Long-term selection experiments provide a powerful approach to gain empirical insights into adaptation, allowing researchers to uncover the targets of selection and infer their contributions to the mode and tempo of adaptation. Here we implement a pooled genome re-sequencing approach to investigate the consequences of 39 generations of bidirectional selection in White Leghorn chickens on a humoral immune trait: antibody response to sheep red blood cells.MHC, SEMA5A and TGFBR2, are also presented.We observed wide genome involvement in response to this selection regime. Many genomic regions were highly differentiated resulting from this experimental selection regime, an involvement of up to 20% of the chicken genome (208.8 Mb). While genetic drift has certainly contributed to this, we implement gene ontology, association analysis and population simulations to increase our confidence in candidate selective sweeps. Three strong candidate genes, The extensive genomic changes highlight the polygenic genetic architecture of antibody response in these chicken populations, which are derived from a common founder population, demonstrating the extent of standing immunogenetic variation available at the onset of selection.The online version of this article (doi:10.1186/s12864-016-3414-7) contains supplementary material, which is available to authorized users. A key aim in the study of evolutionary genetics is to identify loci that facilitate adaptation. The importance of de novo mutations and standing genetic variation to the mode and tempo of the adaptive process has been debated, as well as the importance of fixation for adaptation –3. ThrouDrosophila have demonstrated genome-wide involvement in response to selection [Drosophila melanogaster, nearly 5% of the genome, and 42 genes, were suggested to be involved in an 150% increase in parasitoid resistance [Drosophila C virus [The number of genes underlying an adaptive process often belies the complexity of the selective environment. For traits with a complex genetic basis, such as aging and courtship, experimental evolution studies in election , 13. Forsistance , whereasFST). Due to the bidirectional selection regime, the identified selection signatures would result primarily from selection on standing genetic variants [2 intercross between the HAS and LAS lines at generation 32.Here we use a genomic approach to investigate the consequences of long-term, bidirectional selection on a single immune trait from a base population of randombred White Leghorn chickens . In brievariants , 19. Rel2 intercross between the selected lines, used enrichment analysis to detect regions with immune genes and identified overlaps with immune associations mapped in other studies/populations. In this way, a high-confidence set of candidate selective sweep regions may be inferred. Three particularly relevant candidate genes, MHC, SEMA5A and TGFBR2, are presented in more detail.We observed over 200 regions across the genome affected by the selection regime, characterized by spans of extreme differentiation between HAS39 and LAS39. These would include true selective sweeps and those regions displaying this pattern due to the random genetic drift experienced by the small selected populations. To identify strong candidate sweeps from this large set of differentiated regions, we overlapped regions with associations detected to antibody response in an FPopoolation2. A summary of the population-statistics for the four analyzed populations is provided in Table FST) was also greater between the S39 than the R16 lines.A total of 711,149 1000 bp windows were analyzed in After clustering of windows located less than 0.5 Mb apart, and removing sweep-regions with a single 1000 bp window or only 2 SNPs, 224 highly differentiated regions were retained  region.The 224 highly differentiated regions contained a total 3511 annotated genes. To identify potential candidate adaptive genes, as well as to indicate individual candidate sweeps that are more likely to have reached high frequency by selection rather than drift, we subjected these to a Gene Ontology (GO) analysis. Using the DAVID GO analysis, 67 gene IDs were reported to be associated with immune system processes . Among the markers selected by the 5% FDR threshold, rs14799859 is located on GGA1 between a sweep region ending at 15,887,000 and another beginning at 27,122,500, marker rs15307852 is located just outside the sweep region at GGA1: 67,820,500–69,251,000, and marker rs14242328 falls within the sweep region on GGA2: 119,248,000–119,841,000, none of which contains annotated immune genes. The marker rs14096690 showed only 20% FDR significance in the model, but is located within the sweep region GGA16: 2000–323,000 and notably within the candidate MHC region (see below section).As the linkage disequilibrium is extensive in the F2 population, the candidate sweep region GGA2: 78,748,000–78,800,500 .Previously, we observed a correlation between the length of selective sweeps and their contribution to adaptation in a similar long-term, single-trait selection-experiment for 8-week body weight in chickens . TherefoTGFBR2 . The haplotype that appears fixed in LAS39 has a large deletion overlapping exons 4-5 of this duplicated region. By comparing de novo assemblies of LAS39 and HAS39 genomes, the deletion was localized to GGA2: 40,414,509–40,418,21 and results in a 3712 bp deletion , and second, that selection acts on these standing variants in opposite directions.These extensive genome changes in this long-term selection experiment suggest that SRBC antibody response is a highly polygenic trait. The 6.5 fold difference in antibody titers between the divergently selected lines has been facilitated by large differentiation at many loci with standing genetic variants. This finding is consistent with those in the Virginia body weight lines, which has been bidirectionally selected for juvenile body-weight for over 55 generations. There, it has been shown that selection on standing variation in a highly polygenic genetic architecture was the main contributor to the long-term selection response in body weight .Drosophila melanogaster and Saccharomyces cerevisiae were used to model adaptation in vertebrate populations have rarely detected complete selective sweeps [Genetic drift in these relatively small breeding populations would have contributed not only to random divergence between the selected lines, but also to the fixation of selective sweeps. Previous studies where e sweeps , 9. Furte sweeps , 34. In Antibody production is a major defense mechanism of the humoral immune response. It involves a variety of different cell types with specific functions, including immunological cascades involving the recognition of the foreign cells by T and B immune cells, activation and differentiation of B cells into plasma cells and secretion of specific antibodies . MoreoveOrnithonyssus sylviarum) and Newcastle Disease, but more susceptible to the bacterial pathogens Escherichia coli and Staphylococcus aureus when compared to LAS [Earlier works with the Virginia Antibody lines have, however, shown that the level of antibody-response does not have a direct connection to a general greater immunological efficacy. For example, when the lines selected for high (HAS) and low (LAS) antibody production were faced with different immunological challenges, the HAS were less susceptible to the northern fowl mite , this restricted our genome coverage and would have limited the opportunities of identifying associations to day 5 antibody titers.In the remaining sections, we discuss a number of particularly promising candidate functional genes detected in the identified sweep regions.2 intercross between the HAS and LAS lines in generation 32 overlaps the first 7 of 18 exons  of Semaphorin-5A . Semaphorins are a large family of secreted and membrane bound proteins, originally discovered in the nervous system with a role in axon guidance  with the strongest association to day 5 antibody titers in the Frized in ). SEMA5Adiseases . It has diseases , chronicdiseases , and neu07559 witrleukins .SEMA5A gene is present in at least three different haplotypes: the major haplotypes referred to here as Haplo1, Haplo2, and Haplo3. Haplo3 is present only in LAS39, suggesting that this variant involved in low antibody production was purged in relaxed line LAR16. This is reflected in the population differentiation between the lines as well, as LAS39 is most differentiated of the lines. The extent of differentiation at many markers suggests that these haplotypes were present as standing variants in the base population, as opposed to novel variants emerging in the LAS lineage after generation 24 and the founding of the relaxed line. Although we have no information for earlier generations, we can speculate on the driving forces acting on Haplo3. Given the complete purging of this haplotype from the relaxed lines, it seems reasonable to assume that this haplotype has a fitness cost; a fitness cost that may not have been evident in the base population or present as a cryptic variant. In the context of the LAS line, selection for Haplo3 progressed slowly (no fixation after 39 generations), for which this inferred fitness cost is a possible explanation. As Haplo3 is expected to segregate at an intermediate frequency at generation 24, this fitness cost would purge Haplo3 quickly from the LAR line once selection is relaxed.In the Virginia Antibody lines, the sweep region overlapping the SEMA5A, with the majority of sequence polymorphism between haplotypes contained in intergenic and intronic regions. Taken together, this implies that the involvement of SEMA5A to the differential antibody response in the Virginia Antibody lines would be due to regulatory polymorphisms where the two haplotypes produce different expression levels of the SEMA5A protein, from different promoter or enhancer sequences. This could impact the expression of cytokines within this regulatory network and thus influence an effective and specific immune response.We observed only three synonymous SNP sites in the coding region of TGFBR2), making it a logical candidate gene for further study. TGFBR2 is a member of the serine/threonine protein kinase family, encoding a transmembrane protein that binds the secreted protein TGF-beta. The TGF signaling pathway regulates diverse biological processes during all stages of life and plays a key role in growth and the immune response [Earlier work with the Virginia body-weight selected lines, which have undergone a similar long-term experimental selection regime, has shown that the length of a selective-sweep is correlated with its contribution to the adaptive phenotype . Hence, response , 44.TGFBR2 haplotypes in the selected lines. In-depth analyses comparing the sequence data for HAS39 and LAS39 suggest that these haplotypes differ by a 3712 bp deletion  fixed in LAS39, which encompasses exons 4 and 5 of the TGFBR2 gene prediction. Fixation of these haplotypes in the respective relaxed lines implies that the selected lines were already fixed (or nearly fixed) by generation 24, when the relaxed lines were founded. The length of this sweep, together with the rapid fixation, suggests that the selection acted strongly and fixation occurred swiftly, before multiple recombination events could break down the linkage within this region and leave a shorter sweep.We observed fixation for different TGFBR2 haplotypes might have played a major role in determining antibody response, most likely due to its key function in the TGF-beta pathway. Evidence is lacking, however, that this large sweep region was associated with 5 day antibody titers in the F2 intercross between HAS32 and LAS32, despite being covered by 7 segregating SNP markers . It should be noted, however, that these SNP markers were fixed or near fixed for alternative alleles in generation 39. As the population used for association analysis was small, the power in this analysis was low. Therefore it difficult to make strong conclusions as to whether these haplotypes had no or only minor effects on antibody response, or whether their functional significance was reliant on the genetic background of the population.The strong selection signature suggests that the TGFBR2 can result in serious debilitating and fatal diseases in humans, most notably the autosomal dominant aortic aneurysm syndrome, Loeys-Dietz syndrome, as well as an increased risk of certain cancers [TGFBR2 protein, but with less splice variant options than the HAS haplotype.Defects in  cancers , 46. ThaMHC) is the most well studied immune gene complex in vertebrates due to its key role in the pathogen surveillance [MHC has previously been investigated in the Virginia Antibody lines, albeit not at a sequence level [MHC region is relatively small, simple and tightly-linked, compared to that of mammals, and thus termed the minimal essential MHC [MHC was evident as early as generation 12 in the Virginia Antibody lines, and alternative haplotypes were fixed by generation 32  and immune regulation (TGFBR2 and SEMA5A). Further work to investigate the large numbers of additional immune genes that are likely to have contributed to the response to selection in this experimental population would be useful to improve our understanding of selection on immune traits and to unravel the complexity of their interactions. These experimental lines continue to present a unique opportunity towards understanding the mode and tempo of selection in a higher vertebrate organism.Adaptation to long-term, bidirectional selection for antibody response in this experimental chicken population has been facilitated by standing genetic variation across many regions of the genome. Although selective-sweep studies by themselves were unable to quantify the contribution by individual genes and polymorphisms to adaptation, data from earlier association and functional studies highlight three particularly interesting candidate sweeps and underlying candidate genes involved in immune surveillance  using BWA [http://broadinstitute.github.io/picard/) was used to sort genomes and to mark and remove duplicates. GATK [FST between all populations were calculated. PoPoolation2  using two lanes on an Illumina Hiseq 2500. Reads were aligned to the sing BWA . Picard es. GATK  was usedes. GATK , 60) was2  was use method,FST cutoff (>95% percentile FST = 0.946) was used when defining the sweeps to limit the number of candidate regions due to drift. Windows with FST values above this cutoff were clustered into candidate sweep regions when they were less than 0.5 Mb from one another (custom R scripts). Clusters containing only a single 1000 bp window, or less than 2 SNPs, were removed from the dataset.A stringent Ne(1-10) = 22.4 and Ne(11-39) = 25.6; calculated Ne ~ 4*Nef*Nem/(Nef + Nem)), whereby genetic drift could be a prominent force affecting allele frequencies which would confound the identification of selective sweep signatures. Simulations were carried out for a 5 Mb locus using SFS_CODE [Our experimental populations were small (SFS_CODE  taking iSFS_CODE ), recombSFS_CODE ) change FST between the two simulated populations. Average FST was calculated per 1000 bp window with 50% overlaps then regions of differentiation were clustered together if distance between differentiated SNP sites was less than 50 kb, following a similar methodology applied to the real genomic data.Resulting allele frequencies from simulations were used to calculate We used GO analysis to identify functional candidate sweeps that were enriched for immune-related genes. BEDOPS  was usedhttp://www.ncbi.nlm.nih.gov/sra; runs SRR2889287, SRR2889288, SRR2889289) and aligned using STAR . Genome.2 intercross between the HAS and LAS populations. Data and results from these studies were used to increase our confidence in individual candidate sweep regions. We reanalyzed the SNP association data from Dorshorst et al. [2 intercross generated by intercrossing birds from HAS32 and LAS32 that were genotyped by a custom GoldenGate® Genotyping assay containing 3072 SNP markers as described by Dorshorst et al. [A number of QTL and GWAS studies have been performed for immunological traits in chickens, including an analysis of an Ft et al.  to compat et al. . Brieflyt et al. .2 transformed day 5 antibody titers, and the final model from the backward elimination analysis was decided using an adaptive 5–20% FDR criterion [In order to focus on the most informative markers that tag divergent selective sweep regions, we used a subset of SNP markers that possessed an allele frequency difference > 0.7 in the pooled genome sequence between HAS39 and LAS39. To further refine this subset, neighboring, linked markers were clustered together . Backward elimination was applied to clusters with more than one marker to select the most significant marker as representative of the cluster region for use in the genome-wide analysis. This final refined SNP subset was analyzed using the same backward elimination process as in the within-cluster analysis. All analyses used a standard linear model framework, starting with a full model including the fixed effects of sex and the additive effects of the highly differentiated markers. These were regressed onto logriterion , 74.We do not have DNA and thus genomes from the line founders, so software estimating haplotype frequencies within pooled sequencing samples  cannot b


Diastatic Power (DP) is an important quality trait for malt used in adjunct brewing and distilling. Substantial genetic variation for DP exists within UK elite barley cultivars, but breeding progress has been slow due to the limited demand, compared to the overall barley market, and difficulties in assessing DP. Estimates of DP  from a collection of UK elite winter and spring varieties were used to identify contrasting sets of high and low DP varieties. DNA samples were pooled within sets and exome capture sequencing performed. Allele frequency estimates of Single Nucleotide Polymorphisms (SNPs) identified from the sequencing were used to identify genomic locations associated with differences in DP. Individual genotypes were generated from a set of custom KASP assays, both within sets and in a wider germplasm collection, to validate allele frequency estimates and marker associations with DP. QTL identified regions previously linked to variation in DP as well as novel associations. QTL colocalised with a number of genes annotated as having a diastase related function. Results indicate that winter barley is more genetically diverse for genes influencing DP. The marker assays produced by this work represent a resource that is available for immediate use by barley breeders in the production of new high DP varieties. Hordeum vulgare L.) worldwide is in the production of alcohol following malting, with about 20% of annual barley production being used for processing (FAOSTAT: http://www.fao.org), the vast majority of which is used by maltsters. During the malting process, endogenous proteolytic and amylolytic enzymes (either present within the mature grain or generated during germination) are released and modify the barley endosperm, with starch being converted into fermentable sugars. The breakdown of starch is catalyzed by a group of enzymes known as diastases and their combined ability to do so is called diastatic power (DP). Whilst most barley varieties can produce malt with sufficient diastase activity to convert all its starch into fermentable sugars during malting and mashing, specific malting applications, such as the use of unmalted grain as an adjunct source of starch in brewing and distilling, require the use of barley malt with higher levels of diastase activity. DP is subject to considerable environmental influence, in particular from differences in nitrogen management regime  of allele frequencies from phenotypically contrasting sets. Specifically, the study aimed to identify QTL and genes responsible for genetic variation in DP in both spring and winter elite UK cultivars, enabling tools for breeders to produce new varieties that are specifically targeted at the production of high DP malt.DP data for a collection of 592 UK barley varieties (367 spring and 225 winter) was taken from historical recommended (RL) and national list (NL) trial results over the period 1994-2012 as part of the IMPROMALT project (BBSRC: BB/K008188/1). This panel was largely the same as that used in Thomas et al. , with thContrasting sets of high and low DP lines were selected from each of the two germplasm collections  according to the manufacturer's instructions. Extracted DNA was quantified using a Qubit fluorometer (Thermo Fisher) and an equal quantity of genomic DNA was combined for each line in each set (12 lines × 4 sets). Combined DNAs for each sample pool  were used for individual exome capture and sequencing. A custom barley exome capture SeqCap EZ library was used throughout, representing approximately 62 Mbp of the barley reference exome . The raw SAM output was filtered with the bamtools toolkit (https://github.com/pezmaster31/bamtools) to remove reads that contained more than 4% mismatches, based on their alignment score (AS) flag. Mismatch cut-offs in read mapping are essential for the accuracy of downstream analysis as read mismapping caused by overly relaxed mismatch parameters can lead to dramatically increased false positive rates in variant calling  allele in each contrasting set. Markers were considered to be fixed if they had a reference allele frequency (RAF) less than 0.01 in either set. In addition, only variants with at least 100 reads in each set were included. This filter was applied to limit the effect of sampling error in the estimates of allele frequencies.Allele frequency estimates for each marker within each set were calculated directly from read numbers of each allele. Allele frequency differences (AFD) for each marker were calculated as the reference allele frequency of the high DP set minus the reference allele frequency of the low DP set, giving a possible range between −1 and 1. Genomic locations with an absolute estimated allele frequency difference greater than 0.75 were chosen as putatively associated loci. Whilst it was not possible to derive a null distribution for allele frequency differences , such a difference corresponds to a minimum −log10(p) of approximately 3.8 .Specific winter and spring barley associations were identified independently from either the winter or spring contrasting sets. QTL were considered independent if associated markers were separated by at least 400 Mbp in centromeric regions, or 10 Mbp in non-centromeric regions   was genotyped. Details of all the lines used for this genotyping are given in Supplementary Table On average there were 25 sites across both seasonal growth habits with DP data for each year for the 592 lines in the historical data set. Repeatability was moderately high for DP for each growth habit (0.53 in springs and 0.61 in winters). In addition, DP estimates based on 2 years of field trial data/ micro-malting were taken from 100 spring and 100 winter varieties but the structure of this experiment did not permit a true estimate of the repeatability across the whole data set. There was considerable overlap between the lines for each data source so the total number of lines for which data was available was 602 (371 spring and 231 winter barley lines), from which an overall mean was derived by a REML analysis .The Illumina HiSeq sequencing run generated approximately 720 M reads, with read numbers for the four pools (high versus low DP × winter vs. spring lines) varying between ~170 M and 185 M reads . A final filtering step to exclude SNPs with fewer than 100 reads left a final set of 77,523 SNPs. For the spring set the corresponding numbers were: 859,511 variants, of which 778,676 had a phred score greater than 50; 526,234 were polymorphic, and 83,568 had more than 100x read coverage.Allele frequency estimates from the filtered SNPs were made from the read counts of each allele from each set. These were used to identify markers with highly differentiated frequencies between high and low sets. In the winter sets, 66 markers with an absolute allele frequency difference greater than 0.75 were identified, corresponding to 6 distinct physical positions. These QTL (Wnt-1-6) were located on chromosomes 1H, 2H, 4H, and 7H Figure . In the R2 = 0.93).In order to test the robustness of the estimates of allele frequencies made from exome capture sequencing, a subset of exome capture SNPs that overlapped with those present on the iSelect 9k chip  was identified. A total of 4,986 iSelect SNP markers could be mapped unambiguously to the Morex reference sequence. Of these, 460 unique SNP markers  were also identified as variants in the exome capture set. A linear model describing a one to one relationship between estimated and true reference allele frequencies accounted for a high proportion of the variance in estimated allele frequencies   using a more relaxed differentiation threshold. Individual genotyping of the lines selected for the contrasting sets showed a strong correlation between absolute values of the estimated allele frequency differences from the exome capture and sequencing and absolute values of the true AFD (7) Table . Individ7) Table . In almo7) Table .p < 0.001), with the exception of SNP assay 5 (Wnt-4), SNP assay 9 (Spr-1) and SNP assay 11 (Spr-2) , with nine validated from the 19 markers tested in total. For each marker tested, varieties carrying the high allele (as identified in the contrasting sets) had a higher estimated DP than varieties carrying the low allele. In most cases, this difference was highly significant  will help to address whether variation at these genes is responsible for differences in DP seen within the cultivar collection.The availability of full sequences for each of the barley chromosomes, now allows QTL regions to easily be placed in the context of the underlying gene content. Homology based searching of the Bmy1 & Bmy3). Previous studies have linked variation at the Bmy1 locus with variation in DP  may also be present in this region, although no differentiated SNPs were identified in this gene from the sequence data generated in this project. Variation at these genes may be associated with differences in DP in both spring and winter barley and therefore represent good candidates for detailed characterization. However, the ability of bi-parental QTL mapping studies to distinguish between these linked loci is low due to limited recombination during the generation of mapping populations . Nevertheless, the SNP marker designed in the Bmy1 gene (SNP Assay 9) was not as highly associated with DP as a closely linked marker (SNP Assay 10). This observation could either be interpreted as reflecting a gene (linked to Bmy1) influencing the trait, or being caused by genetic diversity within a linkage block containing both markers, causing a lower correlation between the causal variant and Bmy1 SNP. Given the small number of candidate markers being considered, it is not possible to distinguish between these explanations given the current data. There are 97 reported high confidence gene models reported across the QTL interval , suggesting that genetic variation at endogenous amylase inhibitor loci may also influence malt diastase activity. Indeed, a SNP assay designed in this gene (SNP Assay 7) showed a stronger association with DP in the wider variety collection than the peak marker identified from the exome capture data (SNP Assay 6). Due to this, the gene represents an extremely strong candidate for follow-up studies. Variation in expression of an α-amylase/subtilisin inhibitor has previously been described as correlating with β-amylase activity  structural diastase genes. One of the candidate QTL (at 75 Mbp on chromosome 7H) is linked to a gene encoding a putative α-amylase inhibitor  were present between lines carrying alternative alleles at candidate markers. This result confirms that the QTL detection methodology used in the study is an effective method for identifying genuine marker associations with quantitative traits, irrespective of their genetic architecture. This can be applied to historical or This study demonstrates that the use of NGS techniques on pooled DNA from phenotypically contrasting sets represents a powerful method for conducting GWAS studies at extremely high marker density. This can directly lead to the identification of candidate genes for quantitative phenotypic traits, provided that appropriate methods are used for controlling against population structure and spurious associations from experimental sources of error. Applying this to spring and winter cultivar collections, has led to the identification of at least six novel QTL for the genetic control of DP in UK barley and provided candidate genes that can be followed up in subsequent studies.ML contributed to experimental design, analyzed and interpreted data, and wrote the manuscript. MB advised on experimental design, conducted bioinformatics analysis and contributed to manuscript preparation. HB contributed to the analysis of historical data, coordinated preparation of variety collections for sequencing and genotyping and edited the manuscript. LR contributed to the experimental design and edited the manuscript. WT contributed to the experimental design, analyzed historical malting quality data and contributed to manuscript preparation. AB conducted genotyping and preparation of DNA for sequencing. CD conducted genotyping. JM prepared libraries for sequencing. PH advised on experimental design, coordinated exome capture and sequencing, and contributed to manuscript preparation. JR designed the experimental approach, coordinated the research and contributed to manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Babesia. In their host vector, Babesia spp. undergo sexual reproduction. Therefore, the development of sexual stages and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle. HAP2/GCS1 is a protein identified in plants, protozoan parasites and other organisms that has an important role during membrane fusion in fertilization processes. The identification and characterization of HAP-2 protein in Babesia would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future.Bovine babesiosis is a tick-borne disease caused by the protozoan parasites of the genus hap2 gene DNA from an infected bovine with Babesia bigemina was purified. The hap2 gene was amplified, cloned and sequenced. The sequences of hap2 from four geographically different strains showed high conservation at the amino acid level, including the typical structure with a signal peptide and the HAP2/GSC domain. Antisera anti-HAP2 against the conserved extracellular region of the HAP2 amino acid sequence were obtained from rabbits. The expression of hap2 in the host and vector tissues was analyzed by using semi-quantitative RT-PCR, and the protein was examined by western blot and immunofluorescence. Based on the RT-PCR and WB results, HAP2 is expressed in both, sexual stages induced in vitro, and in infected ticks as well. We did not detect any expression in asexual erythrocytic stages of B. bigemina, relevantly anti-HAP2 specific antibodies were able to block zygotes formation in vitro.To isolate and sequence the Babesia bigemina HAP2 is expressed only in tick-infecting stages, and specific antibodies block zygote formation. Further studies regarding the function of HAP2 during tick infection may provide new insights into the molecular mechanisms of sexual reproduction of the parasite.The online version of this article (10.1186/s13071-017-2510-0) contains supplementary material, which is available to authorized users. Babesia, which infect a wide range of domestic animals and occasionally humans. The species affecting cattle are B. bovis and B. bigemina. Current methods for controlling bovine babesiosis are based on two aspects: one is the control of ticks and the second one is the use of therapeutic chemicals to eliminate the pathogen from the infected animal. Both methods have drawbacks, generating resistance [Babesiosis is a tick-borne disease caused by intraerythrocytic protozoans of the genus sistance –3 and hisistance .Plasmodium spp. These vaccines aim to interfere with and block pathogen development within the vector. These vaccines are based on identifying surface-expressed proteins during the life-cycle stages of parasites inside the vector. In Plasmodium, different surface proteins of gametes, such as Pfs48/45 and Pfs230, have proven to be good immunogens [Arabidopsis thaliana [Plasmodium spp. [hap2 gene is absent or mutated, the zygote formation is completely blocked indicating its relevance in this event [Plasmodium berguei, HAP2 is essential for the fusion of gamete surface membranes but not necessary for the adhesion of male and female gametes, and specific antibodies anti-HAP2 block its transmission in vivo and in vitro [Among the many strategies directed at controlling vector-borne diseases is the advancement of transmission-blocking vaccines (TBVs), which have been developed to interrupt the life-cycle of some protozoan parasites, such as munogens . HAP2 prmunogens . HAP2/GCthaliana  and lateium spp. –12. Therium spp.  and it hium spp. , 15. Difis event , 16, 17.Babesia parasites have a complex life-cycle, including asexual stages in the bovine host and sexual stages in ticks. The development of sexual stages of Babesia spp. and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle, nevertheless very little is known about the molecular events involved in the sexual reproduction of the parasite and sexual stage proteins. There are a few reports in Babesia spp. of sexual stage-specific proteins; two proteins encoded by a six-cysteine (6-Cys) gene family Bbo CysA and B have been found to be expressed during sexual stages in B. bovis [B. bigemina sexual stages, the expression of the family of multidomain adhesion CCp proteins (CCp 1–3) has been demonstrated in vitro [B. bigemina would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future. In this study, we isolated and characterized the hap2 gene of B. bigemina, its expression profile in the host and vector infected cells and we also tested if anti-HAP2 specific antibodies were able to block zygote formation in vitro.B. bovis . Meanwhiin vitro . The ideBabesia-free colony of Rhipicephalus microplus (Media Joya strain) was maintained under laboratory conditions. Rhipicephalus microplus larvae hatched from 0.5 g of eggs, were placed on an intact calf and 21 days later replete female ticks were collected. To obtain infected ticks, concurrently, R. microplus larvae from 0.5 g of eggs were placed on a splenectomized calf. Fourteen days later, the calf was intravenously inoculated with 5 ml of blood infected with B. bigemina (Chiapas strain) previously maintained in liquid nitrogen and 21 days later replete female ticks were collected. Replete female ticks fed on infected or uninfected blood were collected a part of the ticks used to obtain total extracts for RNA and also to obtain their midguts at 0, and 72 h post-repletion as previously reported [A reported . To confhap2 in the genome of B. bigemina, we used the reported sequence for hap2 in Plasmodium berguei (EU369602) [http://www.sanger.ac.uk).To identify U369602)  as a proDNA extraction was performed with a Gentra Puregene kit , according to the supplier’s instructions. The final pellet was resuspended in 100 μl of the hydrating solution and incubated for 5 min at 65 °C, to subsequently be stored at -20 °C until use.hap2 is conserved among geographically different strains we used DNA from the Chiapas-México, Kayseri-Turkey, Rondonia-Brazil, and Seed-México strains. The hap2 gene of each strain was amplified by PCR using oligonucleotides hap2F (5′-GAT AAG AAT TCA TGA CGC ATG CCG TGC TGA AC-3′) and hap2R (5′-GAT AAG AAG CTT CTA CAC CTC GTC GCT ATG GC-3′) and Platinum® Taq DNA Polymerase High Fidelity . The conditions of PCR were as follow: an initial denaturing step at 94 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 60 °C for 30 s and 72 °C for 2.5 min, with a final extension step at 72 °C for 7 min. The amplicons were cloned into the TOPO TA vector (Invitrogen), according to the manufacturer’s instructions. The inserts of the selected clones (3 of each strain) were sequenced by the dideoxy chain-termination method [To analyze if n method  in an Auhap2 gene and the amino acid sequence, the GeneWise tool was used (http://www.ebi.ac.uk/Tools/psa/genewise), considering the amino acid sequence of the putative membrane protein reported in GenBank as a reference (accession number: XM_012913569.1) and nucleotide sequences obtained in this work. The predicted amino acid sequence of B. bigemina HAP2 was used to determine the E-values of related proteins by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). HAP2/GSC1 domain sequence of Chiapas-México strain was aligned with the following sequences: Babesia bigemina BBOND strain (XP_012769023.1), Plasmodium berguei (EU369602), Toxoplasma gondii VAND strain KFH08353.1, Babesia bovis strain T2Bo strain (XP_001611806.1) using Clustal Omega .To deduce the putative introns of the http://www.cbs.dtu.dk/services/TMHMM/To identify the signal peptide, a bioinformatics analysis was performed by using the SignalP Server v 4.0 and TMHMM program. 2.0 was used to identify transmembrane helices http://www.ebi.ac.uk/Tools/msa/clustalo/).The alignment of HAP2 amino acid sequences of the different strains was performed with Clustal Omega  and antibiotic-antimycotic (Invitrogen) and placed in 75 ml culture flasks . Cultures were incubated at 28 °C for 18 h. Sexual stages induced in vitro were centrifuged and separated over a 47% percoll gradient. The interface was collected and washed; smears were made and stained with Giemsa to corroborate sexual stages.Sexual stages of ed below . BrieflyThe midguts from infected or uninfected ticks of replete females were obtained as we reported previously . ProteinB. bigemina infected erythrocytes, induced sexual stages of the parasite, pool of infected and uninfected ticks 0 h and 72 h post-repletion and from the midgut of infected ticks, 72 h post-repletion was purified using TRIzol (Invitrogen). Then, 1 μg of total RNA was reverse-transcribed with ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis (Invitrogen), and used for amplification with Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) following the supplier’s instructions. Two μl of cDNA of each sample was used for PCR. The oligonucleotides used for amplification of 375 bp hap2 were pF (5′-GAC AAA TTC ACC GAC ACG TC-3′) and pR (5′-CTA CAC CTC GTC GCT ATG-3′). Amplification conditions for hap2 were: an initial denaturing step at 94 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 58 °C for 30 s and 72 °C for 40 s, with a final extension step at 72 °C for 7 min and 4 °C indefinitely. To obtain a 155 bp amplicon of the gapdh as a reference gene, we designed a pair of primers from the sequence BBBOND_0202520: forward (5′-CCG CAC CAT CAA ATT GTA C-3′) and reverse (5′-CGG AGA TGA TGA CCA ACT TG-3′). RNA from uninfected erythrocytes and uninfected ticks was used as negative control.RNA from http://www.imtech.res.in/raghava/abcpred/), BCEPred (http://www.imtech.res.in/raghava/bcepred/) and IEDB (http://www.iedb.org/). Two peptide sequences with the highest value in all three B cell epitope and antigenic algorithms were selected  of twenty-one and sixteen amino acid residues respectively which were used for rabbit immunization to generate two monospecific polyclonal antisera. The peptides were designed by using the bioinformatics programs ABCPred  was added. Pre-immune serum (1:80) was used as a control in each case. Slides were incubated at 37 °C for 30 min, washed three times with PBS-Tween 0.05% for 5 min and then allowed to dry at 37 °C for 15 min. Thereafter, the slides were incubated with 20 μl of Alexa-488 conjugated anti-IgG rabbit antibody  diluted 1:200 in PBS-Tween 0.05% for 1 h, washed 3 times with PBS-Tween 0.05% for 15 min and left to dry. The slides were mounted with glycerol phosphate (1:10) and visualized under a fluorescence microscope  with a 100× objective and a specific filter to the spectrum of the Alexa-488 fluorochrome.For indirect immunofluorescence assays, One μg of protein extracts from uninfected and infected erythrocytes, sexual stages, infected and uninfected tick midguts were separated on 10% SDS-polyacrylamide gels and then electrotransferred onto nitrocellulose membranes . Non-specific binding was blocked by incubating the membranes overnight in 5% non-fat dried milk in TBS containing 0.05% Tween-20% (TBS-T). The membranes were then washed and incubated overnight at 4 °C with rabbit antisera anti-synthetic HAP2-peptide 2 diluted 1:2000 in TBS-T containing 1% of non-fat dried milk. Membranes were washed again and incubated with an anti-rabbit IgG conjugated with peroxidase (1:500) (Jackson ImmunoResearch) at room temperature for 1 h. After further washing with TBS-T, proteins of interest were detected by chemiluminescence by using ECL-Prime Western Blotting Detection Reagent . Pre-immune serum diluted 1:2000 was used as a negative control.B. bigemina-infected Rhipicephalus microplus larvae. Twenty-nine days later when the infected bovine presented clinical signs and the parasitemia (determined by Giemsa-stained blood smear counts), raised more than 3%, whole blood was obtained from the jugular vein, defibrinated with glass beads and used for sexual stages induction using a previously described protocol [B. bigemina infected erythrocytes were cultured using induction medium and then monitored by Giemsa-stained smears each hour until zygote formation was confirmed at 36 h. At the same time, additional cultures were set up adding rabbit antiserum against two HAP2 peptides  at a 1:10 proportion. Each experiment was performed in triplicate and serum from rabbits immunized only with adjuvant was used as negative control.An eight-month-old splenectomized bovine was infested with 10,000 protocol  with modWhen zygotes were detected in the normal induction cultures, all the cultures were centrifuged and the supernatant discarded, zygotes were recovered using a Percoll purification gradient as described before . The celpost-hoc test using the software Graph Prism 5.0 with F = 86.71. Comparisons were considered significant at P ≤ 0.0001. To analyze the inhibition of zygote formation, all data were expressed as means percentage considering as a 100% all the cells counted including zygotes, extra and intraerythrocytic parasites ± SEM of three replicas. A one-way ANOVA analysis was performed using the statistical software SPSS v.22 with F = 69.574 with a significance level of P ≤ 0.05.All data for semiquantitative RT-PCR were expressed as the means ± SEM of three independent experiments in each case. The significance of the difference between means was determined by one-way ANOVA with Bonferroni P. berguei was used to search for homologous genes in the B. bigemina genome. A hap2 hypothetical gene was identified, with a size of 2458 bp located on chromosome III, on the complementary strand, in the BBOND_0307410 locus -terminus Fig. .hap2 gene is conserved in B. bigemina strains among geographically different regions, we sequenced the whole gene of Seed-Mexico , Rondonia-Brazil and Kayseri-Turkey. The nucleotide sequence analysis of the strains  showed the presence of four introns at the 5′ end, and one open reading frame encoding a protein of 778 aa for the Rondonia-Brazil strain and 776 aa for the rest of the strains. The protein codified by the hap2 gene of the four strains shares the domain and a high level of identity from 97.82% between Rondonia-Brazil strain and BBOND strain reported in GenBank, to a 99.15% between Chiapas-México strain and Kayseri-Turkey strain. The C-terminus of the protein has a high level of basic amino acid residues, mainly Arg  was lower, 34.78 and 38.10%, respectively.A BLASTp analysis of the Chiapas-México strain showed a HAP2-GSC1 domain (pfam_pfam10699) of 46 aa. The multiple sequence alignment of Chiapas HAP2-GSC1 domain revealed a high identity among vis Fig.  althoughBabesia species so far. Interestingly, the anti-peptide antibody specific to HAP2 recognized the protein in sexual stages induced in vitro at 22 h post-induction .To explore if HAP2 of Sexual stages were observed forming aggregates in cultures treated with anti-HAP2 antibodies Fig. B. bigemina [The identification and study of proteins with a potential role during fertilization in sexual reproduction is vital and has been shown to be considered as potential candidates for the development of transmission-blocking vaccines , 24. Nevbigemina .hap2 gene of B. bigemina which is only expressed in sexual stages induced in vitro and in infected ticks. Relevantly specific antibodies anti-HAP2 inhibited the zygote formation in vitro. The function of HAP2 in gamete fusion and fertilization is extensively documented from plants to unicellular eukaryotes [This study describes the isolation and characterization of karyotes –11, 25. karyotes , 26 and karyotes .B. bigemina genome has a functional hap2 gene which is highly conserved among geographically different strains. The amino acid sequences of the four B. bigemina strains have a conserved HAP2-GSC1 domain in the extracellular portion of the protein, a signal peptide and a transmembrane domain (TDM), which is consistent with the structure of HAP2 previously reported [Here, we demonstrated that the reported , 24. HAPreported . The HAPreported .B. bigemina has a C-terminus rich in basic amino acid arginine. In plants, the predominant basic amino acid is histidine while lysine and arginine are enriched in other species [HAP2 (GCS1) requires a C-terminus domain positively charged for the fusion of gametes . Our stu species , 26, 30. species , althoug species .B. bigemina, an in vitro model for sexual stage development [B. bigemina within the vector ticks. The results presented here show that hap2 gene is expressed in B. bigemina sexual stages induced in vitro and infected ticks. Interestingly, there is no expression of the gene in infected erythrocytes either in the mRNA or protein levels. Similar results were obtained in studies with P. berguei where hap2 transcripts were not present in asexual erythrocytic stages [Plasmodium [P. berguei [To investigate the expression and function of HAP2 of elopment  was usedc stages . Studiesasmodium , 12 bein berguei .Chlamydomonas and Plasmodium [Tetrahymena thermophila [B. bigemina sex-specific markers have not been reported yet, therefore is of great importance to further investigate if HAP2 is only expressed in male gametes.Interestingly, we found that antibodies specific to HAP2, recognized the protein in both sexual stages and in zygotes induced in vitro. It is well known that HAP2 is a gamete-specific protein active in male gametes in asmodium  and indirmophila . In B. bP. berguei it has been shown that specific antibodies anti-HAP2 significantly inhibited ookinete formation in vitro, suggesting that anti-HAP2 antibodies have a transmission blocking effect during fertilization or during the transformation of the zygote to the ookinete [We found that specific antibodies against HAP2 significantly decreased the zygote formation in vitro. Similarly, in ookinete .B. bigemina that might be playing a key function during sexual development of the parasite, hence the relevance to further study this protein as a possible candidate for a transmission-blocking vaccine.In summary, our findings indicate that HAP2 is a specific sexual stage protein in hap2 is a functional gene of B. bigemina highly conserved among different strains and that it is expressed in sexual stages of the parasite and within the vector tick. Besides specific antibodies, anti-HAP2 were able to inhibit the zygote formation in vitro. Altogether, our results suggest that B. bigemina HAP2 could be playing the same role as it has been reported in other apicomplexan species and this protein should shortly be considered as a potential candidate for a transmission-blocking vaccine against Babesia spp. Moreover, more work is needed to investigate whether HAP2 is a male-specific protein in Babesia and its function in the parasite biology.This paper shows that


Pears with red skin are attractive to consumers and provide additional health benefits.Identification of the gene(s) responsible for skin coloration can benefit cultivarselection and breeding. The use of QTL-seq, a bulked segregant analysis method, can beproblematic when heterozygous parents are involved. The present study modified the QTL-seqmethod by introducing a |Δ(SNP-index)| parameter to improve the accuracy of mappingthe red skin trait in a group of highly heterozygous Asian pears. The analyses were basedon mixed DNA pools composed of 28 red-skinned and 27 green-skinned pear lines derived froma cross between the ‘Mantianhong’ and ‘Hongxiangsu’red-skinned cultivars. The ‘Dangshansuli’ cultivar genome was used asreference for sequence alignment. An average single-nucleotide polymorphism (SNP) indexwas calculated using a sliding window approach . Ninescaffolds within the candidate QTL interval were in the fifth linkage group from 111.9 to177.1 cM. There was a significant linkage between the insertions/deletions andsimple sequence repeat markers designed from the candidate intervals and the red/greenskin (R/G) locus, which was in a 582.5-kb candidate interval that contained 81 predictedprotein-coding gene models and was composed of two subintervals at the bottom of the fifthchromosome. The ZFRI 130-16, In2130-12 and In2130-16 markers located near the R/G locuscould potentially be used to identify the red skin trait in Asian pear populations. Thisstudy provides new insights into the genetics controlling the red skin phenotype in thisfruit. Pyrus L. , is animportant temperate fruit crop.1 In China (2014),the area used for pear production exceeded 1.12 million ha with a harvest of 18.10 milliontons, accounting for 70.1% of global pear production . Pear fruit color is amajor characteristic feature and is an important marketable trait.2 Green-, yellow- and russet brown-skinned pear cultivars are commonin China, but red-skinned cultivars are rare.3–5 Previous research has indicated that consumers have astrong preference for red-skinned pears.6 Inaddition to the aesthetic appeal of their color, the anthocyanins in the red skin of pearshave been shown to provide antioxidant benefits for human health.8 Compared with European pearcultivars, there are few full red-skinned Asian pear cultivars,9 and the red color usually appearson the side of the fruit that is exposed to the sun in these cultivars.Pear, 10 and is primarily because of the presence ofanthocyanins.11–13 Thebiosynthesis and accumulation of anthocyanins are mainly determined by genetic factors andare influenced by light, temperature and other environmental factors.14 The peak of anthocyanin biosynthesis occurs in themiddle stage of fruit development in P. communis L. and some Asian pearcultivars15–17 and atharvest in other Asian pear cultivars, a trend similar to that in apples.19 In pears, most of thestructural and regulatory genes in the anthocyanin biosynthetic pathway have beenidentified and functionally characterized.20–24 Phenylalanineammonia-lyase (PAL) and UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT) participatein the first and final steps of anthocyanin biosynthesis, respectively. The correlationbetween anthocyanin biosynthesis and PAL and UFGT activity is obvious in blushed pears butnot in full red European pear fruits.25 In P. pyrifolia red-skinned‘Mantianhong’ and ‘Aoguan’ cultivars, chalcone isomerase (CHI)activity, not PAL, was found to be closely related to anthocyanin biosynthesis andaccumulation.17 A study on two otherred-skinned pear cultivars, ‘Meirensu’ and ‘Yunhongli No. 1’,revealed that PAL induced anthocyanin biosynthesis and that UFGT was closely related toanthocyanin accumulation.18 Research hasdemonstrated that the varying levels of expression of anthocyanidin synthase(ANS), UFGT and tryptophan-aspartic acid repeat protein (WD40)genes and the coexpression of MYB10 and bHLH33 (basichelix–loop–helix 33) genes in anthocyanin biosynthesis form the basis forthe differences in coloration between Asian and European pears.12 Additionally, the differentialimpact of the MYB–bHLH–WD40 complex on the expression of pear anthocyaninstructural genes led to differences in the color pattern of red-skinned pears. Acorrelation between hypermethylation and the green-skinned pear phenotype was found bycomparing the methylation levels in the PcMYB10 promoter in ‘Max RedBartlett’ (P. communis L.) and its green-skinned mutant.25Although there is definite genetic governance of the trait, the extent of the red skincolor of pear fruit is largely dependent on environmental conditionsC.26 Pierantoni etal.24 mapped the position ofPcMYB10 (LG9), noting that it corresponded to the same location asMdMYBa and MdMYB10 and that it controls the pigmentation of appleskin. PcMYB10, however, was not directly responsible for the red or yellow skincolor in the above pear cultivars because the mutation underlying this color differencemaps to a different region of the pear genome. Similarly, in the F1 hybrids of‘BonRouge’ and ‘Packham's Triumph’ cultivars, the ratio ofred- to green-skinned fruits was 1:1, which obeys Mendel's laws ofinheritance.27 In addition, Dondini etal.13 analyzed seven crosses from red-and green-skinned European pears, reporting that the red-skinned phenotype in full-coloredpears was controlled by a single dominant gene and that the dominant gene Red waslocated on linkage group four (LG4) of ‘Max Red Bartlett’. In contrast,researchers studied the F1 hybrids of two crosses from ‘Huobali’, ared-skinned Asian pear cultivar that is considered to have the genetic predisposition forrusset brown, yellow and green skin coloration to be dominant over red skincoloration.28 Volz etal.29 analyzed red skin colorsegregation ratios in 10 Asian pear hybrid families derived from ‘Huobali’and suggested that genetic control of blush color in this cultivar may be due to thecomplimentary action of at least two dominant genes. These results indicate that thegenetic basis and mechanism for red skin color may differ in Asian and European pearcultivars.The full red-skinned phenotype of the European pear ‘Max Red Bartlett’, ared bud mutant of ‘Bartlett’, is controlled by a dominant geneet al.30 developed a bulkedsegregant analysis (BSA) method for rapidly identifying markers linked to any specificgene or genomic region and used this method to identify three randomly amplifiedpolymorphic DNA markers linked to downy mildew resistance in lettuce. The BSA method isapplicable to qualitative trait gene and quantitative trait loci (QTL)analyses.31 With the development andwidespread use of high-throughput whole-genome sequencing technology, BSA analysis hasbecome a fast, efficient and widely used method for identifying gene mutations and theirlocations in the genome.32 Advances in the useof X-QTL,34MutMap,35 MMAPPR32 and QTL-seq36 have beenrecently reported. Takagi et al.36identified QTLs for partial resistance to rice blast disease and seedling vigor usingQTL-seq on recombinant inbred lines and F2 populations of rice. They used whole-genomeresequencing of two DNA bulks of progeny  that exhibitedextremely contrasting phenotypes. The DNA bulks were aligned to the reference sequence ofone parental line to calculate the single-nucleotide polymorphism-index (SNP-index), andQTLs were identified as peaks or valleys in the SNP-index plot. This method can begenerally applied in population genomic studies to rapidly identify genomic regionsassociated with specific traits. QTL-seq has been successfully applied inrice,37foxtail millet,38 cucumber,39 tomato,40chickpea41–43 andpeach.44Michelmore Little information has been reported on the hereditary law of the red color trait inAsian pears. Because there is no significant correlation between the red-skinned trait andthe color of young shoots and leaves, it has not been possible to make an initialselection for red-skinned fruit at the seedling stage by examining shoot or leaf color.Unfortunately, there are no molecular markers for the red- or green-skinned phenotypes.Therefore, the present study used a modified QTL-seq method to map the red or green skintrait in a segregating population of two red-skinned Asian pears. The initial focus of thestudy was to identify molecular markers that could be used in a marker-assisted breedingprogram. This study also provides a solid foundation for identifying the gene responsiblefor red-skinned fruit and the mechanisms involved in the development of red skin in Asianpears.P.pyrifolia), ‘Hongxiangsu’ (P. pyrifolia White Pear Group)and ‘Yuluxiang’ (P. pyrifolia White Pear Group) were grown as theparental lines in the orchard of the Zhengzhou Fruit Research Institute (ZFRI), ChineseAcademy of Agricultural Sciences (CAAS) in Zhengzhou . One F1hybrid population resulting from a cross between ‘Mantianhong’ and‘Hongxiangsu’ (n=348) and another F1 hybrid population resultingfrom a cross between ‘Yuluxiang’ and ‘Mantianhong’(n=458) were used as the study material. The populations were established in2009 and grown at the affiliated experimental orchard of the ZFRI, CAAS in Xinxiang. Most individuals bore their first fruit in 2013. Trees bearingone or more fruits with a red blush surface (>0%) were considered the red-skinnedphenotype, and trees with green to yellow coloration on all fruit surfaces wereconsidered the green-skinned phenotype. Individuals with extreme red or green skincoloration in the population, resulting from the cross between the‘Mantianhong’ and ‘Hongxiangsu’ cultivars, are shown inThe red-skinned Asian pear cultivars ‘Mantianhong’  and simple sequence repeat analyses.The first few young leaves that appeared at the end of the current shoot of eachindividual were collected at the beginning of the vegetative period of growth. Theleaves were immediately frozen in liquid nitrogen and transferred to a−80 °C laboratory freezer. The genomic DNA was isolated using thecetyltrimethylammonium bromide method,A red-skinned pool was constructed from equal amounts of DNA from 28 red-skinned pears,and a green-skinned pool was constructed from equal amounts of DNA from 27 green-skinnedpears. Libraries with 350-bp inserted fragments were constructed using a TruSeq Nano DNAHT Sample Preparation Kit  following themanufacturer’s protocol and sequenced using an Illumina HiSeq 4000instrument  to obtain 150-bp paired-end reads.46 was used to align the clean reads from each sample against thepublic reference genome for pears47 using‘mem 4−k 32−M’, where k is theminimum seed length and M is an option used to mark shorter split alignmenthits as secondary alignments. The resulting alignment was used to identify SNPs andInDels. Variant calling was performed on all samples using the GATK’s UnifiedGenotyper 3.3,48 and SNPs and InDels werefiltered using GATK’s Variant Filtration with appropriate parameter settings. Variants exhibiting segregation distortion or sequencingerrors were discarded. The ANNOVAR Software Tool 49 was used to align and annotate SNPs and InDels and todetermine their physical locations in the genome.Quality trimming was conducted to generate high confidence variant calling. The rawreads were processed to obtain high-quality clean reads by (1) removing reads with⩾10% unidentified nucleotides and (2) removing reads with >50% bases havinglow Phred quality scores (<5). The Burrows-Wheeler Aligner36 Inhomozygous crops, changes in the SNP-index and Δ(SNP-index) in the offspring poolare usually caused by the exchange and recombination of homologous chromosomes when theQTL-seq analysis of the genome of one of the parents is taken as the reference sequence.Using the Δ(SNP-index) for sliding window analysis, changes in the SNP-index andΔ(SNP-index) of the SNP sites within the candidate interval are consistent withinthe recombination section, and the candidate section and the different phenotypescorrespond to the sign of the Δ(SNP-index) window  window . When QT) window .47 cultivar was used as the reference to calculate the SNP-indexand Δ(SNP-index) between the red- and green-skinned pools to save labor. Theaverage SNP-index of the SNPs in the window was calculated and used for the slidingwindow plot. Sliding window analysis was performed on the scaffold sequences in thegenome file 225117_ref_Pbr_v1.0_chrUn.fa (ftp://ftp.ncbi.nlm.nih.gov/genomes/Pyrus_x_bretschneideri/CHR_Un/) andapplied to the SNP-index, Δ(SNP-index) and |∆(SNP-index)| plots with 200-kbwindows and 20-kb increments.In this study, both parental lines belonging to the red-skinned pears were highlyheterozygous, which was not ideal for calculating the SNP-index of the DNA pool of theF1 generations because the origin of the SNPs in the target area could not be estimatedbased on the positive/negative sign of the Δ(SNP-index) window. Therefore, thegenome sequence of the ‘Dangshansuli’ 47 rather than the genome of the parental line, the origin of eachSNP or the linkage around its neighboring region could not be estimated. Positive ornegative values of the |Δ(SNP-index)| did not correspond to the phenotype of theparental lines. Reorganization and ‘pseudoexchange’ can change thepositive and negative direction of the Δ (SNP-index) but cannot change itsabsolute value |). Although use of |Δ(SNP-index)| in asliding window analysis ignores the positive and negative direction ofΔ(SNP-index), it had good positioning ability, especially in locating trait lociin heterozygous crops provided the ability to determine the linkage groups of the scaffolds that containcandidate intervals based on their SNP markers in ‘50This information was compared with the corresponding positions for the red-skinned traitloci in Asian pears reported by Wu et al.50 to confirm the results.The location of the scaffolds containing candidate gene intervals was determined usingthe high-density genetic map of‘Bayuehong’×‘Dangshansuli’ published by Wu 51 The primers for these markers were then screened onnine red-skinned and seven green-skinned genotypes from the red-skinned andgreen-skinned pools, respectively. Twelve markers that were polymorphic between the ninered-skinned and seven green-skinned genotypes were selected and successfully amplifiedin the F1 population resulting from the‘Mantianhong’×‘Hongxiangsu’ cultivar cross. Thepolymerase chain reactions were carried out in a final reaction volume of20 μL, containing 20 ng of DNA, 1× polymerase chain reactionbuffer (Mg2+ plus), 0.2 mmol/L of each dNTPs, 6 pmol of eachprimer and 0.5 U Taq DNA polymerase , using the following amplification program: initial denaturation for 3 minat 95 °C, 30 cycles for 30 s at 95 °C, 30 s at55 °C and 45 s at 72 °C, and a final 10-min extension at72 °C. The amplicons were evaluated using 8% non-denaturing polyacrylamidegel electrophoresis. Joinmap 4.052 was used toperform the linkage analysis. A logarithm of odds score of 4.0 was set for grouping, andthe genetic distances were calculated using the Kosambi function.To verify the accuracy of the candidate interval from QTL-seq using linkage analysis,several primers for InDel and simple sequence repeat markers within the candidateintervals were designed using the Primer3 Software .The red- and green-skinned phenotypes segregated in the F1 populations of the two Asianparental lines. An analysis of the 2014 and 2015 data indicated that there were 117 and182 red- and green-skinned pear individuals, respectively, among the 299‘Mantianhong’×‘Hongxiangsu’ F1 hybrids. Among the 310‘Yuluxiang’×‘Mantianhong’ F1 hybrids, there were 148and 162 red- and green-skinned individuals. The segregation data are presented in47 and SNPs were identified. AnSNP-index was calculated for each identified SNP. Low-quality SNPs with an SNP-indexvalue <0.3 and a read depth <7 or those without an SNP-index in one of the F1hybrid pools were excluded to decrease the influence of sequencing and alignment error.A total of 2 280 509 SNPs were identified within the two DNA pools.Illumina high-throughput sequencing resulted in 78 220 374 and93 410 888 reads from the red-skinned (19.65×depth coverage or 94.74%coverage) and green-skinned (22.98×depth coverage or 95.15% coverage) DNA pools,respectively. The resulting reads were aligned to the pear referencegenome,The top 0.25% and lowermost 0.25% threshold lines for the ∆(SNP-index) plot were setThe candidate intervals derived from thresholds of −0.280 and 0.215 based on the∆(SNP-index) resulted in the partial overlap of some intervals from the|∆(SNP-index)| using a threshold of 0.33. Based on the |∆(SNP-index)|method, the candidate interval was 2.18 Mb. However, based on the∆(SNP-index) method, the candidate interval was 1.72 Mb, indicating a1.1 Mb overlap. Although both methods had a similar distribution on somescaffolds, the candidate interval estimated by the ∆(SNP-index) method wasrelatively smaller, and most intervals were in the subset of the |∆(SNP-index)|method. The influence of the ‘pseudoexchange effect’ caused many of theintervals predicted by the |∆(SNP-index)| method and verified in the linkageanalysis as candidate intervals to be missing in the ∆(SNP-index) method results.This was the case for the intervals located between 500 001 and580 000 bp and between 760 001 and 960 000 bp onscaffold NW_008988130.1 and between 880 001 and 940 000 bp onscaffold NW_008988141.1 , which iMost scaffolds containing candidate intervals were located on the bottom portion of thefifth linkage group (LG5). The exceptions that did not map to a linkage group includedNW_008988041.1, NW_008988581.1 and NW_008989660.1. The remaining nine scaffolds,NW_008988039.1 (scaffold1.0.1), NW_008988076.1 (scaffold40.0), NW_008988091.1(scaffold56.0.1), NW_008988126.1 (scaffold92.0), NW_008988130.1 (scaffold97.0.1),NW_008988141.1 (scaffold110.0), NW_008988461.1 (scaffold430.0.1), NW_008988478.1(scaffold448.0.1) and NW_008989715.1 (scaffold1650.0), were adjacent to each other onLG5 and concentrated in the area between 111.9 and 177.1 cM . These rInDel (231) and simple sequence repeat (55) primer pairs  were useThe R/G locus in Asian pears was located between marker In5039-33 and markersZFRI130-16, In2130-12 and In2130-16, which were identified in scaffolds NW_008988039.1(scaffold1.0.1) and NW_008988130.1 (scaffold97.0.1), respectively. These two scaffoldswere adjacent to each other on the bottom of LG5. Markers In2039-2 and In5039-33 fromscaffold NW_008988039.1 and markers ZFRI130-16, In2130-12 and In2130-16 from scaffoldNW_008988130.1 determined the direction of the two scaffolds. Therefore, the R/G locusshould be in the 582.5 -kb candidate interval comprising two subintervals at thebottom of LG5: a 528.5-kb interval from 3 639 340 to4 167 794 bp in scaffold NW_008988039.1 and a 54-kb interval from908 857 to 962 926 bp in scaffold NW_008988130.1 . This caPAL, CHS, CHI, DFR (dihydroflavonol4-reductase), ANS and UFGT, were found within the candidate region.The MYB transcription factor that forms the MBW complex, the basichelix–loop–helix (bHLH) and the WD40 classes were not detected within thecandidate region. Several genes, including the NAC domain class transcription factor,the WRKY domain class transcription factor and ABC transporter B family member 9-like,all of which are related to anthocyanin synthesis and accumulation, were found in thecandidate region but require further identification and confirmation.Based on the reference pear genome, 81 gene models were predicted in the candidate582.5 kb genomic region .Among t53–56 Thepool sequencing (Pool-seq) strategy, which combines BSA and whole-genome sequencing, canrapidly identify linked genomic regions to target trait loci in mutationdiscovery58 and has been used in model organisms including Arabidopsisthaliana,60 nematodes,62 zebrafish63–65 and mice.66Most studies using Pool-seq identified regions of mutations based on allele frequency andSNP density, but the SNP-index approaches used in rice were simpler and moreefficient.35–37 Theparental lines used in Pool-seq studies have typically been homozygous, and thus thelinkage relationships among SNPs have been unambiguous. Variant regions have beenidentified by determining which SNP genotypes originated from each parent. A few studiesused Pool-seq without a parental genome sequence because the genome in question was highlyheterozygous or for other reasons. For example, Hill et al.32 used the Euclidean distance to reduce the noiseinherent in RNA sequencing data sets when identifying recessive mutations in zebrafishwithout parental strain information. In the present study, we used the absolute value of∆(SNP-index) to shield against background noise caused by the highly heterozygousparental lines and multiple levels of sampling. Both the introduction of the Euclideandistance and |∆(SNP-index)| reduced the dependence of Pool-seq on parental genomesequences and may thus be used in a wider scope of applications.Whole-genome sequencing is a fast and cost-effective approach for identifying geneticvariants.67 In our study, we were not ableto identify the linkage among filial and parental SNPs because the parental genomes werehighly heterozygous. Estimating the parental origins of the SNPs according to the∆(SNP-index) could generate errors. The positive or negative sign of the∆(SNP-index) may have been opposite to the actual condition, resulting in a∆(SNP-index) with an opposite direction offset within the window, with the∆(SNP-index) value of the window approaching 0, reducing the QTL-seq detectionsensitivity (detectability) of the candidate interval with target phenotypes  for the DNA pool from filial generations based on the genome of one ofthe parental lines revealed the parental origins of the SNPs in a hybrid pool, which areeasy to relate to the target phenotype.enotypes . For exaIf candidate intervals are dispersed on the scaffold, the target trait intervals areinterlaced with non-target trait intervals, which decreases the average value of the|∆(SNP-index)| in a window. In the current study, predicting the interval linkedwith target traits proved impossible. The downstream 2 500 000 bpsection on scaffold NW_008988039.1 fluctuated widely, and the peaks were near thethreshold. Therefore, this section could not be the candidate interval . To avoiet al.13 analyzed sevencrosses in European pears with full red bud sport and green-skinned phenotypes. Theirstudy showed that the former phenotype in European pears was controlled by a singledominant gene, Red. This gene was located between two amplified fragment lengthpolymorphism markers on the LG4 of the ‘Max Red Bartlett’ map, which was13.5 cM from marker E31M56-7 and 18.2 cM from marker E33M48-5.13 In ‘Bayuehong,’ a red-blushed cultivarderived from a cross between ‘Clapp’s Favorite’ and‘Zaosu,’ four QTLs for red skin from ‘Clapp’s Favorite’were on LG4, LG13 and LG16.50 The Redgene was located at 64.0 cM of LG4, whereas Pyb04_016, a QTL for red skin from‘Clapp’s Favorite,’ was at 4.8 cM, indicating that they werefar apart. The present study used the Asian pear cultivars ‘Mantianhong’ and‘Hongxiangsu’ as parental lines. Their red skin trait was inherited from the‘Huobali’ sand pear cultivar and the ‘Korla Xiangli’ Xinjiangpear cultivar. We located the R/G locus in a 582.5 -kb candidate interval at thebottom of LG5. This finding is not in agreement with the findings reported by Dondiniet al.13 and Wu etal.50 These varying mapping resultsindicate that there are different mechanisms for the control and regulation of the redskin trait in Asian and European pears and different types of red-skinned phenotypeswithin European pears.Dondini et al.29 analyzed the segregation of red characters in theprogenies of 10 Asian pear hybrids and suggested that it could be controlled by twodominant gene loci, which is consistent with the findings in apples.68 In our study, the segregation ratios of red to greenwere 2:3 (Mantianhong×Hongxiangsu) and 8:9 (Yuluxiang×Mantianhong), which werelower than the minimum segregation ratio  in thehypothesis assuming two independent dominant gene loci. The ratio of red to green is aslow as 1:1 only when the two loci are closely linked or their Ab|aB alleles arecosegregated, the segregation type of these two loci is hk×hk and the homozygous andheterozygosity genotypes of progeny correspond to the green- and red-skinned phenotypes,respectively. However, in this study, the Asian pear red skin trait loci were in acontinuous interval on the fifth chromosome, and the segregation types of three closelylinked molecular markers with red-skinned traits were nn×np, not hk×hk.In the hybrid group of the red-skinned Asian pears, the coloration of the progeny showedthe character of the alternative quality traits. Volz 69 Deviation from Mendel's geneticseparation in the hybrid progeny of the red-skinned Asian pear with bias toward greenseparation appears to be a common phenomenon 55 and may be a result of the linkage of red skin trait loci withlethal or semilethal gene loci.29 A higherdistorted segregation ratio of molecular markers was also detected when we identified thehybrid progeny of ‘Mantianhong’ and‘Hongxiangsu’.70 Additionalwork is needed to fully understand the genetic regularity of the red-skinned trait ofAsian pears, including analysis of hybrid progeny segregation and genotypes, developmentand application of molecular markers, determination of the mechanism of distortedsegregation and validation of gene function.Our findings support the hypothesis that the trait is controlled by a single dominantgene that tends toward green-skinned segregation. This same confusion was observed inapples. At present, only one locus has been found to control the red-skinned trait inapples.72 An expression sequence tag encoding the transcriptionfactor NAC domain protein was found to be selectively induced by cold in blood oranges butnot in common oranges.73 A report onblood-fleshed peaches suggested that the heterodimer of two NAC transcription factors (BLand PpNAC1) can activate the transcription of PpMYB10.1, resulting in anthocyaninaccumulation, and that BL is the key gene for the blood-flesh trait inpeaches.74 Interestingly, there are two R2R3MYB transcription factor 10 genes in scaffold NW_008988130.1 near the candidateregion (listed in MYB genes and the NAC transcription factors in the candidateintervals merits additional investigation.The color area and degree of redness in the skin of Asian pears are influenced byenvironmental conditions and show the characteristics of quantitative traits. Thestructure gene and transcription factor gene of the anthocyanin biosynthetic pathway playimportant regulatory roles in this process. However, it is unclear if the quality traitgenes that control the coloration of the red-skinned pear are derived from the structuralgene or transcription factor gene of the anthocyanin biosynthetic pathway, and no suchgenes were found in the known gene model within the candidate intervals in this study.Therefore, it is not possible to exclude the presence of a gene that acts as a switch inthe coloration of red-skinned pears upstream of anthocyanin biosynthetic pathway-relatedgenes or transcription factors. The most interesting candidate genes identified within theregion containing the R/G locus were the NAC domain class transcription factor, the WRKYdomain class transcription factor and the ABC transporter B family member 9-like gene,which have all been associated with anthocyanin synthesis and accumulation. The NACtranscription factors play important roles in plant stress responses, flowering andsecondary cell-wall biosynthesis.75 some WRKY family genesplay an important role in regulation of anthocyanin accumulation. For example, the WRKYtranscription factor AtTTG2 has been reported to regulate the accumulation ofproanthocyanidins and anthocyanins in A. thaliana.77 The TTG2-like homolog proteinVvWRKY26 is also involved in the regulation of vacuolar transport and flavonoidaccumulation in grape berries.78 In addition, astudy on A. thaliana photomorphogenesis and root development revealed that twoATP-binding cassette transporter proteins, AtMDR1 and AtPGP1, are required for normalaccumulation of anthocyanins.79 The results ofthe present and previous studies clearly indicate the need for additional and moredetailed work to identify candidate genes associated with anthocyanin synthesis andaccumulation. Molecular analyses should be conducted on more taxa. Gene sequences andexpression should also be compared between red- and green-skinned genotypes. Theseadditional studies will elucidate the genetic control and regulatory processes responsiblefor the red-skinned trait in Asian pears.Although WRKY transcription factors play an important role in the plant stress responseand hormone signaling,