PKD2 gene mutations in human preimplantation embryos in vitro. To achieve this goal, a proband family with a heterozygous deletion of c.595_595 + 14delGGTAAGAGCGCGCGA in exon 1 of the PKD2 gene was studied. A total of 10 samples were analyzed, including 7 embryos. An array-based gene chip was designed to capture all of the exons of 21 disease-related genes, including PKD2. We performed Sanger sequencing combined with targeted haplotyping to evaluate the feasibility of this new method. A total of 7.09 G of data were obtained from 10 samples by NGS. In addition, 24,142 informative single-nucleotide polymorphisms (SNPs) were identified. Haplotyping analysis of several informative SNPs of PKD2 that we selected revealed that embryos 3, 5, and 6 did not inherit the mutation haplotypes of the PKD2 gene, a finding that was 100% accurate and was consistent with Sanger sequencing. Our results demonstrate that targeted NGS combined with targeted haplotyping can be used to identify PKD2 gene mutations in human preimplantation embryos in vitro with high sensitivity, fidelity, throughput and speed.Here, we evaluate the applicability of a new method that combines targeted next-generation sequencing (NGS) with targeted haplotyping in identifying  To perform PGD, a small number of cells are taken from the embryos and are tested for certain genetic changes. Only normal embryos are implanted in the uterus to initiate a pregnancy [http://ghr.nlm.nih.gov/handbook/testing/uses]. Since the world’s first birth of healthy baby girls by PGD in 1990Preimplantation genetic diagnosis (PGD) is a specialized technique that can reduce the risk of having a child with a particular genetic or chromosomal disorder. PGD is used to detect genetic changes in embryos that are created by using assisted reproductive techniques such as PKD2 (MIM:173910) gene is located on the long (q) arm of chromosome 4 at position 22.1. The official name of this gene is “polycystic kidney disease 2 ”. More than 75 mutations in the human PKD2 gene have been identified in patients with polycystic kidney disease. These mutations are responsible for approximately 15% of all cases of autosomal dominant polycystic kidney disease (ADPKD), the most common type of PKD (http://ghr.nlm.nih.gov/gene/PKD2). ADPKD, which is generally a late-onset multisystem disorder characterized by bilateral renal cysts, affects 1 in 500 to 1,000 people. Cyst growth leads to enlarged kidneys, which results in kidney failure. The signs and symptoms of ADPKD typically begin in adulthood, and approximately 50% of individuals with ADPKD have end-stage renal disease (ESRD) by the age of 60 years. Except for hemodialysis and kidney transplantation, which are used in treating ESRD, there is no effective therapy to delay PKD progression (http://www.ncbi.nlm.nih.gov/books/NBK1246/). Therefore, the selection of healthy embryos for uterine transfer by PGD has become the most effective way to avoid ADPKD transmission.The in situ hybridization (FISH), array comparative genomic hybridization (aCGH), single-nucleotide polymorphism (SNP) arrays and next-generation sequencing (NGS). The disadvantages of single-cell PCR and FISH are laborious, expensive and time consuming and usually require a specific locus and pedigree. The aCGH, SNP arrays and NGS are the latest single-cell genomic methodologies with high sensitivity, throughput and speed and can replace PCR and FISHCurrent and emerging technologies for PGD include single-cell PCR, fluorescence Because only one or a few cells from each human preimplantation embryo can be used for embryo biopsy, problems such as contamination, allele drop-out (ADO), amplification failure and preferential amplification (PA) are inevitable in single-cell PCR45de novo segmental copy-number aberrations, de novo base mutations and mitochondrial mutations. NGS has been conducted with high accuracy, good stability, high throughput and lower cost101112In recent years, NGS technology has developed rapidly and has established various applications in the field of medical testing that can be applied to aneuploidy screening, DNA copy-number aberrations, carriership of balanced chromosome rearrangements, single-gene disorders, PKD2 gene mutations in preimplantation embryos from a proband family in vitro. To evaluate this technology, we used the Sanger DNA sequencing method to confirm the results obtained from targeted NGS and targeted haplotyping.Therefore, we considered using NGS to detect mutations with the WGA products of a single cell from human preimplantation. An array-based gene chip was designed to capture all of the exons of several genes of interest associated with heritable genetic disorders. This gene chip also allowed us to obtain information on approximately 2,000 SNP  loci regions around each gene and the X and Y chromosome-specific regions. To avoid of inevitable ADOs, we subsequently combined targeted NGS with targeted haplotyping to identify We obtained 7.09 G of raw data from 10 samples. On average, 93.3% of the data were high-quality data (>Q20 and N ≦ 5), and approximately 78.8% of the data yielded clean reads that could be uniquely matched to the NCBI human genome reference build 37. The average utilization rate of the data was 76.6%. In addition, the average coverage and depth of the targeted region were 92% and 108.7-fold, respectively. On average, we detected 24,142 SNPs .PKD2 gene. Among these sequences, 24 were informative SNPs, whereby the SNP genotype of father was heterozygous, and both the paternal grandmother and mother were homozygous but had different SNP genotypes. The remaining 99 informative SNPs had the same SNP genotypes. The results of the targeted NGS and the haplotype analysis of the pedigree are shown in For our analysis, we selected SNP loci in which the husband was heterozygous and both the mother and wife were homozygous. The pedigree of the family was shown in Seven informative SNP candidates were selected from the SNPs in targeted NGS  to perform Sanger DNA sequencing. However, only 5 informative SNPs  were stable and were of high quality for haplotype analysis in embryos. The results from Sanger DNA sequencing of the 5 SNP informative loci are shown in As illustrated in PKD2 gene c.595_595 + 14delGGTAAGAGCGCGCGA are shown in PKD2 and in combination with haplotype analysis is more effective for PGD.The results of direct mutation detection of the PKD2 gene in 7 embryos were consistent with the results of the traditional Sanger sequencing method. We demonstrated that targeted NGS combined with targeted haplotyping could be used in the identification of PKD2 gene mutations in human preimplantation embryos in vitro. Because our capture custom array contained thousands of SNPs with a wide distribution across the genome, it was very suitable for haplotype analysis without having to screen for genetic markers in different people. The analysis is also automated. In addition, we designed X and Y chromosome-specific regions in our chip for the sex identification of some X-linked genetic diseases. In doing so, we accidentally found that embryo 6 was missing an X chromosome, a finding that was consistent with the results of aCGH. This finding suggested that we should focus on the X and Y chromosome-specific regions to avoid sex chromosomal aneuploidy abnormalities in embryo screening. In addition, the method we used in our research is also suitable for the detection of a large number of samples from multiple different families with diseases in a single experiment. These samples can be simultaneously sequenced as long as each sample is ligated to a different library adapter. Each gene chip can capture more than 10 samples, and each sample only requires approximately 1 GB of sequencing data. However, this new method requires approximately one week to complete sequencing and data analysis, which cannot be used for fresh embryo transfer.In this study, we used targeted NGS to perform tests on 10 samples from one family and analyzed the data with targeted haplotyping using the informative SNPs as genetic markers. The haplotypes of the PKD2, targeted NGS combined with targeted haplotyping can be used in other single-gene disorders for PGD, such as DMD or PKD1  to extract the genomic DNA. The cell from the embryo used for PGD was biopsied from a 6- to 8-cell human cleavage-stage embryo on day three after IVF. Then, we used laser-assisted microdissection of the zona pellucida for embryo biopsy and performed WGA with a Qiagen WGA kit (150345#REPLI-g Single Cell Kit (96), QIAGEN/A) as soon as possible.A custom capture array  was used for the PGD of single-gene disorders. The total capture region was 3.5 M, including 21 common genes associated with genetic disorders. The capture region of this gene chip was divided into three parts. The first part was the coding region, including the alternative splice region and other gene regions associated with genetic diseases. This grouping was applied to direct mutation detection. The second part was a region with an approximately 2,000 SNP (MAF > 0.3) locus within a 2 M range on each side of the targeted gene. The SNPs (MAF > 0.3) were derived from the international 1000 Genomes Project  databaseEscherichia coli DNA polymerase to fill 5′ overhangs and remove 3′ overhangs. Terminal A residues were added following a brief incubation with dATP and the Klenow 3′–5′ exo-enzyme using standard Illumina protocolsFragmentation of the DNA into 200- to 300-bp pieces was completed using an ultrasonoscope . Next, 1 μg of purified DNA (quantified via NanoDrop) was treated with T4 DNA polymerase, T4 phosphonucleotide kinase and the Klenow fragment of the After sequencing, the primary data were obtained using the Illumina Pipeline, and the indexed primers were used to identify the different reads from different samples in the primary data. Only the reads that were perfectly matched to the theoretical adapter-indexed sequences and reads that matched the theoretical primer-indexed sequences with a maximum of three mismatches were considered to be acceptable reads. We then removed a few unqualified sequences from the primary data using a local dynamic programming algorithm, which included low-quality reads, which were defined as reads that contained more than 10 percent Ns in the read length, 50% reads with a quality value of less than 5 and with an average quality of less than 10 and adapter sequences that included the indexed sequence. The remaining sequences were considered to be clean reads for further analysis.29 and the GATK Indel Genotyper , respectively. Previously identified SNPs were determined using the NCBI dbSNP or HapMap databases. Known disease-causing mutations were identified using the Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff  or from mutations previously reported in the literature.The clean reads with a length of 90 bp were then compared to the reference human genome from the NCBI database (Build 37) using the BWA (Burrows Wheeler Aligner) Multi-Vision software packageSNPs from the paternal and maternal haplotypes were analyzed to identify informative SNP loci. Informative SNP loci were those loci in which the SNPs of the two parents were in the same location and that one parent was homozygous and the other was heterozygous. This method was used to identify unique haplotypes. The informative SNPs were then assembled to form a unique haplotype according to the SNP information from the child’s samples based on Mendelian analysis. According to the linked informative SNPs of the embryo, we could identify the paternal or maternal origin of each haplotype of the embryo and map the inheritance of these haplotypes. In addition, we could determine whether the embryo inherited the mutation haplotype . FurtherHow to cite this article: Chen, S.-C. et al. Identification of PKD2 mutations in human preimplantation embryos in vitro using a combination of targeted next-generation sequencing and targeted haplotyping. Sci. Rep. 6, 25488; doi: 10.1038/srep25488 (2016).


Verticillium dahliae. On the one hand, V. dahliae is a haploid organism that reproduces clonally. On the other hand, single-nucleotide polymorphisms and chromosomal rearrangements were found between V. dahliae strains. Lineage-specific (LS) regions comprising about 5% of the genome are highly variable between V. dahliae strains. Nonetheless, it is unknown whether horizontal gene transfer plays a major role in generating genetic variation in V. dahliae. Here, we analyzed a previously sequenced V. dahliae population of nine strains from various geographical locations and hosts. We found highly homologous elements in LS regions of each strain; LS regions of V. dahliae strain JR2 are much richer in highly homologous elements than the core genome. In addition, we discovered, in LS regions of JR2, several structural forms of nonhomologous recombination, and two or three homologous sequence types of each form, with almost each sequence type present in an LS region of another strain. A large section of one of the forms is known to be horizontally transferred between V. dahliae strains. We unexpectedly found that 350 kilobases of dynamic LS regions were much more conserved than the core genome between V. dahliae and a closely related species , suggesting that these LS regions were horizontally transferred recently. Our results support the view that genetic variation in LS regions is generated by horizontal transfer between strains, and by chromosomal reshuffling reported previously.There are major gaps in the understanding of how genetic variation is generated in the asexual pathogen  Verticillium dahliae is strictly asexual . We found highly homologous elements in LS regions of each strain; LS regions of V. dahliae strain JR2 are much richer in highly homologous elements than the core genome. We also found unexpectedly that 350 kilobases of dynamic LS regions were much more conserved than the core genome between V. dahliae and a closely related species , suggesting that these LS regions were horizontally transferred recently. In addition, we discovered, in LS regions of JR2, several structural forms of nonhomologous recombination, and two or three homologous sequence types of each form, with almost each sequence type present in an LS region of another strain. A large section of one of the forms is known to be horizontally transferred between V. dahliae strains. We determined the mating type of each V. dahliae strain to study the relationship between the mode of reproduction and the extent of variation between strains.There are major gaps in the understanding of how genetic variation is generated in the asexual pathogen Recombination between homologous chromosomal segments is called homologous recombination, whereas recombination between nonhomologous ones is called nonhomologous recombination. For nonhomologous recombination, the sequences of two chromosomal segments may differ for most of their lengths, but the fragments at the sites of recombination may be similar. A structural form refers to a sequence structure produced by nonhomologous recombination. The structure consists of two sections combined at the site of recombination.Short DNA sequences are assembled into sets of overlapping sequences called contigs. A supercontig is a set of contigs ordered and oriented with respect to one strand of the genome. A complete set of supercontigs is called an assembly. After an assembly for a strain is produced, SNPs between this reference strain and other strains can be found by generating much shorter sequences called reads and mapping the reads onto the reference assembly for each of the other strains. Note that there is no biological basis for the notion of reference strain; the reference strain is introduced because of the limitations of the current genome sequencing technology.V. dahliae strains were downloaded from the NCBI Sequence Read Archive (SRA) under BioProject PRJNA169154  of concordantly mapped pairs of paired-end reads and a significant number of discordantly mapped pairs of paired-end reads. Such a region was the site of recombination for a potential nonhomologous recombination event. Genes in recombination forms were found by performing database searches with Blastx ; transpoV. dahliae were compared to the VdLs17 and VaMs102 genome assemblies with the AAT package , CG (20), and TA (22) in strain JR2, formed by two close type 2 SNPs T/C  and G/A (56/30) at positions 1,033,835 and 1,033,865 bp of VdLs17 supercontig 1.9. After observing the high coverage depths (80 and 86) by reads for the two type 2 SNPs, we found that 64 was the average depth of coverage for the 612 type 2 SNPs in LS regions, 25 was the average depth for the 261 type 2 SNPs in the core genome, and 28 was the average depth for the 3031 type 1 SNPs in the whole genome. In sum, the type 2 SNPs in the core genome were dispersed with low depths of coverage in sequence contexts of extended runs of a single base. In contrast, the type 2 SNPs in the LS regions were concentrated with high depths of coverage often in sequence contexts without extended runs of a single base. This contrast revealed a major difference in the chromosomal structure of JR2 between the core genome and LS regions: the LS regions are rich in highly similar but unidentical sequences, whereas the core is poor in such sequences. In the above example, the three types of highly similar sequences were found in the LS regions of JR2. To see whether the three sequence types were present in the other strains, we examined the read mappings for the other strains at the two positions of VdLs17 supercontig 1.9. Only the sequence type TA  was found in strain St.100, formed by the REF allele T (27) and a type 1 SNP G/A (0/22) at the positions, and only the sequence types TG (23) and CG (22) were found in strain DVD-s26, formed by a type 2 SNP T/C (29/35) and the REF allele G (65) at the positions. Both TG (16) and CG (43) were found in strain St14.01, as in DVD-s26. Only the type CG (15 to 25) was found in strains DVD-3, DVD-31, DVD-161 and DVD-s29. There was no coverage of the region by reads from DVD-s29. Similarly, another example with type 2 SNPs at positions 615,713 and 615,747 bp of VdLs17 supercontig 1.9 illustrates that JR2 was composed of three types of highly similar sequences, with one or two out of the three types found in the other strains. In summary, the type 2 SNPs in LS regions are often in sequence contexts without extended runs of a single base, have a much higher average depth of coverage than the type 2 SNPs in the core genome and the type 1 SNPs in the whole genome, and are mostly due to the presence of at least two types of highly similar sequences in LS regions, not a result of sequencing errors in reads.V. dahliae population, and DVD-s26 is also far distant from JR2 . The AVE1 gene is located in a 51.4-kb section of an LS region of JR2 chromosome 5 (CM001867). The 51.4-kb section is highly conserved between JR2 and St14.01  of the 51.4-kb section. In the read coverage of the 5.6-kb section, 28 type 2 SNPs were found, revealing at least three types of highly similar sequences. In addition, the 5.6-kb section is also highly conserved between JR2 and St14.01, as indicated by 24 common type 2 SNPs in the 5.6-kb section and two common types of sequences between the two strains; the average depth of coverage by reads from St14.01 was 39 over the 51.4-kb section and 51 over the 5.6-kb section. For example, three sequence types AGC (linked by 20 reads), ACT(17) and GGC (21) were found in the JR2 read coverage of a 33-bp fragment in the 5.6-kb section, formed by three close type 2 SNPs A/G (55/28), G/C (66/20) and C/T (61/21) at positions 691,591, 691,617 and 691,623 bp of JR2 chromosome 5. Only two types AGC (17) and ACT(21) were found in the St14.01 read coverage of the fragment, formed by REF allele A, type 2 SNP G/C (23/21) and type 2 SNP C/T (21/21) at the three positions of JR2 chromosome 5. An important observation about multiple types of highly similar sequences in JR2 LS regions, as revealed by type 2 SNPs in JR2 read coverages, is that one of such sequences was linked to a horizontal gene transfer into the JR2 lineage, through the physical connection between the 51.4-kb section and the 5.6-kb section.V. dahliae strains, some of them are more conserved than the core genome between V. dahliae and V. albo-atrum. A database search using Blastx  of JR2 chromosome 5, to a region (form 2) of VaMs102 supercontig 1.19, to a region (form 3) of VaMs102 supercontig 1.20, or to an initial section of form 2 combined with a terminal section of form 1 (form 4). Forms 1 through 4 are shown in A comparison of the four structural forms revealed that they resemble results produced by nonhomologous recombination. Forms 1, 2 and 4 share nearly identical 320-bp segments (with at most one nucleotide substitution) at their sites of recombination; form 3 has a 280-bp segment at the site of recombination that is similar to the 3′ portion of the 320-bp segment with 11 nucleotide substitutions and with no gaps. Note that the length of the common segment in each form is at least 180 bp smaller than the average insert size of 500 bp for paired-end reads. In addition, each of forms 1 through 3 has a unique section, and form 4 has a unique combination of sections. Thus, although a read completely inside the common segment can have matches to more than one form, a pair of paired-end reads (read pair) outside and across the common segment can not have matches to more than one form. On the basis of these observations, the read pairs with both reads mapped onto the sections of a form outside and across its common segment were used as evidence to support the presence of the form in the reads. In particular, form 4 was found to be present in the reads of JR2 solely based on read pairs; form 4 was not found in any of the three assemblies as a reference. For each form, the number of read pairs that support the form, along with the 5′ position of the common segment in the reference sequence, is given in V. dahliae population, forms 2 and 3 probably occurred before form 4, which shares the RT section with form 2. Then, form 1 with the AVE1 section was found only in JR2 and St14.01, with only one SNP between JR2 and St14.01 in the AVE1 section, as indicated previously. Forms 1 and 4 share the BRCT1 section as well as the site of recombination. Thus, form 1 was probably produced recently by nonhomologous recombination from form 4. Next, form 1 was linked to a horizontal transfer event involving the AVE1 gene. Forms 2 and 3 were parts of LS regions horizontally transferred between V. dahliae and V. albo-atrum, as indicated in the previous section. Finally, forms 1, 3 and 4 all contain a BRCT domain involved in cell cycle checkpoint functions responsive to DNA damage  was mostly covered by concordantly mapped reads from St.100. The region was searched for SNPs and was found to contain only one SNP. Thus, the region is remarkably conserved between the two species, considering a genome-wide SNP rate of 2.99 per kb between strains St.100 and VdLs17 of the same species.V. dahliae strains. Four base positions in form 3 were found, at each of which two alleles occurred in the JR2 read coverage. For example, consider position 40,089 bp of VaMs102 supercontig 1.20, one of the four positions in form 3. The REF allele A at this position was confirmed by 43 concordantly mapped reads from strain St.100. However, a type 2 SNP A/C (58/34) was found in the JR2 read coverage of this position with 89 concordantly and 3 discordantly mapped reads. Interestingly, only the ALT allele (C) was observed in the DVD-s26 read coverage of this position with 24 concordantly and 7 discordantly mapped reads, and in the St14.01 read coverage of this position with 19 concordantly and 5 discordantly mapped reads. The same kind of observation was made at each of the three other positions of form 3  of the VdLINE1 section are present in JR2. Only the REF sequence type, probably the ancestral sequence type, is present in St.100; only the ALT sequence type is in St14.01 and DVD-s26. Each REF allele is present in a large number of reads in JR2 & St.100, and so is each ALT allele in JR2, St14.01 & DVD-s26. Thus, it is unlikely that these alleles are the result of sequencing errors. These observations show that the strains differ in sequence type composition.Similar observations were made with 12 SNPs in form 2 . AlthougV. dahliae strain DVD-s29; the site of recombination and nearby sections of each form were sparsely covered by discordantly mapped reads from DVD-s29. The same observation was made for each of strains DVD-3, DVD-31, DVD-161 and DVD-s94, which form a separate cluster , 82 (DVD-31), 160 (DVD-161), and 199 (DVD-s94). The four strains in the cluster share 60 type 2 SNPs . Of thesV. dahliae strains except DVD-s29 have the same mating type, supports that many of the REF and ALT alleles were present during the period of sexual reproduction and that the strains in the cluster or their ancestors acquired their REF or ALT alleles at the positions of these SNPs by exchanging DNA over LS regions with other strains of the same mating type.Then we checked if any of the sequence type variations is present outside the cluster. None of the 50 type 2 SNPs was found in strain St.100. Only one of the 50 type 2 SNPs  is shared by strains St14.01, JR2, DVD-s26 and DVD-s29, indicating that the corresponding sequence type variation is conserved in these strains outside the cluster. The SNP occurred in the coding region of a short new hypothetical protein (of 53 residues) with 83% percent identity to hypothetical protein VDBG_10182; it was a synonymous substitution at the third base of the codon for residue 52. Another of the 50 SNPs  is shared by strains JR2 and DVD-s26. A third of the 50 SNPs  was found in strain JR2. None of the 50 type 2 SNPs was found as type 1 in any strain except DVD-s29, indicating that the sequence types in strains St14.01, St.100, JR2 and DVD-s26 contain no ALT alleles at the positions of all or most of the 50 SNPs, so the REF allele at each position was the ancestral allele for the cluster. On the other hand, 7 of the 50 type 2 SNPs were found as type 1 in strain DVD-s29; the sequence types in DVD-s29 contain only the ALT alleles at the positions of these 7 SNPs. Note that for each of the 50 type 2 SNPs, each strain in the cluster has one sequence type with the REF allele and another sequence type with the ALT allele at the SNP’s position. For each of the 7 SNPs, strain DVD-s29, unlike any strain in the cluster, contains only the sequence type with the ALT allele at the position of the SNP. This observation, combined with the information (given below) that all V. dahliae strain VdLs17 and V. albo-atrum strain VaMs102. When the VaMs107 assembly instead of the VdLs17 assembly was used as the reference, 43 type 2 SNPs in the LS region of VaMs102 supercontig1.19 were shared by all the four strains in the cluster. Of the 43 type 2 SNPs, 42 are on the list of the 50 type 2 SNPs. This was unexpected, considering that the average nucleotide identity of the VdLs17 and VaMs102 genomes is about 92%  in 1994 (SNPs between reference strain VdLs17 and other  in 1994 . The gro in 1994 . Althoug in 1994 , the numSNPs were used to find a potential homologous recombination event in the past. A large variation in the number of SNPs unique to DVD-s26 or DVD-s29 was found in two adjacent regions with their common border at position 400,000 bp of VdLs17 supercontig1.3. The region of length 70 kb immediately upstream of the border contains 52 SNPs unique to DVD-s26, 7 SNPs unique to DVD-s29, and 2 SNPs common to both strains. On the other hand, the region of length 100 kb immediately downstream of the border contains 3 SNPs unique to DVD-s26, 4 SNPs unique to DVD-s29, and 14 common SNPs. The variation in the numbers of common and unique SNPs indicates a potential recombination event in the core genome between DVD-s26 and DVD-s29.V. dahliae switched from the past mode of sexual reproduction to the present mode of asexual reproduction to become adaptable to hundreds of plant species. Here, we investigated the genetic variation in a V. dahliae population with both mating types from different geographical locations. Our results show that asexual reproduction is divided into two main modes of reproduction: clonal and aclonal. Aclonal reproduction generates genetic variation through horizontal recombination, a nonsexual mechanism involving horizontal transfer and chromosomal reshuffling of which a special case is nonhomologous recombination. Evidence for genetic variation in the form of multiple types of homologous sequences was found in the sequence reads of every V. dahliae strain. The finding of the low SNP rate between some of the strains in the core genome confirms that they propagate by asexual reproduction at the present time (The prevailing wisdom concerning asexual reproduction is that strictly asexual organisms lack mechanisms to produce genetic variation and cannot adapt to changing environments. However, this view cannot explain why ent time . Becauseent time , horizonV. dahliae had produced significant genome-wide diversity among strains. On the other hand, aclonal reproduction generates a significant depth of localized variation by horizontal recombination. The current aclonal reproduction in V. dahliae has produced both point and structural changes in LS regions in the same strain. The finding of a conserved LS region in V. albo-atrum strain VaMs102 and the finding of common types of sequences in DVD-s26, St14.01 and JR2 suggest that LS regions are transferred between strains and between species. The diversity among strains in the core genome and homologous sequences in the LS regions of the same strain might be what V. dahliae needs to become adaptable to hundreds of plant species.Aclonal reproduction complements sexual reproduction by generating homologous sequences with single-nucleotide and structural differences in LS regions. Sexual reproduction produces, by random mutation and homologous recombination, genome-wide variation. The past sexual reproduction in The finding of multiple types of homologous sequences in the LS regions of the same strain provides support for balancing selection. Each of three of the four recombination forms in strain JR2 was found in another strain or species, so the form was viable on its own. In addition, multiple sequence types of a recombination form were found in the same strain. Multiple types of homologous sequences were conserved in a cluster of four strains. Possible explanations for maintaining multiple types of homologous sequences in the same strain are that they have a higher adaptive value than any single type of sequence and that this benefit is achieved without increasing the genome size significantly. Note that multiple types of homologous sequences are limited to smaller LS regions; the larger core genome lacks such sequences. The lack or shortness of a diploid phase in this asexual fungus and the constraint on its genome size might have caused the fungus to come up with the current strategy for achieving some benefits of a diploid organism.V. dahliae was reported (Magnaporthe oryzae (V. dahliae generates genetic variation mostly in LS regions; lack of type 2 SNPs in the core genome indicates that no horizontal recombination took place in the core genome. In addition, our results confirm that horizontal recombination is commonly used in nature to produce genetic variation. As LS regions are known to harbor effector genes (Our study provides evidence at the nucleotide level for mitotic genetic exchange known as parasexual cycle. Evidence for the parasexual cycle in laboratory populations of reported . The hypreported . Althougreported , an isole oryzae . Parasexor genes , our stuOur results raise the possibility that recombination between DNA molecules is a nearly universal mechanism for generating genetic variation in reproduction. There are two complementary types of recombination: recombination between homologous chromosomes in sexual reproduction and recombination between nonhomologous segments in aclonal reproduction. Homologous recombination occurs within the same species and generates slightly different recombinant chromosomes, making it suitable for gradual evolution in stable environments. Nonhomologous recombination, on the other hand, can occur between different species, and generates hugely different recombinant segments, making it suitable for rapid adaptation to changing environments.V. dahliae strains shows that every strain has its unique combination of homologous sequences in LS regions. We speculate that the unique combination of homologous sequences in LS regions provides the genetic variation needed for the adaptation of the strain to its environment. Genetic variation is generated in V. dahliae by horizontal transfer and incorporation of small LS elements into chromosomes. In contrast, genetic variation in Fusarium oxysporum is generated by horizontal transfer of whole LS chromosomes (Fusarium oxysporum can be twice as large as that of V. dahliae. It remains to be seen whether the mechanism for generating genetic variation in V. dahliae is used in other filamentous pathogens such as Phytophthora infestans (The analysis of the genetic variation in the omosomes . For thinfestans .The difference in the rate of type 2 SNP between the core genome and LS regions can be used to check on the neutral theory of molecular evolution  which clV. dahliae population and V. albo-atrum strain VaMs102, with both species being strictly asexual plant pathogens. Our results revealed that LS regions are highly enriched for homologous sequences, whereas the core genome lacks this feature. In particular, the LS regions of V. dahliae strain JR2 are comprised of homologous sequences with single-nucleotide differences and with various structural forms like those produced by nonhomologous recombination. Some of the sequence types and structural forms in JR2 were found in other V. dahliae strains or the V. albo-atrum strain. The extraordinary conservation of some homologous sequences in LS regions between V. dahliae strains and between the two species, as compared to that in the core genome, suggests that those homologous sequences were acquired by horizontal transfer, consistent with a previous finding of a horizontal transfer event (V. dahliae (V. dahliae LS regions is generated by horizontal transfer and chromosomal reshuffling between strains and between species. Understanding the evolutionary mechanism of LS regions is an important step in finding an effective way of controlling V. dahliae.We have performed an analysis of genome sequence data from a er event . Buildin dahliae , we conc10.7717/peerj.650/supp-1Table S1Click here for additional data file.


Arabidopsis halleri and associations with climatic variation among populations in the Alps. Using a pooled population sequencing (Pool-Seq) approach, we discovered more than two million SNPs in five natural populations and identified highly differentiated genomic regions and SNPs using FST-based analyses. We tested only the most strongly differentiated SNPs for associations with a nonredundant set of environmental factors using partial Mantel tests to identify topo-climatic factors that may underlie the observed footprints of selection. Possible functions of genes showing signatures of selection were identified by Gene Ontology analysis. We found 175 genes to be highly associated with one or more of the five tested topo-climatic factors. Of these, 23.4% had unknown functions. Genetic variation in four candidate genes was strongly associated with site water balance and solar radiation, and functional annotations were congruent with these environmental factors. Our results provide a genomewide perspective on the distribution of adaptive genetic variation in natural plant populations from a highly diverse and heterogeneous alpine environment.Natural genetic variation is essential for the adaptation of organisms to their local environment and to changing environmental conditions. Here, we examine genomewide patterns of nucleotide variation in natural populations of the outcrossing herb  Studies of genes and genomic regions underlying adaptation are at the heart of ecological genomics  is effective for SNP discovery and provides accurate estimates of allele frequencies  O'Kane & Al-Shehbaz  and higher, including mountain slopes at more than 2300 m asl, grassy meadows, forest margins and rocky crevices  what proportion of SNPs show highly elevated differentiation across the studied populations, 2) what functional categories are overrepresented among the genes containing highly differentiated SNPs, 3) what proportion of these genes are associated with the studied abiotic topo-climatic factors, and 4) whether such genes also have functions consistent with their associations with environmental factors.In the present study, we investigated geographically close natural populations of A. halleri were collected from five locations in the south-eastern Swiss Alps representing a wide variation in environmental factors  were extracted for 21 topo-climatic factors collected over a 30-year period (1961–1990) at 25-m resolution (Zimmermann & Kienast r) with IBM SPSS Statistics 19 to remove highly correlated factors (|r| ≥ 0.8). This led to the selection of five topo-climatic factors, describing precipitation (PRECYY), slope (SLP25), solar radiation (SRADYY), site water balance (SWB) and temperature . DNA quality was checked on 1.5% agarose gels stained with GelRed (Biotium) on a UV-Vis Spectrometer . DNA quantity was measured with a Qubit fluorometer . Equal amounts of high-quality DNA from 20 individuals per population were then pooled, resulting in a pool size of 7 μg RNA-free genomic DNA per population. Library preparation  and sequencing on an Illumina HiSeq2000 were performed by GATC Biotech .http://hannonlab.cshl.edu/fastx_toolkit). Files containing forward and reverse reads were resynchronized with an in-house perl script, and only paired sequences were used for further analysis.Forward and reverse reads were screened for tags and adaptors and trimmed with CUTADAPT Martin . Phred-tA. thaliana reference genome  and ambiguously mapped reads (2.54–2.97%) were rare. The latter were removed before the remaining high-quality reads were sorted with SAMTOOLS v0.1.18  was calculated with fst-sliding.pl in POPOOLATION2 according to Hartl and Clark  of single SNPs for each pairwise population comparison by adding or subtracting the deviation of the pairwise comparison from the mean neutral population differentiation based on all >2 million SNPs , this provided only a weak correction but may reduce the rate of false positives as a consequence of population structure  for identifying highly differentiated SNPs to reduce the rate of false positives.Highly differentiated SNPs were identified in a three-step procedure. First, genetic differentiation (nd Clark  using a http://www.arabidopsis.org) and NCBI (http://www.ncbi.nlm.nih.gov) reference databases containing 33 323 genes . We identified 25 764 genes in our A. halleri data set that fulfilled these criteria for at least part of each gene.To assess the number of genes covered by our Pool-Seq approach, we used the mpileup file produced for SNP identification, filtered for all positions that had a minimum coverage of 20x for each of the five populations and annotated them using an in-house perl script with the TAIR10  as a threshold criterion. We first performed partial Mantel tests between 20 000 matrices with simulated pairwise FST values  and the five environmental factors , again controlling for population structure using  genomewide pairwise FST. Then, we used the 99% quantile of the resulting 100 000 r values (i.e. 0.7573) as our threshold for r. Above this threshold, a specific SNP locus was considered to be associated with the respective environmental factor. In addition to counting the number of associations between environmental factors and individual SNPs, we assessed the numbers of associations with particular genes, that is, those that could be annotated to the A. thaliana genome (see above) and for which we found at least one SNP being associated with a particular environmental factor.Partial Mantel tests were performed with Ecodist 1.2.7 (Goslee & Urban The top candidate genes for associations with climate were selected from Gene Ontology (GO)-categories with biological functions relating to our five abiotic environmental factors, including response to temperature stimulus (GO:0009266), water stimulus (GO:0009415), osmotic stress (GO:0006970) and radiation (GO:0009314). Genes without significant association with at least one environmental factor were removed. This stringent approach did not identify genes associated with biotic factors or some of the excluded topo-climatic factors, as well as genes with unknown functions, but allowed us instead to identify candidate genes whose functions correspond to the environmental factors they are associated with.A. thaliana counterparts, and noncoding regions were removed using an in-house workflow and the MAFFT sequence alignment program . These represent the consensus multilocus genotype for each candidate gene and population and were created by an in-house perl script, taking the nucleotide at each position with the highest frequency within a population pool (i.e. >50%). MACHs were aligned to their et al. A. halleri genes were used as background reference. Significance for each individual GO-identifier was computed with Fisher's exact test and a significance threshold of 1%. As GO terms are nonindependent, we used the ‘elim’ method in topGO, which integrates GO graph topography to improve enrichment analysis by iteratively removing genes mapped to significant GO terms from higher  terms and reduces the rate of false positives  with a median Phred-score of 37 . This corresponds to an average 99x coverage per population pool.A. halleri reads to the A. thaliana reference genome with low variation between the five population pools (range: 17.9–18.7%). One reason for this moderate proportion of mapped reads is that the A. halleri genome is more than twice the size of the A. thaliana reference genome. Consequently, we can expect to map less than 50% of our reads. Further, we discarded all reads mapping to organellar genomes (9.2–14.6% of the reads depending on the population), leaving approximately 35% of all reads. Because of the divergence between the two species, the remaining reads mapped primarily to conserved genic regions rather than to variable intergenic regions, resulting in ∼18% of mapped reads.We could unambiguously map 18.3% of all FST among populations based on all polymorphic SNPs was moderate, with an average of 0.038 and a range of 0.026 (Aha09 vs. 31) to 0.048  is shown along a 5 Mb example section of chromosome 2 .Using the three-step procedure presented above, we identified 4282 strongly differentiated SNPs located within the 0.1% most strongly differentiated genomic regions. This corresponds to only 0.2% of all SNPs in the data set. Average sequence coverage for these SNPs was 60.3x (range: 51.5–70.3x). 204 SNPs could not be annotated and may be located in intergenic regions. The remaining SNPs resided in 571 unique genes , 139 (24.3%) of which are assigned unknown function in TAIR10. The 0.1% most strongly differentiated sliding windows contained on average 12.96 SNPs. Polymorphism in these windows was thus significantly reduced compared with the average of 20.3 SNPs across all windows  showed A. halleri . Of these, four genes had a clear association with environmental factors directly related to their putative biological function and were therefore considered as top candidate genes for adaptation to the abiotic environmental conditions studied in P-GLYCOPROTEIN 1 (PGP1)/ATP-BINDING CASSETTE B1 (ABCB1) (AT2G36910), contained 64 strongly differentiated SNPs, and the second, ATGLR3.6 (AT3G51480), contained 25 strongly differentiated SNPs (TableGLUTATHIONE PEROXIDASE 3 (GPX3) (AT2G43350), contained ten strongly differentiated SNPs that were all associated with SWB, and the second, LOW OSMOTIC STRESS 5 (LOS5)/ABA DEFICIENT 3 (ABA3) (AT1G16 540), contained a single strongly differentiated SNP associated with SWB (TableTwo of these genes in the GO category ‘response to radiation’ were associated with radiation (SRADYY). The first, ABCB1) ATG36910, cABCB1) ATG36910, cFST and sliding-window analyses and environmental association plots are shown in Fig.PGP1 and for the three other top candidate genes in GPX3 and ATGLR 3.6 were among the highly differentiated SNPs that are associated with the corresponding environmental factors.Results of the Among the 571 genes containing strongly differentiated SNPs, we identified 16 significantly overrepresented biological processes Table. ResponsA. halleri. Five geographically close populations from habitats characterized by diverse and distinct abiotic environmental conditions were investigated using a Pool-Seq genome resequencing approach. We observed more than 2 million SNPs among the populations and found a complex and highly diverse footprint of selection across the A. halleri genome. In a stepwise approach that included the identification of highly differentiated SNPs and genomic regions, environmental associations and GO term analyses, we identified a set of candidate genes associated with climatic variation. For four genes, genetic differentiation between populations and population allele frequencies, as well as functional annotations, was congruently associated with solar radiation and water availability. Our results are consistent with a scenario in which these genes are associated with adaptation to local abiotic environmental conditions in the investigated A. halleri populations and provide testable hypotheses for further experimental analyses of the ecological role of these genes.We studied genomewide polymorphisms in association with climatic variation in alpine populations of the emerging model plant et al. et al. A. halleri by Rellstab et al. (in press), where the average difference in population allele frequencies derived from pooled or individual samples was only 3.8% ± 0.8 SE.Pool-Seq genome resequencing used in this study was a cost-effective and powerful approach to assess genetic variation in natural populations and to identify genes that are potentially under selection, as previously suggested by both theoretical . Consequently, FST for these SNPs was about 15 times higher than the average genomewide value (FST = 0.038). Moreover, SNPs of interest had to be located in highly differentiated genomic regions identified through sliding-window analysis. We thus focused on 4282 strongly differentiated SNPs that represented only 0.2% of all the SNPs detected and were located in 571 genes. The more than two million SNPs detected in this genome resequencing study therefore allowed us to be much more conservative than earlier genome scan studies based on AFLPs or SSRs, which often inferred 5–10% of markers to be under selection , as well as solar radiation and temperature, affected adaptation in the A. halleri populations studied . Gene LOS5/ABA3, associated with ‘osmotic stress’, is involved in the synthesis of the plant hormone ABA and appears to function in regulation of osmotic stress response , which provides the mechanistic basis for hitchhiking, is expected to be low. Our observation that patterns of elevated genetic differentiation often decay after a few thousand base pairs argues against hitchhiking (e.g. see Fig.A. halleri is available.We hypothesize that genetic variation in the top candidate genes identified in this study has been shaped by natural selection. Alternatively, the identified genes could be hitchhiking genes (Strasburg et al. It is generally difficult to assess the potential role in adaptation of genes identified in population genomic analyses. To illustrate this point, more than 23% of the genes in our study containing highly differentiated SNPs have unknown functions, even in the most recent TAIR update (Lamesch A. halleri. The genomic distribution and assigned functions of the identified genes were as heterogeneous as the environmental conditions under which the sampled populations live. This may indicate that the evolution of geographically proximate populations that are potentially connected by gene flow, but are adapted to local environmental conditions, can have a complex genetic basis. Ideally, studies of adaptation should not solely rely on genetic variation, even on a genomewide scale, but should include experiments to study gene function at the molecular level and field transplant experiments to assess the adaptive value of candidate alleles and genes under realistic environmental conditions. This study helps formulating testable hypotheses for future experimental analyses of the ecological role of the identified genes in natural populations of alpine plants.The study of plant adaptation and genome evolution is an emerging field that is developing rapidly and offers countless opportunities to investigate adaptive processes. Here, we used a stepwise approach to identify genomic regions and candidate genes potentially involved in adaptation. The identified genes showed footprints of selection and were correlated with environmental factors that differed between sites, as expected under a scenario of environment-mediated selection in natural populations of


Taurine cattle breeds in Burkina Faso are trypanotolerant. Zebuine cattle, which are also kept there are susceptible to trypanosomosis but bigger in body size. Farmers have continuously been intercrossing Baoule and Zebu animals to increase production and disease tolerance. The aim of this study was to compare levels of zebuine and taurine admixture in genomic regions potentially involved in trypanotolerance with background admixture of composites to identify differences in allelic frequencies of tolerant and non-tolerant animals. The study was conducted on 214 animals , genotyped with 25 microsatellites across the genome and with 155 SNPs in 23 candidate regions. Degrees of admixture of composites were analyzed for microsatellite and SNP data separately. Average Baoule admixture based on microsatellites across the genomes of the Baoule- Zebu composites was 0.31, which was smaller than the average Baoule admixture in the trypanosomosis candidate regions of 0.37 (P = 0.15). Fixation index FST measured in the overall genome based on microsatellites or with SNPs from candidate regions indicates strong differentiation between breeds. Nine out of 23 regions had FST ≥ 0.20 calculated from haplotypes or individual SNPs. The levels of admixture were significantly different from background admixture, as revealed by microsatellite data, for six out of the nine regions. Five out of the six regions showed an excess of Baoule ancestry. Information about best levels of breed composition would be useful for future breeding ctivities, aiming at trypanotolerant animals with higher productive capacity.Trypanosomosis is a serious cause of reduction in productivity of cattle in tsetse-fly infested areas. Baoule and other local  Trypanosoma parasites: T. congolence, T. vivax, and T. brucei. Trypanosomosis is responsible for the deaths of millions of livestock each year and a reduction in the productivity of many more. With no vaccine available, and with heavy expenditure on trypanocidal and vector control, trypanosomosis is estimated to cost over 4 billion US dollars each year in direct costs and lost production  is a severe disease caused by three species of Bos indicus cattle and is dependent on admixture proportion in hybrid breeds  are original indigenous cattle of Africa which entered into the African continent before Zebu cattle, around 8000 years ago for longhorn and around 4750–4500 years ago for shorthorn, while the humped zebu populations (Bos indicus) were brought only later into the African continent, via the Horn of Africa  and trypanosusceptible improved Kenya Boran (Bos indicus) cattle, and mapped QTLs associated to trypanotolerance on 18 autosomes. Results suggest that selection for trypanotolerance within F2 cross between N'Dama and Boran cattle could produce a synthetic breed with higher trypanotolerance levels than currently exist in the parental breeds. Noyes et al.  is much more susceptible to the disease, but still preferred by farmers because of body size and suitability as draft animal. With the intention of having both big and tolerant animals, many farmers use composites, continuously mating Zebu, Baoule and their crosses. The preference for larger animals means that Zebu ancestry is predominant among the admixed animals. In genomic regions responsible for trypanotolerance however, higher levels of Baoule ancestry are expected. In a paper on approaches to detect signatures of selection from genome wide scans, Oleksyk et al.  describeAim of this study was to compare levels of zebuine and taurine admixture in candidate regions for trypanotolerance with the “background” admixture levels, to identify differences in allelic frequencies of trypanotolerant and non-tolerant breeds, and to assess individual differences in admixture for particular animals. Regions potentially responsible for trypanotolerance were identified based on composite log-likelihoods of the differences in allelic frequencies of trypanotolerant and non-tolerant breeds, using Bovine HapMap data  and SW (n = 36) regions and 34 were Baoule-Zebu composites from SW. The North of Burkina Faso is part of the Sahelian region with no threat of trypanosomosis, while SW is a Sudanese region that is heavily tse-tse infested.Blood was taken from 214 animals in total out of which 90 were Baoule from South West (SW) Burkina Faso, 90 were Zebu from the North  and Sheko (N = 19), either pooled or separate, were compared to the allelic frequencies of the entire population (N = 497) in the study and nominal P-values were calculated for the differences in frequencies at each SNP. The nomimal P-values were then used to calculate composite log-likelihoods (CLL) for sliding windows of 9 SNP across the genome. To determine statistical significance, permutation testing was employed by comparing the CLL to the distribution of 50,000 permutations of CLL obtained with random samples of animals .To identify putative selection signatures, allelic frequencies of the N'Dama (P < 0.01 in each breed) were chosen. Within each region, 4–10 roughly equally spaced SNPs were selected from the Illumina data base for genotyping. The rationale for this approach was that signatures of selection are likely linked to trypanosome tolerance in the African taurine breeds. Also, the signals were narrow compared to the results of QTL analyses available at the time, allowing targeted SNP selection. Furthermore, the signatures targeted were observed in both the N'Dama and Sheko, perhaps suggesting that they arose in a past ancestral population of all trypanotolerant breeds and were likely to be present in the Baoule as well.The signals typically pointed to narrow regions (0.2–0.4 Mb), with an average of 254,841 bp. A total of 158 SNPs from 23 regions with strong signals (genome-wide http://www.ncbi.nlm.nih.gov/) database. The selected microsatellites covered 22 autosomal chromosomes.To reflect the admixture levels in the background genome of the animals in this study, a total of 25 autosomal microsatellites were chosen, giving a preference to FAO recommended markers FAO, , withoutGenomic DNA was isolated from white blood cells according to a modified protocol of Whatman (Whatman FTA Protocol BD09). Genotyping of the 25 microsatellites was performed on a MegaBACETM 500 genotyping device. The PCR reaction mixture with the final volume of 22 ɥl included 10 ng template genomic DNA was used in autosomes amplification. 8.05 ɥl of double distilled water, 3.20 ɥl of 10 × Buffer B 2SO4, 0.2% w/v Tween-20), 2 ɥl of 2 mM dNTP-Mix, 1.60 ɥl of 25 mM MgCl2, 0.5 ɥl of each forward and reverse primers and 0.15 ɥl of 5 U/ɥl FIREoL® DNA polymerase. One primer in each pair was labeled FAM or TET. The 155 selected SNPs were were multiplexed and genotyped on the Sequenom MassARRAY system. The choice of SNPs within the regions of interest was guided by a bioinformatic protocol optimizing the multiplexing strategy. We tried to space the SNPs equally across the 200–400 Kb regions of interest. The total number of SNPs targeted for genotyping was 150 with a minimum of 5 per region. The mastermix for multiplying SNPs comprised 0.50 ɥl of each forward and reverse primers and 0.20 ɥl of 5 U/ɥl HotStar Taq DNA polymerase plus 3 ɥl (3 ɥg) of Salmon sperm to the all mastermix. Total volume of tubes was 4 ɥl. A digestion was made after the PCR with shrimp alkaline phosphate (SAP). The SAP cleaves a phosphate from the unincorporated dNTPs, converting them to dNDPs and rendering them unavailable to future reaction. The SAP mix has been made from 1.5 ɥl of water (HPLC grade), 0.17 ɥl of 10 × SAP buffer, 0.30 ɥl of 1.7 U/ɥl SAP enzyme. 2 ɥl of SAP mix was added to the normal PCR product for digestion. The digestion was followed by ani PLEX PCR. The iPLEX mix was made of 0.619 ɥl of water, 0.20 ɥl of 10 × iPLEX buffer, 0.2 ɥl od iPLEX Termination mix, 0.041 ɥl of iPLEX Enzyme and 0.940 ɥl of the extent primer. 2 ɥl of the iPLEX mix was added to digested product. The following cycling program was run for amplification: 5 min initial denaturation at 95°C followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1:30 min, extension at 65°C for 3 min and final extension step of 65°C for 5 min using Applied Biosystems 96-Well GeneAmp® PCR System 9700 thermal cycler.The normal PCR of SNP study was run according the following protocol: 2 min initial denaturation at 95°C followed by 45 cycles of denaturation at 95°C for 0:30 min, annealing at 56°C for 0:30 min, extension at 72°C for 1 min and a final extension step of 72°C for 5 min and 4°C for 5 min. The digestion was run on 45 min: 40 min at 37°C and 5 min at 85°C. While the iPLEX PCR run as followed: 030 min of initial denaturation at 94°C, denaturation again at 94°C for 0:05 min, annealing at 52°C for 0:05 min, extension at 80°C for 0:005 min. Annealing up to extension (80°C) was repeated 5 times, from the second denaturation to the extension 40 repeats as well. A final extension step of 72°C was run for 3 min ended by 4°C forever. The normal PCR, the SAP digestion and the iPLEX PCR were performed on using Applied Biosystems 384-Well GeneAmp® PCR System 9700 thermal cycler.SNPs were positioned according to Btau 4.0. Monomorphic SNPs and SNPs with more than 10% of missing values were excluded, data analysis was performed with the remaining 135 SNPs. Average linkage disequilibrium levels, calculated as R-squared values of SNPs within candidate regions were 0.091, with 5% and 95% quantiles of 0.00003 and 0.478.K = 2; cv = 0.48. PLINK  for every microsatellite, SNP and haplotype derived from candidate regions. This calculation was based on variance in allele frequencies , smaller than the average Baoule admixture in the AAT candidate regions (assessed by SNP markers), 0.37.Crosses/composites of trypanotolerant and trypanosusceptible cattle were the focus of this analysis. STRUCTURE results indicate that the information acquired from farmers about pure Baoule and Zebu breed types is reasonably accurate with 0.87/0.89 and 0.07/0.06 Baoule ancestry proportions for these two breeds based on Microsatellite/SNP markers , the total frequency of two haplotypes (out of 22) was 83.34% for Baoule, in composites the frequency of these haplotypes was 51.52%  whereas their frequency (16 haplotypes) in Zebu animals was 22.03% . Differences were insignificant after applying Bonferroni correction in the region of Chr 18 (12.40–12.60 Mb) for Zebu and composites and region from CHR 22 (51.20–51.40 Mb) of Baoule and composites. FST calculated for haplotypes was greatest for CHR 22 (20.60–20.90 Mb), with a value of 0.51.Admixture proportions were also determined for each genomic region potentially implicated in AAT tolerance Table . Regionses Table . In ordewww.ensembl.org) are provided in Supplementary Table Genes found in candidate genomic regions studied as recovered from Ensembl (P = 0.15). Admixture was measured with two distinct types of markers, justification for the process can be found in a study of Schopen et al. , see Table n et al. , showingn et al.  when anan et al.  who founST in some candidate regions, STRUCTURE was not able to separate pure breeds in those regions. Overall FST calculated with SNPs from candidate regions (FST = 0.14) matches that from other studies, see Dayo et al.  was lower. When looking at FST for single SNPs, the highest value was 0.70 for a SNP on CHR 26 while very low FST values were found in almost every region (results not shown). FST values below 0.05 indicate low differentiation, 0.20 moderate to strong and values above 0.65 indicate extreme differentiation  showed significantly lower Baoule admixture than average admixture measured by microsatellites and SNPs. In a mapping study with experimental crosses of trypanotolerant N'Dama and trypanosusceptible Boran cattle, Hanotte et al.  had founIn this study admixture in genomic regions potentially related to trypanotolerance was compared with admixture in the background genome. A non-significant trend of higher proportions of Baoule admixture in the candidate regions was found and a majority of regions (5 of 6) with admixture levels significantly different from background admixture indicated high levels of Baoule ancestry.In this study, the discovery of trypanotolerance candidate regions was performed via a selection signature approach based on differences of allelic frequencies of trypanotolerant African taurine breeds versus other breeds around the world, using data from the Bovine HapMap consortium. Targeted SNP genotyping in candidate regions was the method of choice. Given the reduction of cost for high density SNP chip genotyping, part of the samples used in this study are now being genotyped with the commerical chip of Illumina Inc.,covering almost 800,000 SNPs. A large number of markers will also allow estimation of individual age of admixture and therefore the number of generations of natural selection acting on the composites. Targeted re-sequencing approaches of interesting candidate regions that can identify both common and exceedingly rare causal variants could potentially give more insight into trypanotolerance mechanisms.T. vivax, T. congolense, and T. brucei) from blood samples of most of the animals involved in this study. Zebus were twice as often infected (21.74%) compared to Baoule (9.70%) and composites (9.57%). Phenotypic measures oftrypanosomosis by routine checking of infection status will help to identify best composites (Soudré et al., Silbermayr et al.  developeSTF analysis was performed by ASm who also drafted the manuscript. Results were interpreted by all authors, PB, JS, GM, and ASm provided the biggest contributions in manuscript revision. All authors read and approved the final manuscript. ASm – Anamarija Smetko; ASo – Albert Soudre; ASt – Alessandra Stella. The views expressed in this publication are those of PB and do not necessarily reflect the views or policies of FAO.JS conceived the study, with the support of OH. ASo collected samples and background information, as suggested by MW, GB, and MM provided genotyping facilities and support. The study performed to find trypanosoma tolerance region was performed by PB and ASt. Genotyping of bovine microsatellites was performed by ASo and SM, SNPs were genotyped by JB and ASo while KS genotyped parasites. Admixture and 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.


Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae, horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen. Vibrio vulnificus is a gram-negative halophilic bacterium that is naturally found in marine and estuarine environments the world over V. vulnificus are classified into three biotypes based on phenotypical characteristics and differences in host range V. vulnificus species Tilapia fish. This biotype has been shown to possess biochemical properties that differ from those of biotypes 1 and 2 V. vulnificus has led to attempts to find indicators of its virulence and pathogenicity V. vulnificus isolates (composed of 63 and 115 strains), dividing the species into a few distinct evolutionary lineages with indications of different pathogenic potential Vibrio in particular, contributing to the formation of new strains and species Recently, studying the genetic relationships of 188 environmental and clinical isolates based on variations at 12 variable number tandem repeat  loci Salmonella serovar Typhi The GoldenGate SNP genotyping assay from Illumina V. vulnificus by high-throughput genotyping of a large and diverse panel of V. vulnificus isolates using genome-wide coverage. A custom SNP-genotyping microarray implemented on the Illumina GoldenGate platform was developed based on in silico data mining; 254 environmental and clinical V. vulnificus isolates were genotyped at 570 SNPs. The genotyping results were then used to infer the phylogenetic relationships between various V. vulnificus strains and specifically to follow the possible genomic origin of the recently emerged biotype 3.In this work, we studied the evolution of the human pathogen V. vulnificus isolates were tested in this study  over the last 30 years (1979–2009). Most of the Israeli environmental strains were isolated between 2004 and 2009. Of the 254 isolates tested, 114 were recovered from clinical sources (wound infections and blood) and 140 from environmental sources .The collection contained isolates from all three biotype groups identified in V. vulnificus strains were grown on thiosulfate–citrate–bile salts–sucrose (TCBS) agar and stored at −80°C in Luria Broth (LB) supplemented with 50% (vol/vol) glycerol.All High-quality DNA extraction from pure cultures was performed using a DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer's instructions. DNA concentrations were quantified using the Quant-IT kit (Invitrogen).The microbial samplings were taken from dead Tilapia fish at a local fish store in “Ein HaMifratz”, as part of the food chain while following the origin of the artificial pond where the fish were grown. No approval was required from any animal ethics committee since the tests were under the category of fresh sea food sampling, thus the fish were not scarified for the purpose of this study. The fish origin was from aquaculture fish pond of Kibbutz “Ein HaMifratz” and “Kibbutz Afek”, private land, in the western Galilee region of Israel . Also we confirm that the field studies did not involve endangered or protected species.V. vulnificus isolates: random comparative SNP data mining and selection from targeted genes.Two strategies were adopted to select informative SNPs that would differentiate among a large and diverse panel of V. vulnificus biotype 1 whole-genome sequences: YJ016 (NC_005139.1) An in-house genome-wide sequence comparison computer program was used to mine SNPs from two available V. vulnificus biotype 1 strains: YJ016 and CMCP6. These included 11 virulence-associated genes that were selected from the Virulence Factors of Bacterial Pathogens database (VFDB) (http://www.mgc.ac.cn/VFs/) and 13 plasmid and unique biotype 3 genes (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) and The RAST Server http://rast.nmpdr.org/ , followed by multiple alignment using ClustalW . Sequences were then converted to Excel using an in-house ‘convert to Excel’ program followed by SNP selection from the aligned sequences.Twenty-four targeted genes were selected from the draft genomic sequence of biotype 3 strain VVyb1(BT3)  Twenty candidate genes were selected and sequenced on a subset of 30 isolates that included strains of the three biotypes as well as the clade A group, from clinical and environmental sources. The genes included seven previously studied housekeeping genes , of which the top-ranked 574 were selected for genotyping. Thirty-one of these SNPs, whose sequences were also determined by sequencing in a panel of 30 strains included in the array (see above), served for array quality control. The full list of SNP loci and alleles used in the analysis is given in V. vulnificus isolates The custom Illumina GoldenGate assay was used to genotype 254 V. vulnificus is haploid, only homozygous clusters are expected.A total of 46 BeadChip arrays were utilized and scanned with the Illumina BeadStation 500G. The GoldenGate genotyping assay is based on a highly multiplexed, allele-discriminating, extension reaction Thus here, samples with no signal for both alleles are assigned to a third allele. To fit the ‘no signal’ cluster in place of the heterozygous cluster, signals from the GoldenGate assay were analyzed using Illuminus-P as previously described Two phylogenetic analyses were performed for the array data: parametric and nonparametric.The array data of the 254 strains at 570 SNPs were concatenated to give a specific sequence for each isolate. The same was applied to the MLST data for a panel of strains at 20 specific genes, and multiple alignment of the sequences was performed using SeqScape software (Applied Biosystems). The output files of both the array and MLST (FASTA format) were converted to MEGA format, and a phylogenetic tree was constructed with MEGA 5 A nonparametric analysis of allelic variation was used for phylogenetic analysis of the array data from the SNP alleles. The data for all genotypes were scored as present (1) or absent (0) for each of the alleles  at a specific SNP locus. Genetic relationships among strains were inferred from the GoldenGate data using Jaccard similarity coefficient, and the distance matrix was generated with PAST software Version 1.94b V. vulnificus biogroups and phylogroups was analyzed by PowerMarker V3.25 Variation among A heat map of the 570 SNPs was calculated by using the ‘heat map’ module of the statistical software environment R, version 2.14 to generate cluster analysis of 68 biotype 3 isolates and 59 representative biotype 1 isolates. Biotype 1 strain selection was based on the parametric phylogenetic tree. 40 unique biotype 3 SNPs were excluded from all further analyses (as these were present only in biotype 3 isolates), leaving 530 SNPs as the full genotyping dataset .The GoldenGate genotyping datasets of biotype 1 isolates were processed in relation to a biotype 3 synthetic haplotype. The original SNP-genotyping data was transformed so that instead of each of the alleles, a value that represents whether that allele is similar to the parallel allele in the biotype 3 synthetic haplotype  was placed. In total, three matrices were constructed: for each chromosome separately and for both chromosomes together.Principal component analysis (PCA) The biotype 3 synthetic haplotype was marked according to the three origin groups revealed by the PCA analysis. The occurrence of each allele was counted in each of the three biotype 1 groups. The origin of the allele of biotype 3 was determined based on the relatively higher appearance of that allele in one of the three biotype 1 groups , resulting in a group origin map using the developed algorithm (written in Perl and Excel).V. vulnificus in a large and diverse panel of V. vulnificus isolates. The first step was construction of a SNP-variation database using two approaches for SNP selection: random comparative SNP data mining and selection from targeted genes. 472 SNPs were selected by in silico genome-wide comparison of genes from two V. vulnificus biotype 1 genomes (YJ016 and CMCP6) V. vulnificus genome.A high-throughput custom SNP-genotyping microarray was developed to study the evolution of the human pathogen V. vulnificus isolates representing the three biotypes and phylogroup clade A recovered over a 30-year period from a wide variety of hosts and habitats all over the world. A unique pattern was achieved for every strain based on the response for each SNP locus interrogated. Genotyping revealed one of the two alleles for each strain based on the designed probe  concordance score of 29.2 (compatibility of 94% of the SNPs). Concordance scores of 28.7 and 28.9 were obtained with Illumina GenomeStudio software and Calc Allele, respectively . Thus Illuminus-P was used throughout the study to analyze the array data.V. vulnificus. The final genotyping results were obtained for each of the 254 V. vulnificus strains at 570 SNP loci, determined according to three replications, using the Illuminus-P calling algorithm.Genetic relationships among the strains inferred from the quality control data  showed cV. vulnificus isolates. Two approaches were used to analyze the SNP data: parametric and nonparametric.The array results matrix was used to infer phylogenetic relationships among all studied V. vulnificus phylogenetic studies. Both dendrograms showed that isolates of biotype 3 form a separate, clear cluster, whereas biotype 1 was highly diverse and dispersed throughout the dendrogram. Biotype 2 was heterogenic as well and clustered into subgroups according to serovars. The dendrogram showed grouping into three main clusters, which were assigned as the three main evolutionary lineages of V. vulnificusV. vulnificus strains  representing the three biotypes and the phylogroup clade A were successfully genotyped by the array, yielding a large allele dataset consisting of a unique SNP pattern for every strain at 570 loci. The quality of the genotyping was demonstrated by the similarity of the three replicated samples for each strain. Variation and phylogenetic analysis of GoldenGate data demonstrated the high genetic diversity among The phylogenetic tree based on our relatively large-scale genomic data showed a general subdivision of the species into three main evolutionary lineages, supporting previous studies conducted on a small number of loci V. vulnificus isolates, providing a broad picture of their genetic distribution, haplotype SNP analysis of the array data enabled tracing the evolutionary origin of various genomic segments While the phylogenetic analysis revealed clustering of the various tested V. vulnificus strains and/or other bacterial species in its natural environment, such as ShewanellaV. vulnificus, but this is less likely to have been the main event for biotype 3 creation. The contribution of the different biotypes and their subgroups to creation of the biotype 3 genome could be more precisely determined by extensive full-genome comparison of multiple strains representing the various biogroups.In an attempt to trace the evolution of biotype 3, the alleles of a synthetic haplotype that represents this highly conserved biotype were compared with those of the Israeli biotype 1 strains. The analyzed Israeli biotype 1 isolates clustered into three different groups in relation to biotype 3—LI, LIII and clade B—implying three possible origins for the biotype 3 haplotype. Construction of a group-origin SNP map  for the V. vulnificus strains that includes a large panel of isolates. Aside from the high discrimination power presented by the array, the phylogenetic tree inferred by the SNP-genotyping data showed formation of the main known evolutionary lineages  with an additional cluster, clade B, composed of Israeli environmental and clinical biotype 1 strains. Attempts to follow the evolutionary origin of the newly emerged biotype 3 indicate that its genome may have been created in an as-yet undiscovered event, based on the core genome of a biotype 1 strain most likely belonging to clade B that gained a rather small number of genes by HGT from its natural environment, leading to a change in biotype. Our results emphasize the continuous evolution of V. vulnificus and support the recent emergence of new pathogenic groups within this species as a recurrent phenomenon. Use of the high-throughput SNP-genotyping array enabled the study of a large panel of strains and contributes to our broader understanding of the evolution of this human pathogen.In conclusion, we describe here the first genome-wide SNP-based typing study of S1 FigDistribution of SNPs found between CMCP6 and YJ016 genomes presented along the V. vulnificus YJ016 genome.(TIFF)Click here for additional data file.S2 FigAllelic variation among 30 V. vulnificus strains at SNP locus VV1197_322.(TIFF)Click here for additional data file.S3 FigHeat map generated for the GoldenGate data at 570 SNPs distributed along the bacterial chromosomes of 185 V. vulnificus strains isolated in Israel between 1996 and 2009.(TIF)Click here for additional data file.S1 TableThe V. vulnificus strains used in this study.(DOCX)Click here for additional data file.S2 TableTargeted genes used to mine SNPs in biotype 3 and biotype 1 genomes.(DOCX)Click here for additional data file.S3 TablePCR primers for sequence analysis of 30 strains performed for SNP discovery.(DOCX)Click here for additional data file.S4 TableThe 574 SNP loci and alleles used in this study.(DOCX)Click here for additional data file.