read were kept for further analysis. A modified UNEAK pipeline included an additional step to discriminate allelic tags . BrieflyHR) value  value , which ir2) between an anchor SNP and an unassigned SNP is larger than the background LD, these two SNPs are thought to be linked. The background LD level was set to the 99th percentile of r2 distribution for unlinked anchor markers [Multiple SNPs from a tag should be in a complete LD due to the small insert size (< 420 bp). Thus one SNP was selected from each allelic tag pair to represent the locus to reduce computational load. All discovered SNPs were directly aligned to the BnaNZDH genetic linkage map constructed using SNPs developed with the same ddRAD protocol . The SNP markers .r2) threshold of 0.4 for SNP pairs in a sliding window of 50 SNPs using PLINK [B. napus lines. PIC values were calculated for each SNP using PowerMarker v3.25 [t test in R [To reduce the influence of strong LD on the assessment of population stratification, a set of SNPs were selected by setting an LD (ng PLINK . A Bayesng PLINK  and prinng PLINK  were doner v3.25 . The difest in R .r2 [r2 fell to the background LD (r2 = 0.26).LD was estimated as the correlation coefficient r2  between r2 . LD decaThe estimation of haplotype blocks was performed using PLINK  with a wBroad-sense heritability of seed oil content was calculated as SAS v9.2 . Within SAS v9.2 , represe-5  based on the adjusted Bonferroni correction method [qOC” followed by alphabetical letter of the chromosome, on which the QTL located. For the repeatedly detected QTLs, based on all significant SNPs sorted by physical positions in each locus, the haplotype, which at least had two inbred lines, and corresponding number of inbred lines and average oil content were investigated. Stepwise regression was performed to examine the effect of multiple markers of all QTLs in each year and estimate total variance explained (R2) [F test for each marker with P-value < 0.05.GWAS was performed with the mixed linear model (MLM) with the control of pairwise kinship among 189 inbred lines using Genome-wide Efficient Mixed Model Association (GEMMA) . The genn method . The quaned (R2) . The forB. napus inbred lines were constructed as previously described [The ddRAD libraries for the 189 escribed  and thenHR, we discriminated allelic tag pairs from paralogous or homologous tags in a network as previously described [Firstly, identical 80-bp PE reads are collapsed into tags . These tescribed . Finallyr2) of unlinked marker pairs of the 4,995 anchor SNPs were calculated, and the 99th percentile of the r2 distribution in the association panel was 0.26, which was set as the background LD (r2) were calculated between the 14,332 unmapped SNP and the 4,995 anchor SNPs, and the anchor marker having the highest LD value was selected to represent the position of the unmapped SNP. Using LD-based genetic mapping, we further assigned 10,926 SNPs to the BnaNZDH linkage map. In total, 15,921 SNPs were assigned to the genetic linkage map, with 6,883 (43.2%) and 9,038 (56.8%) SNPs located in the A and C subgenomes, respectively  between pairs of the 10,343 SNPs [r2) was 0.113. The average LD in the A subgenome was 0.075, which is significantly lower than that in the C subgenome (0.132) (P < 0.001). LD levels showed considerable variations between chromosomes. In the A subgenome, the LD varied from 0.028 (A3) to 0.154 (A6), while it varied from 0.039 (C3) to 0.217 (C8) in the C subgenome (r2 > 0.26), significantly lower than the percentage in the C subgenome   .r2 decreased to the background LD (r2 > 0.26). Significant variations in LD decay distances were observed among chromosomes. In the A subgenome, the distances of LD decay varied among chromosomes ranging from 106 kb  to 1,908 kb (A6), with an average LD decay distance of 405 kb. In the C subgenome, the distance of LD decay varied from 227 kb (C5) to 4,089 kb (C2), with an average LD decay distance of 2,111 kb  related to the biosynthesis of glucosinolates (BnaA02g20840D is homologous to AT5G23010 (MAM1) in Arabidopsis and encodes a methylthioalkylmalate synthase that catalyzes the condensation reactions of the first two rounds of methionine chain elongation in the biosynthesis of methionine-derived glucosinolates [BnaA02g20860D is homologous to AT4G03060 (AOP2) in Arabidopsis and encodes a 2-oxoglutarate-dependent dioxygenase which is involved in glucosinolate biosynthesis [P = 0.001). The two SNPs located between BnaA02g20840D and BnaA02g20860D , whilst in the C subgenome the PIC value of P1 was significantly lower than that of P2  (B. napus genome (Genetic diversity levels were measured as polymorphic informative content (PIC) values in the P1 and P2 groups. In the A subgenome, the PIC value of P1 was significantly higher than that of P2  . We then< 0.001) . Furthers genome . The tots genome , suggests genome .P value raged from 3.3×10-4 to 1.6×10-2) and the overall level skewed toward higher values (H2) for oil content was 87.7% , suggesting that these loci were stably expressed in different environments and controlled oil accumulation in seeds. The other two loci, qOCC1 and qOCA6, were specific to the environment of years 2010 and 2011, respectively. In addition, GWAS with the BLUP values of seed oil content across three environments also detected qOCA3 and qOCC6, but missed the two environment-specific loci, qOCC1 and qOCA6  was the major and favorable haplotype with 168 (88.9%) inbred lines and had an average oil content of 40.92±1.88% (P = 1.4×10-4)  being the major and favorable haplotype. Inbred lines conferring the H1 haplotype had an average oil content of 40.8±2.05%, which was significantly higher than that of H2 .The 92±1.88% , which i.4×10-4) . The qOCB. napus is an allotetraploid species that originated from the natural hybridization of B. rapa and B. oleracea. Before this hybridization, the two progenitors had experienced whole genome triplication [B. napus genome. The existence of homologous and paralogous sequences makes it still difficult to discover SNPs even though the draft reference genome sequence of B. napus is available [lication ,39,40, wvailable . To avoivailable . Discardvailable , the modB. napus, which provides deep insights into breeding history in the past century and facilitates understanding the genetic determinants of complex traits in GWAS. Previously, a set of 451 SSR markers were used to evaluate LD level of the association panel, but the resolution of estimated LD level was low due to the limited number of markers [B. napus inbred lines, the whole-genome LD decay distance was estimated to be 1,214 kb at the background level (r2 < 0.26), which was longer than the distances of LD decay estimated in previous studies using different B. napus representative germplasm resources [High-density SNP mapping provides an opportunity to characterize genome-wide LD patterns systematically in  markers . In the esources –17. In aesources –17. In tB. rapa cultivars [B. rapa had largely increased the level of genetic diversity in the A subgenome and thus resulted in less and shorter haplotype blocks in P1 compared to P2. Additionally, the limited number of cultivars introduced from Europe or Canada in different periods were probably the founders of the C subgenome in these semi-winter cultivars. No exotic genome was further introgressed into the C subgenome of the semi-winter Chinese oilseed rape, which resulted in more and longer haplotype blocks in P1 than in P2 in the C subgenome. The understanding of the breeding history of oilseed rape in China would prompt us that it is necessary and urgent to broaden the level of genetic diversity in the C subgenome.The SNP markers clearly classified the association panel into two groups (P1 and P2) and four subgroups. The classification of the 189 inbred lines was basically consistent with that revealed using SSR markers . Ten inbultivars  that hasqOCA3 and qOCC6, were also detected in previous studies using traditional linkage mapping [qOCA3 and H1 (GAAG) at qOCC6, have a frequency of 88.9% and 93.1%, respectively. Both favorable haplotypes had significantly higher oil content than the minor haplotypes. These results suggest that strong selection has been applied to these two favorable haplotypes. In the 200 kb region flanking qOCA3, a gene BnaA03g32620D encoding a lipid transport superfamily protein [qOCC6, a gene BnaC06g22630D encoding an acyltransferase and involving in diacylglycerol biosynthetic process [B. napus, although additional evidence is needed to support the identified loci and candidate genes. The SNPs associated with oil content would be useful for oil content improvement in oilseed rape breeding program.GWAS was performed on seed oil content using the high-density SNP markers. The two stably expressed loci for seed oil content,  mapping , 43. The protein  was iden process  was founS1 Figth percentile of r2 distribution for unlinked SNP pairs based on the genetic map with 4,995 anchor SNPs.The vertical dash red line indicates the 99(TIF)Click here for additional data file.S2 Fig(A) Estimation of LnP(D) and Δk in the whole panel. (B) The 189 diverse inbred lines were classified into two groups P1 and P2 by Structure analysis. (C) PCA of the entire panel, blue and red dots represent the inbred lines from P1 and P2, respectively, and the values in parenthesis represent the proportions of variances explained by the first two principal components.(TIF)Click here for additional data file.S3 Figk for P1 (A) and P2 (B) in Structure. Subgroups were derived from P1 (C) and P2 (D).Estimation of LnP(D) and Δ(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.