Arctocephalus gazella). SNPs were discovered from a combination of genomic and transcriptomic resources and filtered according to strict criteria. Out of a total of 85,359 SNPs tiled on the array, 75,601 (88.6%) successfully converted and were polymorphic in 270 animals from a breeding colony at Bird Island in South Georgia. Evidence was found for inbreeding, with three genomic inbreeding coefficients being strongly intercorrelated and the proportion of the genome in runs of homozygosity being non-zero in all individuals. Furthermore, analysis of genomic relatedness coefficients identified previously unknown first-degree relatives and multiple second-degree relatives among a sample of ostensibly unrelated individuals. Such “cryptic relatedness” within fur seal breeding colonies may increase the likelihood of consanguineous matings and could therefore have implications for understanding fitness variation and mate choice. Finally, we demonstrate the cross-amplification potential of the array in three related pinniped species. Overall, our SNP array will facilitate future studies of Antarctic fur seals and has the potential to serve as a more general resource for the wider pinniped research community.High density single nucleotide polymorphism (SNP) arrays allow large numbers of individuals to be rapidly and cost-effectively genotyped at large numbers of genetic markers. However, despite being widely used in studies of humans and domesticated plants and animals, SNP arrays are lacking for most wild organisms. We developed a custom 85K Affymetrix Axiom array for an intensively studied pinniped, the Antarctic fur seal ( Single nucleotide polymorphisms (SNPs) have become one of the most popular genetic markers in evolutionary and conservation biology (Two of the most common approaches for genotyping SNPs in non-model organisms are genotyping by sequencing (GBS) methods such as restriction site associated DNA (RAD) sequencing  or they appear monomorphic and are consequently for most purposes uninformative. Among recent efforts to develop SNP arrays for wild organisms, the proportion of tiled SNPs converting into high quality polymorphic genotyping assays has varied from just over 50% to at most around 80%  is a prime example of a species that would benefit from the development of a SNP array. On Bird Island in South Georgia, a breeding colony of fur seals has been intensively monitored since the 1980s and genetic, phenotypic and life-history data have been collected for around ten thousand animals. This information has provided the foundation for elucidating the species’ mating system  behind the population decline as well as more generally to explore the genetic architecture of fitness-related traits, we developed a genome-wide panel of nuclear SNPs based on RAD sequencing data from a recent study . Finallyn = 57), Cape Shirreff in the South Shetlands (n = 6), Bouvetøya (n = 5), Îles Kerguelen (n = 5), Heard Island (n = 5) and Macquarie Island (n = 5). RAD libraries were prepared using a protocol with minor modifications as described in process_radtags in STACKS v1.41 . The latter filter was applied in order to prioritize SNPs that were polymorphic in our study population.Priority four status was assigned to the remaining RAD loci with an Affymetrix recommendation of “recommended” in either the forward or reverse direction, that mapped to no more than three different locations in the reference genome and that were neither an A/T nor a C/G SNP.Priority five status was assigned to any high quality A/T or C/G SNPs that were assigned an Affymetrix recommendation of “recommended” in either the forward or reverse direction and that mapped uniquely and completely to the reference genome.Priority six status was assigned to all of the remaining RAD loci with a “neutral” recommendation in either the forward or reverse strand, that mapped to no more than two different locations in the reference genome, that were neither an A/T nor a C/G SNP and that had no secondary SNPs present within the flanking sequence.Priority seven status was assigned to all remaining RAD SNPs with neutral recommendations for either the forward or reverse strand.Any SNPs remaining after these prioritization steps were assigned a priority of zero and were no longer considered for array design. After determining the priority of each SNP, we then thinned the dataset so that all RAD derived SNPs with a priority greater than or equal to three were at least 1 kb from the next adjacent SNP, and all SNPs with a priority of one or two were at least 100 bp apart. Finally, we removed 289 duplicate SNPs that were discovered by more than one approach. The final set of 87,608 SNPs was submitted to Afymettrix for Axiom myDesign chip manufacture.We took our combined list of probe sequences, comprising genomic and transcriptomic SNPs together with pre-validated and MHC-derived SNPs, and evaluated their suitability for inclusion on an Affymetrix Axiom SNP genotyping array. First, we assessed the genomic context of each SNP by blasting their flanking sequences against the fur seal reference genome using BLASTn v2.2.30 with an e-value threshold of 1en = 276) were collected from Antarctic fur seals at Bird Island, South Georgia as part of a long-term monitoring study conducted by the British Antarctic Survey. These were made up of females born between 1984 and 2016 and included 53 mother-offspring pairs. Additionally, we evaluated cross-species amplification by genotyping four samples each of three pinniped species including one phocid  and two otariids . DNA was extracted using a standard phenol-chloroform protocol (n = 7) or between 20 and 40 ng/µl (n = 8). These were included to evaluate how samples with suboptimal DNA concentrations would perform on the array.To assess the performance of the genotyping array, a total of 288 samples on three 96 well plates were genotyped on a Gene Titan platform by the Beijing Genomics Institute (BGI). To estimate the overall genotyping error rate, a single fur seal individual was genotyped three times, once on each plate. The majority of samples  command line software. We applied two workflows to the data, the first to assess the performance of the array in the Antarctic fur seal, and the second to quantify rates of cross-species amplification. For the former, we excluded samples belonging to the three additional pinniped species so that their inclusion did not impact overall cluster quality, and then filtered out samples with dish QC scores less than 0.82 and with call rates below 97%. For the latter, we excluded samples with dish QC scores below 0.82 but did not filter on the basis of call rate in order to retain as many samples from the other pinniped species as possible.apt_genotype_axiom function in APT, with quality metrics and classifications being assigned to individual SNPs using the Ps_Metrics and Ps_Classification functions respectively. We then used the OTV_Caller function in the SNPolisher R package to recover SNPs that were originally classified as “off-target variants”. The resulting output was then re-classified using the APT functions Ps_Metrics and Ps_Classification. To estimate the genotyping error rate, we quantified the probability at each typed locus of both alleles being IBD between replicate samples using the Z2 score output of the –genome command in PLINK v1.9 , pre-validated and originating from the Illumina Canine BeadChip , transcriptomic SNP pre-validated on the GoldenGate assay , RAD SNP pre-validated using Sanger sequencing , mapped uniquely and completely to the reference genome  and originated from RAD loci . For each of the predictors we reported model estimates and confidence intervals (CIs) from parametric bootstrapping on the log-odds scale. To translate log odds into probabilities, we took the inverse logit of the log odds estimate for a given combination of variables. For example, we calculated the probability of success for SNPs that were recommended by Affymetrix (affy = 1), mapped uniquely and completely to the reference genome (unique = 1) and originated from RAD loci (rad = 1), but were not pre-validated  as follows:affy, canine, goldengate, sanger, unique and rad respectively.We quantified the effects of our selection criteria on SNP conversion success , for which the X chromosome is well assembled, using BWA MEM with the default parameters , a measure based on the correlation of uniting gametes (FROH). sMLH was calculated using the R package inbreedR . A window was defined as homozygous when it contained no more than one heterozygous site (–homozyg-window-het 1) and no more than five missing sites (–homozyg-window-missing 5). If at least 5% of all windows containing a given SNP were defined as homozygous, the SNP was presumed to lie within a homozygous segment (–homozyg-window-threshold 0.05). Homozygous segments were then called as ROH when they contained at least 20 SNPs (–homozyg-snp 20) and no more than one heterozygous site (–homozyg-het 1). Furthermore, to ensure that incomplete marker information did not bias ROH detection, segments were only called as ROH when they contained at least one SNP per 100 kb (–homozyg-density 100) and were at least one Mb in length (–homozyg-kb 1000). If two SNPs within an ROH segment were further than 1000 kb apart, the ROH was split into two segments (–homozyg-gap 1000). The proportion of the genome in ROH (FROH) was then calculated as the sum of the detected ROH lengths for each individual over the total assembly length (2.3 Gb). In addition to these inbreeding estimators, we also quantified the extent of identity disequilibrium using the measure To calculate 2r = 0.5. We then excluded SNPs that deviated significantly from HWE as described above. Finally, in order to retain a subset of SNPs that contained as much information as possible for inferring relationships among individuals, we filtered out loci with MAF below 0.3 and that had been called in fewer than 90% of individuals. Based on the resulting dataset of 6,575 SNPs, we quantified relatedness among all 270 individuals using three different approaches.Next, we used the SNP dataset to infer patterns of relatedness among the Antarctic fur seal individuals. For this analysis, we pruned the dataset of polymorphic SNPs for linkage disequilibrium using the –indep function in PLINK. We used a sliding window of 50 SNPs, a step size of 5 SNPs and removed all variants in a window above a variance inflation factor threshold of 2, corresponding to First, we used the program NgsRelate v2  to estimWe next inferred relatedness categories for each pair of individuals based on theoretical expectations for different familial relationships. While MaxSibIter to zero. We then ran a second iteration of sequoia to assign siblings and second-degree relationships by setting MaxSibIter to 20. For both iterations, birth year information was provided using the LifeHist parameter. To identify likely second-degree relatives, we ran the function GetMaybeRel.Finally, we used the R package sequoia v2.0.7  to assigFinally, we investigated the cross-amplification potential of the array by quantifying the number of markers that could be successfully called in the gray seal, the Galápagos sea lion and the Steller’s sea lion using the –missing function in PLINK. We furthermore quantified the proportion of called SNPs that were polymorphic in each species.https://doi.org/10.25387/g3.12458255. Code for the downstream genotyping analyses is available at https://github.com/elhumble/Agaz_85K_workflow_2018 Supplemental material available at figshare: https://doi.org/10.25387/g3.12458255.The full dataset of 77,661 SNPs genotyped in 276 Antarctic fur seal samples and flanking sequences for the 85,359 tiled SNPs are available at figshare: We discovered SNPs from a combination of genomic and transcriptomic resources, applied appropriate downstream filters, and then selected the most suitable loci for tiling on a custom Antarctic fur seal SNP array according to the priority scheme described in the Materials and methods. RAD sequencing data from 83 individuals were used to call a total of 797,768 biallelic SNPs with GATK’s best practices workflow . To selen = 1) or fell below the call rate threshold of 97% (n = 3) and were therefore removed from the dataset. The remaining 272 samples were successfully genotyped at 77,661 SNPs, corresponding to an overall success rate of 90.1% , that mapped uniquely and completely to the genome assembly (P = < 2e-16) and that originated from RAD loci (P = 2.32e-16). Surprisingly, SNPs originating from the Illumina Canine HD BeadChip had a lower rate of conversion success (P = < 2e-16). There was no effect on conversion success if SNPs had been pre-validated using the GoldenGate assay (P = 0.62) or Sanger sequencing (P = 0.89). Overall, SNPs that were recommended by Affymetrix, that mapped uniquely and completely to the reference genome, and that originated from RAD loci had an estimated probability of conversion success of 94.1% (CI = 92.9–95.2%).To evaluate the success of our selection criteria, conversion rates  were quantified separately for each priority category. SNPs assigned to priority categories one, five and six had conversion rates in excess of 90% . Conversn = 282, df = 280, P = 0.27). All fifteen of the samples submitted for genotyping with DNA concentrations below the manufacturer’s recommendation of 50 ng/µl had call rates above 98%, whereas the four samples that were excluded from the final dataset on the basis of suboptimal quality or call rates had DNA concentrations above 50 ng/µl.Overall, no relationship was found between genotyping success, expressed as the call rate per sample, and DNA concentration . Empirical MAF was right skewed among the RAD SNPs  and variability inferred from the original genomic and transcriptomic resources . Second, we calculated for each individual (i) sMLH, an estimate of genome-wide heterozygosity; (ii) FROH, an estimate of the proportion of the genome in ROH. FROH was non-zero for every individual  was present at around 0.5, corresponding to 52 mother-offspring pairs . These ci.e., those falling within 0.01 of the inference thresholds), 25 second-degree relatives remained . Third-degree relationships were assigned to 63 pairs of individuals. However, while around 30 of these clustered apart from other relatedness categories in the scatterplots, the remainder were close to the kinship coefficient threshold for unrelated individuals. After removing difficult to call relationships, 24 third-degree relatives remained . The majority of individual comparisons were classified as being unrelated (99.6%) and formed a large cluster in the bottom left  and 74,130  respectively. This is equivalent to a call rate of 96.2% for the Galápagos sea lion and 96.5% for the Steller’s sea lion. Of those SNPs that could be genotyped, 4,480 (6.1%) were polymorphic in the Galápagos sea lion and 4,191 (5.7%) were polymorphic in the Steller’s sea lion.We developed a custom 85K SNP array for the Antarctic fur seal. Our efforts to prioritize high quality SNPs for tiling on the array resulted in a relatively high conversion rate, with 88.5% of the tiled loci generating readily interpretable and polymorphic genotypes. Furthermore, call rates were in excess of 99% for the majority of individuals and the genotyping error rate was low at 0.004 per reaction. Analysis of data from 270 fur seals genotyped at 75,601 polymorphic SNPs provided new insights into inbreeding and the relatedness structure of the population. Although our dataset of individuals is modest, this study provides a first impression of the promise of the array for population genomic studies of an emerging model marine mammal species.Designing SNP arrays for non-model species is non-trivial and conversion rates are not always as high as desired  of the SNPs that successfully converted on the array were polymorphic in the Antarctic fur seal. Moreover, the true rate of polymorphism is probably higher, as several hundred SNPs were included on the array that showed in silico and empirical MAF values of seals genotyped on the array suggests that, at least for moderately variable loci, the array provides a reasonable reflection of the SFS. This in turn suggests that the discovery pool of individuals in the original RAD sequencing study was large enough to estimate MAF reasonably well for the majority of SNPs that we built into the array. In line with this, a much weaker association was observed for the transcriptomic SNPs, which were discovered by sequencing many fewer individuals. Consequently, we do not recommend the array for approaches that may be sensitive to deviations from the true SFS, such as demographic inference. Nonetheless, for most purposes, SNPs with high MAFs are beneficial as they afford greater power for a multitude of applications ranging from parentage and relatedness analysis through linkage mapping to genome-wide association studies. Consequently, we believe this array will open up a wealth of new possibilities for delving into the population genomics of this important Antarctic predator.Ascertainment bias cannot be avoided with SNP arrays because high frequency polymorphisms will always be easier to discover and can be called with greater confidence due to the minor allele being present in more individuals. Nevertheless, the strong positive association that we observed between the FROH). The resulting values were strongly intercorrelated, with r values ranging from 0.62 to 0.88, although associations involving FROH tended to be somewhat weaker. When using incomplete marker information from a SNP chip, short ROH arising from inbreeding in the very distant past cannot be reliably detected due to inadequate SNP densities  and third-degree relationships (such as possible first cousins). However, the classification of relatedness categories based on theoretical criteria is challenging due to variation in IBD sharing between relatives with the same pedigree . This unTo shed further light on the relatedness structure of the study colony would require the construction of a multigenerational pedigree. In the past, we have considered this problematic due to the long generation time of the species relative to the duration of our study and the fact that not all pups are sampled every year. However, the potential for augmenting classical microsatellite-based parentage analysis with genomic information gives us new grounds for optimism.Arctocephalus, some of which diverged from A. gazella as recently as around one million years ago (Finally, we explored the cross-species amplification potential of our array by genotyping a handful of gray seals, Galápagos sea lions and Steller’s sea lions. Although none of the gray seals passed quality control, over 70,000 loci cross-amplified in both otariid species and over five percent of these were polymorphic, yielding over 4,000 polymorphic SNPs per species. This is in line with expectations set out in SNP arrays provide a straightforward and effective solution for generating large genetic datasets encompassing many individuals. As such, they have been instrumental in opening up a wide variety of questions to investigation in natural populations, from population genomics to quantitative genetics. This manuscript describes the successful development and implementation of a SNP array for a model marine mammal species, the Antarctic fur seal. By employing strict filtering approaches incorporating knowledge of the genomic context of each SNP, we were able to achieve comparably high rates of conversion and polymorphism. We also confirmed and built upon the results of previous studies by quantifying both inbreeding and genomic relatedness. We hope not only that our array will open up new avenues in fur seal research, but also that the protocols we developed to improve genotyping outcomes will be applicable to the design of arrays for other species.


Mycobacterium tuberculosis complex (MTBC), is a major global public health burden. Strain-specific genomic diversity in the known lineages of MTBC is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Fast and accurate tracking of MTBC strains is therefore crucial for infection control, and our previous work developed a 62-single nucleotide polymorphism (SNP) barcode to inform on the phylogenetic identity of 7 human lineages and 64 sub-lineages.Tuberculosis, caused by bacteria in the M. tuberculosis var. bovis, M. tuberculosis var. caprae and M. tuberculosis var. orygis). The data was partitioned into training  and test  sets and were analysed using an integrated phylogenetic tree and population differentiation (FST) statistical approach.To update this barcode, we analysed whole genome sequencing data from 35,298 MTBC isolates (~ 1 million SNPs) covering 9 main lineages and 3 similar animal-related species  discriminated perfectly the 86 MTBC isolate (sub-)lineages in the test set and could accurately reconstruct the clades across the combined 35k samples.TB-Profiler informatics platform (https://github.com/jodyphelan/TBProfiler).The validated 90 SNPs can be used for the rapid diagnosis and tracking of MTBC strains to assist public health surveillance and control. To facilitate this, the SNP markers have now been incorporated into the  Mycobacterium tuberculosis complex (MTBC), is a major global burden causing approximately ten million active cases and killing 1.5 million people in 2018 (www.who.int/tb). The MTBC consists of Mycobacterium tuberculosis sensu stricto (Mtb)  and M. tuberculosis var. africanum , which cause human disease, but others including M. tuberculosis var. bovis affect predominantly animals [Mtb lineages  have been proposed [Tuberculosis, caused by bacteria in the There is some evidence to suggest that MTBC lineages can determine the transmission, control, and clinical outcome of pulmonary and extra-pulmonary tuberculosis. In particular, variational phenotypes include differences in the emergence of drug resistance, transmissibility, virulence, host response, disease site and severity , 6. SuchTracking the spread of lineages is of great importance in tuberculosis research and control. Rapid lineage identification enables the analysis of phenotypic associations, informs on likely provenance and can assist in the prediction of potential future outbreaks. The molecular barcoding of lineages and sub-lineages can be used to classify clinical isolates to aid in the evaluation of tools to control the disease, including therapeutics and vaccines, whose effectiveness may vary by strain type , 5. HistM. tuberculosis var. bovis (M. bovis), M. tuberculosis var. caprae (M. caprae) and M. tuberculosis var. orygis (M. orygis), which are similar and sometimes misclassified. The new barcode was validated on the 17,395 (49.3%) remaining MTBC isolates. The ninety SNP markers have been incorporated into the TB-Profiler software (https://github.com/jodyphelan/TBProfiler) [Here, we update the 62-SNP barcode using WGS for 35,298 MTBC isolates. In particular, we use WGS data for 17,903 (50.7%) isolates to reconstruct a global phylogeny, resulting in 30 new (sub-)lineages. This analysis led to the 62-SNP barcode being modified and extended to ninety robust SNPs to cover 90 MTBC (sub-)lineages or species, including animal-related rofiler) , which hMtb lineages , M. africanum (lineages 5 and 6), M. bovis, M. caprae and M. orygis [n = 17,903; 50.7%) serving as a training dataset, and the second set collated subsequently  serving as a testing dataset  and GATK CombineGVCFs (default parameters) to perform joint calling using all samples. The resulting multi-sample vcf file was filtered to remove indels and heterozygous calls and monomorphic SNPs. A multi-FASTA file containing all isolates was generated from the filtered SNP file  and H37Rv reference genome using bedtools (v2.28.0) [delly software [Spades software [spolpred software [All raw sequences were trimmed using software    and in-hsoftware . Spoligosoftware .Plink software  [IQ-TREE (v1.6.12) (http://www.iqtree.org/) [-m GTR+G+ASC), with 1000 bootstrap samples used to measure branch quality and robustness. Phylogenetic trees were generated for all MTBC isolates, as well as for each main lineage separately. The resulting Newick-formatted tree files were visualised and annotated with metadata in iTOL  [Distance matrices and the principal components of the multi-FASTA files were computed with /plink2) . The disee.org/) . A genermbl.de/) . These mETE3 Toolkit (v3.1.1) package in Python3 (http://etetoolkit.org/) [t test assuming unequal variances [P < 0.05) and a Cohen’s d effect size [d > 0.5); (3) the ratio of the branch length of the clade compared to the mean branch length of its descendants (ratio > 1); (4) estimation of the number of clade-informative SNPs, requiring at least 10 SNPs with a fixation index (FST) [FST statistic assigns a strength of association between each SNP and (sub-)lineage, with a score of 1 indicating that the SNP allele is fixed in the sub-lineage of interest and not present outside that group. Using the five criteria led to the addition of 87 (27 new) sub-lineages or lineages (including 8 and 9), or changing the branch position of established others (e.g. 1.2 and 1.1.1.1) , and three barcoding SNPs were required for these mycobacteria. The overall number of (sub-)lineages or species covered was 90.The visual inspection of the phylogenetic trees  revealed that some pre-existing (sub-)lineages (as defined using the 62-SNP barcode) could be merged or split, as well as new ones created. The original 62-SNP barcode was constructed to reflect the original strain-type families used by researchers based on spoligotypes and RDs. We sought to analyse the phylogenetic tree to further divide these clades where obvious splits in the phylogeny existed. To aid in old lineage revision and new lineage identification, phylogenetic trees relating to lineages 1 to 9 and animal strains were analysed using a semi-automated procedure. Each tree was traversed (and each clade inspected) from root to tip using the it.org/) . We idenariances  (P < 0.0ect size  (d > 0.5ex (FST)  value of in MTBC  was applFST = 1) were robust, where possible, we retained synonymous SNPs in essential genes [TB-Profiler [n = 421), a minimal set of one per lineage included preferentially those already present in the 62-SNP barcode [Tuberculist (tuberculist.epfl.ch), and the frequency of ontologies across all potential barcoding, robust and minimal SNPs, was assessed for differences across lineage using the chi-squared tests.To ensure that the required 90 clade-specific mutations  and nonProfiler . From thTB-Profiler [FastTree2 software [ETE3 Toolkit, and test samples were examined for their presence in the clades defined by the training dataset.To validate the final set of robust 421 clade-defining SNPs , 2 (25.2%), 3 (11.1%) and 1 (9.5%), with the frequency of others being at most 1% , N = 17,903, 50.7%; all MTBC; 620,652 SNPs) and test  datasets (Table The 35k isolates were split into training (Mtb was 85 (L (ineage)1 16, L2 7, L3 7, L4 52, L7 1, L8 1, L9 1) and M. africanum was 2  lineage of interest (FST = 1). We identified 8128 potential barcoding SNPs (with FST = 1) across the 90 clades (Table N = 2) and 9 (N = 3) which had small sample sizes, we compared the distribution of gene functions for all potential barcoding SNPs in all characterised genes (7060/7282 SNPs) with only those in essential (and non-drug resistance) loci (790/824 SNPs) lineages, there were no potential barcoding SNPs lying within essential and non-drug resistance genes, so they were identified in non-essential and non-PE/PPE loci  which had representation of 86 of the 90 (sub-)lineages, we found that the minimal set of 90 SNPs had perfect predictive performance for all clades . This analysis excluded four (sub-)lineages , which had no test samples.By considering only the SNPs with synonymous changes, similar to the selection strategy applied in , a totaln = 90) improved the resolution of sub-lineage assignment over the SNP-IT tool. For 14 of the 21 SNP-IT strain types present in our data, the 90-SNP approach provides higher resolution of clades (range 2 to 15 sub-lineages per SNP-IT clade) , which are less common than SNPs, but may have major functional consequences. An analysis of the gene ontologies of the barcoding SNPs revealed some differences across lineages, but there is a need to the characterise functional effects of the lineage-specific SNP variants, as these could provide insights into disease control measures. Overall, we have provided an updated molecular barcode for MTBC strain types, with ninety robust markers that can be detected from applications of WGS or integrated within high-throughput genotyping or sequencing (e.g. amplicon) platforms to inform on-going TB surveillance and control.M. tuberculosis , M. africanum (2 lineages), M. bovis, M. caprae and M. orygis bacteria. The new barcode has been implemented within the publicly available TB-Profiler informatics tool, to assist the rapid, simple and reliable phylogenetic identification of individual MTBC isolates, thereby aiding clinical studies in the tracking, maintenance and phenotypic determination of MTBC pathogens.The use of molecular barcoding of MTBC bacteria causing tuberculosis can provide insights into outbreaks and help to reveal strain types that are more virulent and prone to drug resistance. In an analysis of 35,298 isolates from MTBC, we update an established 62-SNP barcode with a minimal set of 90 genetic markers, which now cover Additional file 1: Table S1. The study samples  used and their lineages. Table S2. Robust barcoding SNPs . Table S3. The ninety minimal barcoding SNPs. Figure S1. Population structure of the Mycobacterium tuberculosis complex isolates by lineage. Figure S2. Examples of discrepancies using the 62-SNP barcode. Figure S3. The genome-wide distribution of barcoding SNPs (FST=1) for each lineage. Figure S4. Functional differences between genes containing lineage-barcoding (FST=1) SNPs. Figure S5. Differentiation of sub-lineages when comparing the 62- versus 90-SNP barcodes. Figure S6. The increased resolution of our 90-SNP barcode (implemented in TB-Profiler software) over the comparable (sub-)lineages of the SNP-IT tool.


Several recent national and international projects have focused on large-scale genotyping of plant genetic resources in vegetatively propagated crops like fruit and berries, potatoes and woody ornamentals. The primary goal is usually to identify true-to-type plant material, detect possible synonyms, and investigate genetic diversity and relatedness among accessions. A secondary goal may be to create sustainable databases that can be utilized in research and breeding for several years ahead. Commonly applied DNA markers (like microsatellite DNA and SNPs) and next-generation sequencing each have their pros and cons for these purposes. Methods for large-scale phenotyping have lagged behind, which is unfortunate since many commercially important traits  are difficult to score. Nevertheless, the analysis of gene action and development of robust DNA markers depends on environmentally controlled screening of very large sets of plant material. Although more time-consuming, co-operative projects with broad-scale data collection are likely to produce more reliable results. In this review, we will describe some of the approaches taken in genotyping and/or phenotyping projects concerning a wide variety of vegetatively propagated crops. Propagation method has profound effects on both plant cultivation and research. Most of the vegetatively propagated crops are perennial, cross-pollinated, and often self-sterile. There is sometimes a progression from seed propagation of landraces to vegetative propagation of more actively selected or intentionally bred cultivars. Seeds derive from crosses between two heterozygous parents and are not genetically identical to the mother plant. Multiplication of selected genotypes with valuable traits is therefore achieved only through vegetative propagation methods. This also affects the research, breeding, and preservation of valuable germplasm, which must be focused on particular genotypes instead of on seedling families as in seed-propagated crops. Genetic research and development of whole genome sequences is further complicated by the fact that these crops are often polyploid  or derive from whole-genome duplication, so-called ancient polyploids (apple and pear).Especially desirable genotypes of, e.g., apple and grapes, have probably remained unaltered for hundreds of years and have been disseminated across large areas, with profound effects on the amount and distribution of genetic variation. Clonal propagation of long-lived crops also involves the emergence of somatic mutants or “sports”, some of which represent an improvement of a particular trait. In some crops like grape, the growing of local varieties in restricted locations like mountain valleys has created locally adapted clones, which have often spread further under different names . The truFruit and nut trees are traditionally conserved in ex situ field collections as budded or grafted cultivars, while other trees, bushes, and large herbs are grown from leaf or stem cuttings, or from suckers harvested at the stem basis. Field-planted collections are required also for tuber crops like potatoes, sweet potatoes and cassava, although tubers may be preserved in cold storage for a limited time. The collections are hosted by a wide range of governmental organizations, universities, private companies, botanical gardens, foundations, public parks and individuals. Many publicly funded collections are focused on the preservation of heritage accessions and include mostly old and/or local cultivars from a defined geographic area. Other collections are designated mainly for genetic studies and plant breeding, and may include both old and new cultivars from various areas, advanced breeding lines and wild relatives. Field collections are sometimes backed up by in vitro cultures and cryopreserved tissue (buds and shoot tips), whereas older and/or local cultivars are sometimes preserved in situ (fields and orchards).Malus/Pyrus, potato, Prunus and Vitis. The Global Crop Diversity Trust has employed specialists to develop global ex situ conservation strategies for some key crops. Thus, Bramel and Volk [The European Cooperative Program for Genetic Resources (ECPGR) has created a network of plant conservationists and scientists, with working groups designated to specific crops, e.g., berries, and Volk  outlinedGlobal germplasm exchange is facilitated by the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), which aims to strengthen the conservation, exchange, and sustainable use of plant genetic resources for food and agriculture. Several public databases for accession-level information sharing are available like the European Search Catalogue for Plant Genetic Resources (EURISCO) and the GRIN-Global database for accessions conserved by the USDA-ARS National Plant Germplasm System. Genesys, initiated by Bioversity International, the Global Crop Diversity Trust, and the secretariat of ITPGRFA, was launched as a global platform in 2011, with cooperation by EURISCO, GRIN, and the System-Wide Information Network for Genetic Resources (SINGER) from CGIAR. Not all crops are, however yet covered by these databases and the information is fast becoming obsolete since accessions continuously enter or leave different plant collections.Each preserved accession in a plant collection should have passport data . The collection becomes infinitely more valuable if the accessions are accompanied by phenotypic data  and data from molecular studies on true-to-type status and markers/genes for traits of interest. Many plant collections were established long before DNA fingerprinting became available. Decisions about what accessions to preserve have therefore relied mainly on morphological traits, information on ecogeographical origin and/or historical importance. The number of redundant (synonymous genotypes) and mislabeled accessions frequently reaches 30% or even higher when these collections are investigated with molecular markers [In order to increase the importance and utilization of the preserved accessions, co-ordinated national and regional efforts are needed to compare collections at the phenotypic and genetic levels, and to confirm cultivar identity and identify valuable genes. The scientific community has produced considerable amounts of data for accessions in various plant collections. Several rosaceous fruit and berry crops were thus investigated in recent multinational research projects; the RosBREED projects in 2009–2019 involved many research institutes in the USA and some overseas . AnotherOf all DNA fingerprint methods emerging in the early 1990s, only simple sequence repeats  remain as a major option for screening collections of plant genetic resources, although other single-locus methods like SCoT (start codon targeted) markers, and multilocus methods like ISSR (inter-simple-sequence repeats), IRAP (inter-retrotransposon amplified polymorphism), REMAP (retrotransposon-microsatellite-amplified polymorphism), and SRAP (sequence-related amplification polymorphisms) are occasionally applied. Large-scale analyses are sometimes also carried out with the diversity arrays technology (DArT), which is based on the hybridization of fluorescent DNA probes to target DNAs spotted onto a microarray.However, with the arrival of next-generation sequencing (NGS) technologies, a rapidly increasing number of studies have instead utilized single nucleotide polymorphisms (SNPs) and various other sequencing-based technologies like DArTseq.SSR have long been the preferred marker for genotyping vegetatively propagated plants, mainly because of their multiallelic and highly polymorphic nature, high discriminatory potential, and good reproducibility . Once thSSR-based analyses usually report very high levels of interaccession polymorphism, and duplicate samples are easily detected. By contrast, somatic mutants, so called sports or clones, are seldom distinguished from their original genotypes although clone-specific mutations are occasionally identified . More thPyrus Genetic Resources set, USPGR) was recently proposed, with longer repeat motifs (3–6 bp) resulting in fewer alleles but also less artifacts such as stutters, split peaks, and binning errors, which are common when using dinucleotide repeat loci as in the ECPGR set [Comparison of results across studies is often hampered by the use of different sets of SSR loci . To remeCPGR set . The ECPmotifs 3– bp resulA standardized and internationally recognized set of SSR loci is used not only for the coordinated characterization of genetic resources, but also as tools for cultivar identification and protection of Plant Breeders’ Rights. Such SSR marker-based genotype data are included in the International Variety Catalog (VIVC) for grapes  and The Several approaches are used to generate SNPs in plant material. Genotyping-by-sequencing (GBS) produces SNPs from the whole genome, and is especially valuable for plants where reference genome sequences have not yet been published. One way to reduce the complexity in GBS is RAD-Seq (restriction-site associated DNA sequencing). Using this technique, 1.25 million SNPs were discovered in a panel of 84 mango varieties and used for development of the genomic resource MiSNPDb . SNPs ar® or Illumina II® platforms. Similarly, a 20K array was developed for apple [® apple 480K SNP array was subsequently developed [® arrays have been developed, including a 20K array for potato [Use of a chip or microarray can reduce time and cost for genotyping large sets of accessions. SNP arrays have been developed for many commercially important crops, such as the 6K cherry , 8K applor apple  but the eveloped . Severalr potato , a 68K ar potato , a 700K r potato , and botr potato ,33. For r potato  and bothr potato .The SNPs must be developed from a genetically relevant material to avoid a biased representation and thus failure to reflect true differentiation among investigated samples. SNP discovery panels can differ considerably between projects aiming to produce a genomic map  and projHighly automated scoring of SNPs can easily generate errors that affect the deductions made from obtained results. A common workflow (using software ASSIsT) was therefore designed for removal of faulty SNPs in apple, peach, and sweet cherry, according to inheritance principles and pre-existing pedigree information . SNP-basIt has been argued that use of GBS is overall less expensive than SNP arrays since marker discovery and genotyping can be achieved in a single step in highly diverse crops like apple , and canAssessments of diversity can be used to reveal genetic identity  and genetic relationships  among samples, thereby facilitating both management and use of the preserved accessions. High levels of polymorphism in, e.g., ancient germplasm indicate the presence of genes that could be valuable in the face of climate change and new market demands. Changes in polymorphism over time may reflect the existence of, e.g., genetic bottlenecks during domestication. Evaluation of genetic diversity and population structure also yields valuable information for plant breeding-related research such as genome-wide association studies (GWAS) and genomic selection (GS).Analyses of genetic diversity in one or several plant collections can provide valuable information, not just on the collections but also on how the various crops have been affected by biological and historical factors such as breeding system (self- or crosspollinated), plant longevity, distribution area, domestication history, and orchard/field management. Vegetatively propagated crops are often characterized by high diversity among accessions due to, e.g., obligate cross-fertilization, multiple hybridization events combined with human activities like selection and breeding, and diversifying selection associated with adaptation to different agricultural environments and uses. By contrast, geographic differentiation is often low since vegetative propagation has enabled dispersal of selected genotypes over wide areas.Many statistical analysis methods are being used but only a few will be mentioned here. SSR data are especially difficult to handle in polyploid crops, but software like SPAGeDI 1.3  and PolySequencing data are also difficult to handle in polyploids, and specialized software may be required like EBG with two models , which was recently developed for estimating genotypes and population genetic parameters .Malus × domestica Borkh., derived mainly from the Central Asian M. sieversii (Ledeb.) M.Roem. but with some input also from the European M. sylvestris Mill. Numerous national germplasm collections have been screened using SSR loci; e.g., Urrestarazu et al. [Apple: Cultivated apple belongs to the hybridogenous u et al.  investigu et al.  investigu et al.  investigu et al.  investigIn the hitherto most wide-ranging study, a common set of 16 SSR loci were used to genotype more than 2400  accessions from 14 collections in 9 European countries, Western Russia, and Kyrgyzstan . A totalM. sieversii, M. sylvestris, and M. orientalis Uglitzk. preserved by the Plant Genetic Resources Unit at USDA, Geneva, USA. A subset of these were subsequently analyzed with 10 additional SSR loci. Diversity estimated as expected heterozygosity (He) and allelic richness was somewhat lower in cultivated apple compared to each of the wild species. Determination of the diversity for 735 cultivars developed at various times (from the 13th century up until now) showed no significant reduction in variability through time during the last eight centuries. In another study based on the USDA apple collection, 1063 diploid, unique accessions of M. × domestica, M. sieversii, M. baccata (L.) Borkh., M. floribunda Siebold ex Van Houtte, M. orientalis, and M. sylvestris were investigated with almost 28,000 GBS-generated SNPs . A Bayesian genetic structure analysis produced three groups, consisting mainly of M. domestica, M. sieversii, and M. floribunda + M. baccata, respectively. Dessert apples were shown to have a relatively higher influence of M. sieversii compared to cider apples, which instead showed more influence of M. sylvestris.Some studies have included wild apple relatives for comparison. Thus Gross et al.  used 9 STwo different genotyping methods were compared by Larsen et al. ; 15 SSR ® SNP array [® apple 480K SNP array [Several kinds of array-based methods have been applied, e.g., DArT, which was used to study genetic diversity and pedigree relationships in over 2000 accessions in the National Fruit Collection (NFC) at Brogdale in Great Britain . InsteadNP array  while moNP array .Pyrus) or “European pears” (P. communis L.). Liu et al. [Pear: Pear fruits have been produced commercially for centuries, and are divided into “Asian pears”  with eight chromosomes at the basic level and high synteny [Prunus germplasm research [P. persica (L.) Batch), European plum (P. domestica L.), apricot (P. armeniaca L.), sweet cherry (P. avium L.), and almond (P. dulcis (Mill.) D.A.Webb).Stone fruits: Stone fruits belong to the genus  synteny . Genome research , and pro® peach 9K SNP array developed by the International Peach SNP Consortium (IPSC) was used for the hitherto most comprehensive peach diversity analysis with 1580 accessions [P. davidiana (Carr.) Franch., P. kansuensis Rehd., P. mira Koehne, P. tangutica  Koehne, and P. webbii (Spach) Vierh.) and outgroup species like P. salicina Lindl., P. mume Siebold and Zucc., and P. avium, and demonstrated the transferability of genetic information among different Prunus species [The peach genome was the first to be sequenced (including repeated updates) ,54, whiccessions . DomestiP. cerasifera Ehrh. and P. spinosa L., and the evolutionary development of P. cerasifera [Several national and international SSR-based genotyping projects in hexaploid European plums have provided an overview of genetic structuring and the grouping of accessions into taxonomic and/or pomological classification systems ,60. Usinrasifera .S) locus sequence [Several SSR-based analyses have investigated diversity in local sweet cherry genetic resources ,63,64,65sequence . Developsequence .Fragaria × ananassa Duchesne) is one of the most widely grown horticultural crops, genetic research has lagged behind due to the high ploidy level (2n = 8x = 56). Over time, a large range of molecular markers has been used for diversity studies [® SNP array [F. vesca L. and F. iinumae Makino to create a road-map for SNP development and screening in high-ploidy species. This SNP array has also been used for cultivar discrimination [F. vesca and its subspecies include the entire northern hemisphere. Nearly 300 genotypes from 274 locations in 31 countries were collected and genotyped using 68 SSR loci [Fragaria, were successfully applied to both Fragaria and Rubus germplasm (including rare cultivars and wild relatives) thus ensuring transferability of markers across genera [Berry crops: Although strawberry  were compared with some widely grown, advanced cultivars established in Europe and the USA [The genus SSR loci . ProblemSSR loci . One wayolius L. . Using S the USA . SignifiSorbus and Crataegus: Some species in the genera Sorbus and Crataegus are cultivated as minor crops and are grown regionally in relatively small volumes for special purposes and products. Research has been limited and the process of domestication is still ongoing. Studies of wild populations are important for the selection and introduction of the most suitable plant material. Thus, 302 and 441 genotypes from 17 wild European populations of Sorbus domestica L. were analyzed using nuclear and chloroplast SSR loci, respectively [Crataegus; 55 accessions of 7 Crataegus species grown in China together with European and American reference samples were analyzed with 20 SSR loci selected from hawthorn transcriptional data [Crataegus accessions were analyzed in Turkey using selected apple and pear SSR loci [ectively . The hignal data . Two groSSR loci , while 2SSR loci .Citrus species have probably originated in southeastern Asia but are now cultivated around the world in warm-temperate to tropical climates, e.g., citron (C. medica L.), clementine (C. × clementina hort. ex Tanaka), grapefruit (C. × paradisi Macfad.), lemon (C. × limon (L.) Burm. f.), lime (C. × aurantifolia (Christ.) Swingle), mandarin (C. reticulata Blanco), pummelo (C. maxima (Burm.) Merrill), sour orange (C. × aurantium L.), and sweet orange (C. × sinensis (L.) Osbeck). Three of these appear to be ancestral  while the remainder have a hybridogenic origin as demonstrated by genome sequencing [Papeda (papedas), Poncirus (trifoliate citrus), and Fortunella (kumquats) are also included among citrus fruits.Most quencing . SeedlinC. maxima was studied in detail using 31 SSR loci applied to 274 accessions including local landraces from 12 provinces in China [C. reticulata, modern mandarins have often been developed by interspecific crosses. Various DNA markers including 50 SSR loci, 24 indel markers, and 67 SNPs were analyzed for 191 accessions from germplasm collections of the Instituto Valenciano de Investigaciones Agrarias (IVIA), Valencia, Spain, and the Station de Recherches Agronomiques (CIRAD-INRA), Corsica, France [C. reticulata but numerous accessions showed some introgression from other ancestral species, especially C. maxima.Genetic relationships and diversity in different citrus crops were investigated with 24 SSR loci in 370 mostly sexually derived accessions preserved at University of California, Riverside, USA . Geneticin China . High le, France . Genetic® array (CitSGA-1) was developed with 384 SNPs mined from sequence-tagged site (STS) fragments amplified from genomic DNA of eight cultivars representing citrus breeding in Japan [Citrus species and Poncirus and Fortunella. A complex genetic background with introgression between both ancestral and hybridogenic species was demonstrated for several important cultivars.An Illumina GoldenGatein Japan . This arin Japan . The genVitis spp.) constitute the fourth most important fruit crop in the world [Vitis research information. Both national and international databases with SSR marker information have been developed for the identification of cultivars . An internationally developed set of nine SSR loci is used as a standard for large-scale screening of Vitis genetic resource collections [V. vinifera L.) is applied also to other Vitis species [V. riparia Michx, V. rupestris Scheele, and V. berlandieri Planch., which are used as rootstocks and donors of important traits. Combinations of SSR, CAPS (cleaved amplified polymorphic sequences), and SCAR (sequence-characterized amplified region) markers have been used to screen germplasm collections for resistance to downy and powdery mildew, which are the major grape diseases [Grapes  demonstrated great marker transferability [V. vinifera subsp. sylvestris and 77 cultivars and non-vinifera accessions, using both the 18K SNP array and nuclear and chloroplast SSR loci, revealed considerable hybridization with introgression of V. vinifera subsp. sativa into subsp. sylvestris [Genetic research was facilitated substantially by access to the whole genome sequence together with the introduction of SNP technologies. A first version of the  in 2007 . Severalfor GWAS . The 18Kfor GWAS , while arability . Analysilvestris .V. vinifera subsp. vinifera and 27 V. vinifera subsp. sylvestris accessions [Vitis accessions representing 48 different species from a wide geographic distribution [SNPlex (Applied Biosystems) and Veracode (Illumina) SNP genotyping with 261 nuclear SNPs was performed on 231 cessions . The resribution . AnalyseOlea europaea L., is an ancient crop with clones that have been multiplied by vegetative propagation for centuries. Domestication is, however, still an ongoing process and seed propagation is common in traditional agroecosystems in the Mediterranean basin (MB). A dataset of 289 different cultivars maintained at World Olive Germplasm Bank of Córdoba, Spain, and two sets of together 96 wild accessions (Olea europaea subsp. europaea var. sylvestris) were genotyped with 25 SSR loci [Olive, SSR loci . The culIn a study of French olive germplasm, 113 accessions in the Olive Germplasm Bank, Porquerolles, France, were characterized with 20 SSR loci . For comDiospyros contains about 500 species, the most important in cultivation being the hexaploid D. kaki L.f. native to China but grown extensively in Asia and in other Mediterranean climate regions. Genetic diversity and relationships among 129 accessions originating from China and Japan were investigated with 17 SSR loci [Persimmon: The temperate-tropical genus SSR loci . High poSSR loci . The fivVaccinium have been domesticated recently. Cultivated blueberries belong to North-American species in the section Cyanococcus, with the tetraploid V. corymbosum L. cultivated in temperate regions around the world. A total of 367 blueberry samples , Corvallis, USA, representing V. corymbosum, V. angustifolium Ait., V. virgatum Ait., and interspecific hybrids) were genotyped with one or both of two multiplex reactions with SSR loci, each consisting of five loci with trinucleotide or longer repeats [Vaccinium: Several species in the genus  repeats . The resV. macrocarpon Ait.) is cultivated on large bogs where accidental seedling establishment may go unnoticed. A total of 271 cranberry plants from 77 accessions representing 66 named cultivars in the NCGR, were investigated with 12 SSR loci to identify TTT material and assess cultivar integrity and relatedness [V. vitis-idaea L.) is still harvested mostly from wild shrubs in Northern Hemisphere forests, and there has been little genetic research. Still, 1586 GBS-derived SNPs were developed to genotype 56 Canadian samples and find correlations with six environmental variables and two phenotypic traits: total phenolic content and antioxidant capacity of fruit [The diploid North American cranberry  C.F.Liang and A.R.Ferguson variety “Hayward”, which originated in New Zealand from Chinese plant material. The germplasm used in breeding has increased considerably and now involves a wider range of genotypes including other Actinidia species. A set of almost 35,000 GBS-derived SNPs were used to investigate genetic diversity in 89 kiwifruit accessions maintained in the germplasm repository in Namhae Branch of National Institute of Horticultural and Herbal Science, Korea [A. chinensis Planch., A. deliciosa, A. eriantha Benth. with wild accessions, female A. arguta (Sieb. and Zucc.) Planch. ex Miquel, and male A. arguta.Kiwifruit: For many years, the international kiwifruit trade was completely dominated by the e, Korea . A clustMangifera indica L., is now cultivated in tropical areas around the world. Fourteen SSR loci were used to screen 387 mango accessions collected from different regions in India and preserved at the Field Gene Bank at ICAR-Indian Institute of Horticultural Research in Bangalore, and ICAR-Central Institute of Subtropical Horticulture in Lucknow [Mango: Several mango species are grown in South Asia but the most important, i.e.,  Lucknow . Two strMusa acuminata Colla), or A in combination with genome B . The most popular types of banana are triploid such as the “Cavendish” clones (AAA) and various plantains (AAB). Breeding programs focus mainly on creating triploid and, to an increasing extent, tetraploid cultivars. Fully or partially fertile diploid genotypes are used to create variable progeny, thus emphasizing the importance of access to well-described wild or semi-wild germplasm.Banana: Originating in South-Eastern Asia/Western Oceania, bananas are of major importance in the tropics. They are usually propagated vegetatively since edible fruits lack seeds. Both dessert and cooking bananas are diploid, triploid, or more rarely tetraploid, and are composed of either genome A  in Leuven, Belgium, were genotyped with 19 SSR loci [A total of 695 accessions, most of which were obtained from the world’s largest ex situ SSR loci . OverallSSR loci  genotypeSSR loci . PCoA anPrunus. Other important nut crops are walnut (Juglans regia L.), cashew , pistachio (Pistacia vera L.), and hazelnut (Corylus avellana L.) [Economically important nut crops are vegetatively propagated and represent a group of botanically unrelated species. Almond, which is the most widely grown nut crop , is mentlana L.) .® 700K SNP array [Walnut: In walnut, large-scale genotyping and genetic research has been promoted significantly by the development of an AxiomNP array  and the NP array . A studyNP array . EfficieNP array .A. humile St.-Hil. [Cashew: Despite its importance, intra- and interspecific genetic structure has been sparsely studied in cashew. Genetic diversity was analyzed using ISSR markers in 240 wild-type accessions , while SSt.-Hil. . SNP marSt.-Hil. .Pistachio: A relatively small number of genotypes have been analyzed in pistachio germplasm studies, based mostly on SSR ,119, ISSC. avellana populations may be very valuable for introduction of agronomically important traits, and for a better understanding of crop development [Hazelnut is native to Europe and Western Asia, where it has been cultivated for a long time. A GBS-based analysis of the European hazelnut collection with 227 accessions, produced a detailed analysis of the germplasm including identification of genetically distant populations . Accessielopment .Solanum tuberosum L.) belong to the economically most important food sources in the world. Genetic research on potato has, however, been restricted because of polyploidy and high levels of heterozygosity [S. tuberosum [Solanum species [Potato: Tuber crops and especially potato  Lam.) is a hexaploid and self-incompatible crop. Traditionally, sweet potato is propagated by cuttings but virus-free tissue culture-derived material has become increasingly desirable due to the high virus pressure [Sweet potato: The sweet potato  and morphoagronomic characteristics [D. alata accessions from four continents were subsequently analyzed with 6017 GBS-derived SNPs [Yams: The genus eristics . A set oved SNPs . DiploidAllium cepa L.) and garlic (A. sativum L.) are widely grown, and breeding and development of new cultivars is carried out as well as conservation and research on genetic resources. Approximately 25,000 accessions of Allium are stored in various germplasm collections around the world [Allium crops are the biannual life cycle, high levels of cross-pollination, and inbreeding depression in onion, and the vegetative propagation and lack of flowering in garlic [Allium has the largest genome among vegetable crops, which complicates sequencing and assembly of genomes, development of markers, genetic mapping, and genomics-assisted breeding [Onions  using high-quality SSR loci . UsuallyR. multiflora Thunb. to identify SNPs within and between rose varieties. SNPs, which detected variation among tetraploid roses, were selected for constructing the WagRh SNP 68K Axiom® genotyping array, which has since been used to genotype 96 ornamental rose cultivars for use in several GWAS [Koning-Boucoiran et al.  made useral GWAS ,157,158.Chrysanthemum are important as ornamentals or medicinal plants and are propagated by cuttings; chrysanthemum ranks second after rose in the cut flower trade. Many chrysanthemum species are highly polyploid and have large genomes, which make them less amenable for genetic analyses. A worldwide collection of 159 cut-flower cultivars of C. morifolium (Ramat.) Hemsl., maintained at Nanjing Agricultural University’s Chrysanthemum Germplasm Resource Preserving Centre in Nanjing, China, were genotyped using 707 informative fragments derived by SRAP, SCoT, and EST (expressed sequence-tagged)-SSR amplicons [Several perennial species of the genus Efforts to screen large germplasm collections, particularly for phenotypic traits, are very costly. A restricted number of accessions can be appointed as a core collection for intensified preservation efforts and research. Definition of a core collection usually involves either (1) some kind of stratification with a cluster analysis or (2) various methods to determine genetic uniqueness. Using the M-strategy method (MSTRAT software), Liang et al.  deviced Choice of accessions for a core collection is sometimes based exclusively on genetic diversity estimates as in the examples provided above. However, expert knowledge on factors like popularity, prestige, role in breeding history, or presence of phenotypic traits of interest, can also be considered. Urrestarazu et al.  evaluateIn crops that have been propagated for a very long time, like apple, pear, grapes and olive, the same cultivar names often turn up in different plant collections. Genotyping often shows that these accessions are not genetically identical. In some cases, the discrepancy is probably due to a chance seedling resembling one of its parents but in other cases there are more wide-ranging problems with mistaken identity and mislabelings. Similarly, it is common to find the same genotype under different names in different collections, sometimes due to mistaken identity or the wish for a more “native-sounding” name for a foreign cultivar.xy (= 2nxy/(nx + ny)), is often used to determine identity by descent (IBD) and thus distinguish between identical and non-identical genotypes. The critical value of course varies between studies depending on methods and material; an IBD value of 0.90 was used as a cut-off point between non-clonality and clonality  for apple cultivars analyzed with DArT [The tried-and-true Jaccard’s similarity coefficient, Sith DArT  or GBS [ith DArT , while aith DArT . IBD valith DArT . In olivith DArT .In polyploid crops, SSR profiles are generally scored as “allelic phenotypes” based on the presence of alleles in each locus but not the (perceived) actual number of copies of each allele. The likelihood of actually overlooking alleles becomes higher with increasing levels of ploidy, which affects the threshold for perceived genotype identity. In a study of hexaploid plum accessions, the cut-off IBD value for clonality was set at 0.88 .Correct pedigrees are valuable for applied breeding programs but many cultivars in, e.g., root and tuber crops are still derived mainly from open pollination and polycross blocks . PedigreUsing SSR, pedigrees were determined for numerous French apple cultivars, with frequent occurrence of “King of the Pippins” and “Calville Rouge d’Hiver” as founders . PedigreM. × domestica accessions at USDA, Geneva, USA, “Golden Delicious” had the highest number of first-degree relatives (66) followed by “Red Delicious” (61) [Screening the apple collection at Brogdale, Great Britain, with DArT markers revealed several parent–offspring relationships including some important triploid cultivars originating in the 18th century . Some ofus” (61) . More th® Apple480K Array for 1425 mostly old European apple cultivars [In the largest apple pedigree analysis presented so far, information was generated with the Axiomultivars . IBD wasultivars  but SNP ultivars  but inst® Pear 70K array were used to reconstruct a pedigree network for a total of 1890 diploid samples in the USDA-ARS Pyrus collection [Pyrus accessions. SNPs were filtered for missing values and then a Mendelian test was run on the known trios in the F1 populations and in the genebank samples, using trio.check as described in Montanari et al. [P. communis germplasm.Data obtained with the Axiomllection . Five F1i et al. , and SNPi et al. ). In totRubus collection [SSR genotyping data and available pedigree information was used to check trueness-to-type of 39 cultivars in the German llection . SSR finPedigree relationships were detected with the triadic likelihood estimator (TrioML) in olive accessions collected from all over the Mediterranean basin, suggesting that most first-degree relationships involve cultivars from the same major geographic group . In keepPhenotyping of PGR is generally undertaken for one  of three major reasons; (1) discrimination among accessions and ascertainment of TTT status, (2) identification of potentially valuable traits for use in plant breeding, and (3) genome-wide association studies (GWAS) for development of genetic markers for marker-assisted breeding (MAB) and marker-assisted selection (MAS).National and publicly funded PGR collections are often screened for the IPGRI  or UPOV  descriptors, which were designed to discriminate among different genotypes. Some of these traits are qualitative and possibly controlled by one or a few major genes whereas others are quantitative and have a more complex genetic background. Plant breeding progress is, however, seldom focused on descriptor traits but instead on traits like yield, precocity, flowering and ripening time, storability, disease resistance, and content of various chemical compounds that affect color, texture, aroma and consumer health. Most of these traits are quantitative, i.e., show a continuous distribution, and are controlled by a combination of several quantitative trait loci (QTL).Some disease resistances are monogenic and easily identified with molecular markers, e.g., the locus-specific apple scab resistance genes. Unfortunately, these resistances are often race-specific and can be overcome by the pathogen or pest at some stage. Genes for broad and more durable resistance are instead quantitatively inherited. Attention has therefore shifted in resistance breeding, from markers for major genes to the development of markers for QTL.Phenotyping of tree and bush crops is usually undertaken by examination of a few trees or bushes (sometimes just one) of each genotype during 1–3 years. Phenotyping of shortlived perennials or tuber crops is instead accomplished by annually planting and phenotyping 10–100 plants of each genotype. Ideally, all phenotyping experiments should be replicated not only during several years but also in several locations, but this is seldom possible due to constraints in time and cost. Multiyear data can sometimes be mined from publicly available phenotypic databases like USDA-GRIN, and used for trait inheritance analysis . Historihttps://www.rosbreed.org/, and publicly available RosBREED-generated phenotypic data can be accessed in the GDR using the “Search Trait Evaluation” page to query qualitative or quantitative traits.The need for plants at a similar developmental stage when assessing morphological traits is usually self-evident although sometimes difficult to achieve when the only material available consists of already existing field plantations. Plant age, and, e.g., type of rootstock and other environmental factors, may however also affect traits like flowering time, chemical fruit contents, and disease and pest susceptibility. Large-scale phenotyping is sometimes attempted by pooling data collected from different genotypes growing in plant collections at different locations. Such a set-up is always problematic because of the considerable environmental variation due to differences in, e.g., field management, soil type and climate. Very detailed methods for standardized phenotyping of apple were developed in RosBREED I . CorrelaPenicillium expansum Link), bitter rot (Colletotrichum spp.), and bull’s eye rot (Neofabraea spp.) have been carried out in collections of cultivated apples and wild germplasm . Corresm et al. .Erwinia amylovora) in apple [Monilinia spp.) in peach [Blumeriella jaapii (Rehm.) Arx) in sour cherry [As part of RosBREED 2, disease resistance was evaluated and standard protocols developed for inoculation and evaluation of, e.g., fire blight , occurrence of polygenic traits  might be more appropriately addressed using genome-wide association studies (GWAS) or genomic selection (GS). GWAS was developed for the detection of QTLs based on associations between genome-wide markers and trait phenotypes. A large number of unrelated genotypes is needed, e.g., germplasm collections, since these can produce a much higher mapping resolution than classical offspring families. In addition, the number of QTLs that can be identified for a given phenotype is not limited by the segregation products of a specific cross, but rather by the number of QTLs underlying the trait and the genetic diversity of the studied germplasm collection.® array and phenotyped with a high-resolution texture analyzer. QTL mapping was undertaken using the seedling families, while a GWAS performed on 233 accessions produced detailed information on associations between allelic configurations and associated traits. A set of 172 apple accessions from the Nova Scotia Fruit Growers’ Association Cultivar Education Trial, Kentville, Canada were genotyped with GBS-derived SNPs and phenotyped during two years for fruit quality traits and resistance/susceptibility to apple scab  G.Winter) [® pear SNP array was applied for screening 188 variable pear accessions and an F1 population with 98 individuals [Apple and pear: Two different approaches were used in a study of fruit texture in apple; (1) a pedigree-based analysis (PBA) using six full-sib pedigreed families, and (2) a GWAS based on a collection of apple accessions, thus making it possible to exploit a larger range of both genetic and phenotypic variation . Both pl.Winter)  and for .Winter) . GWAS re® SNP array [ST values or significant results in a GWAS when bitter versus sweet fruits (as denoted in the literature) were compared. Historical phenotypic data (downloaded from the GRIN-USDA database) were used in a study of 689 apple accessions at USDA, Geneva, New York, screened with 4395 GBS-derived SNPs [MYB1 locus, and novel associations between the transcription factor NAC18.1 and harvest date and fruit firmness. In another study, comparison of genome sequences of M. × domestica and M. sieversii suggested that domestication might have altered the frequency of genes related to stress response [MYB1 gene) and possible selection for firm fruits (NAC18.1) during domestication.Table (dessert) apples differ considerably from cider apples in traits related to fruit quality and taste. Two sets with 48 dessert and 48 cider apple accessions, respectively, in the INRA Angers germplasm collection were genotyped with the 8K IlluminaNP array . The genved SNPs . Signifiresponse . DiffereMdAAT1 and MdIGS. Moreover, analysis of allelic configuration of two loci for fruit texture variation, MdPG1 and MdACO1, revealed an interplay between fruit texture and the production of volatile organic compounds. Larsen et al. [Not just texture and sugar/acid ratio but also aroma is important in apple breeding. The entire apple volatilome and fruit texture was phenotyped in 162 apple accessions, and a GWAS was performed using a 9K SNP array . Marker–n et al.  used GBS® SNP array [Within the FruitBreedomics project, approximately 1200 apple genotypes distributed over six locations in Europe  were phenotyped for various pomological characters and then genotyped with the 480K AxiomNP array . A set oPrunus species with the peach 9K SNP array were used in GWAS, focusing on traits determined by single Mendelian genes, like fruit pubescence, fruit shape, fruit flesh color, non-melting/melting flesh, titrable acidity, leaf gland type, and showy/non-showy flower type [Stone fruits: Data obtained by screening 1580 accessions of peach and some related wer type . Some clwer type . This stwer type .Vitis species, and a series of GWAS were applied to study traits like budburst-to-veraison time, cluster weight, muscat flavor, wine acidity, seedlessness, berry skin color, and sex [Vitis genotypes, a very large number of GBS-derived SNPs were able to reveal associations with berry shape, number of seeds, panicle type, berry sucrose content, berry acid content, and 12 aromatic compounds [Grapes: The GrapeReSeq 18K Vitis SNP array was used to screen 783 accessions of various  and sex . In anotompounds .1 individuals from 35 full-sib families maintained at Institute of Fruit Tree and Tea Science, NARO (NIFTS), Nagasaki and Shizuoka, Japan [Citrus: In a GWAS, 2309 GBS-derived SNPs were used to genotype 110 accessions maintained at Kuchinotsu Citrus Research Station, Nagasaki, Japan, including landraces, modern cultivars and breeding lines evaluated for fruit quality . Seven Qa, Japan . The whoBanana: A set of 105 accessions were genotyped with 5544 GBS-derived SNPs, revealing high levels of admixture in most accessions, except for a subset of 33 samples from Papua New Guinea . A GWAS Cassava: The first GWAS in cassava was conducted with 6128 African breeding lines preserved in Nigeria and Uganda. These were phenotyped for susceptibility to cassava mosaic disease in annual field trials for two years and genotyped-by-sequencing for 42,113 reference genome-mapped SNP markers . One reg® SNP array, and several GWAS were performed. Five genomic regions proved to be associated with the total anthocyanin content, two large clusters were associated with the carotenoid content, and several candidate genes were identified with known functions in either the anthocyanin or the carotenoid biosynthesis pathways [APETALA2/TOE homologue was identified as a major regulator of petal number on chromosome 3 using genetic information acquired in the process of developing a high-quality genome sequence in diploid rose [Chrysanthemum morifolium with 707 markers including SNPs, and 11 quantitative morphological traits, producing 54 significant associations that were consistent over the two years of phenotypic data collection [Ornamentals: An association panel with 96 mostly tetraploid roses was investigated for a number of commercially important traits. Carefully conducted experiments were performed to provide data for concentration of anthocyanins and carotenoids in the rose petals , the cappathways . A totalpathways . For thepathways . Some ofpathways . A rose oid rose . A few allection .Some of our conclusions from the review of large-scale studies of germplasm for vegetatively propagated plants are as follows:Standardization of SSR loci: SSRs are widely applied in all kinds of crops, and they allow cost-effective studies also for smaller number of samples. In addition, they are usually sufficiently diverse for estimation of overall diversity in PGR collections and analyses of genetic structuring, discrimination between sexually and asexually (vegetatively) derived offspring, and pedigree determinations. However, standardization of the loci used should be improved to facilitate the merging of data sets originating from different laboratories. It should also be mentioned that SSRs do not provide sufficient information for fine-scale analysis of marker-trait associations.GBS-derived SNPs: Due to the fast decreasing costs for genetic sequencing, GBS-derived SNPs are increasingly often applied, even in crops that lack an annotated reference genome. In addition to estimating genetic diversity, etc., these SNPs can also yield important information on marker–trait associations. It is, however, difficult to see how SNP data produced in different laboratories could be merged to create larger data sets.SNP arrays: Genome sequencing, assembly, and annotation has contributed to the development of large-scale research methods in some of the more important crops. A number of crop-specific SNP array systems have been developed using these data, and are now being applied for assessment of diversity, etc., and are often used also for analysis of marker–trait associations. These SNP arrays have the added benefit of allowing later additions into a database although the set of new samples should be close to 100 for cost effective laboratory analyses.Phenotyping systems: Modern high-throughput phenotyping systems are available for many crops, concerning, e.g., plant morphology and physiology and chemical contents and disease resistance. Still, the number of phenotyping studies is low and so is the number of accessions involved in such studies. To fully reflect the response of each genotype, data should ideally be collected under different plant management and climatic conditions during several years, but this is seldom achieved. The problems are especially pronounced for tree crops; time and financial constraints seldom allows propagation and planting of a randomized experiment with replicated genotypes. Instead, already existing PGR plantings are often utilized, both for measurements carried out on the plants themselves and for measurements carried out on, e.g., harvested fruit.GWAS: In some crops, functionally important genes have been identified, and marker-assisted breeding methods have been developed for their introduction into new cultivars. Application of GWAS based on large-scale and high-quality genotyping and phenotyping has the potential to reveal the exact position of key genes on the genome, and further improve molecular breeding methods. Presently, the major obstacle seems to be the lack of sufficiently informative phenotypic data. International multilocus trials of a common set of diverse genotypes could be a very valuable resource in future research.


Yr7 and identify associated SNPs in wheat. First, we used a traditional QTL mapping approach using a double haploid (DH) population and mapped Yr7 to a low-recombination region of chromosome 2B. To fine map the QTL, we then used an association mapping panel. Both populations were SNP array genotyped allowing alignment of QTL and genome-wide association scans based on common segregating SNPs. Analysis of the association panel spanning the QTL interval, narrowed the interval down to a single haplotype block. Finally, we used mapping-by-sequencing of resistant and susceptible DH bulks to identify a candidate gene in the interval showing high homology to a previously suggested Yr7 candidate and to populate the Yr7 interval with a higher density of polymorphisms. We highlight the power of combining mapping-by-sequencing, delivering a complete list of gene-based segregating polymorphisms in the interval with the high recombination, low LD precision of the association mapping panel. Our mapping-by-sequencing methodology is applicable to any trait and our results validate the approach in wheat, where with a near complete reference genome sequence, we are able to define a small interval containing the causative gene.We used three approaches to map the yellow rust resistance gene  Puccinia striiformis is one of the most damaging diseases of wheat with 88% of the world's wheat production now susceptible to infection [Yr5 and Yr7, were identified in wheat and mapped to the long arm of chromosome 2B [Yr5 and Yr7 near-isogenic lines (NILs) in the genetic background of Avocet S (AVS) revealed distinct segregation patterns for each gene, indicating probable allelism. This hypothesis was confirmed by a recent study which showed that Yr5 and Yr7 are distinct paralogues arranged in a complex tandem cluster [Yr5 being effective against the pathogen in most wheat growing regions of the world [Yr7 being effective under various environmental conditions including high temperature [Yellow stripe rust (YR) caused by nfection , 2, 3. Tnfection , 5. Two osome 2B , 7. Inte cluster . Both arperature .Genetically dissecting complex responses such as stripe rust resistance is regularly achieved by the analysis of trait-marker associations via QTL and/or genome wide association (GWA) mapping ,11. WhilYr7. Both populations were genotyped with the same high-density iSelect SNP array to show the relative power and precision of these population types in locating Yr7. We go on to show that using mapping-by-sequencing of resistant and susceptible bulks from the bi-parental population we can identify an overlapping interval to that defined by QTL analysis and GWA. In addition, we can populate the Yr7 interval with a much higher density of polymorphisms including a series of SNPs from an NBS-LRR gene which would make a logical positional and biological candidate. Performing mapping-by-sequencing of resistant and susceptible bulks using the recent wheat reference genome sequence (RefSeqv1) we show the power of an ordered reference genome. With no prior knowledge of the gene of interest here, we demonstrate that we can refine a genetic interval of 60Mbp containing 589 genes, only 0.55% of the high confidence genes in the genome. Furthermore, with limited information regarding gene function, e.g. in this case our candidate is likely to be an NBS-LRR, we were able to define a candidate gene list of only 10 genes. Our methodology is therefore broadly applicable for mapping genes associated with a wide variety of traits. Finally, given that wheat is hexaploid, it was notable that our method is perfectly capable of distinguishing between the homoeologs, identifying a clear peak on chromosome 2B that is absent from 2A and 2D.Here, we take advantage of using both bi-parental and GWA mapping populations, phenotyped for severity of infection following inoculation with a pure Pst isolate avirulent on The population of doubled haploid (DH) individuals, derived from F1 progeny of a cross between cultivars ‘Avalon’ and ‘Cadenza’, was developed by Clare Ellerbrook, Liz Sayers and the late Tony Worland (John Innes Centre), as part of a Defra funded project led by ADAS. The parents were originally chosen (to contrast for canopy architecture traits) by Steve Parker (CSL), Tony Worland and Darren Lovell (Rothamsted Research). BS-coded SNP genotypes were imported from supplementary data of .The main criterion for the membership of the YR-GWAS panel was the existence of at least one year of adult plant resistance data as part of evaluations carried out under the auspices of the National and Recommended List trials and UK Cereal Pathogen Virulence Survey. Three hundred and ten winter wheat varieties released between 1990 and 2009 were selected to comprise the core wheat YR-GWAS panel. An additional 9 older winter varieties were included in the YR-GWAS panel based on their importance in the pedigrees of elite wheat germplasm. The full wheat YR-GWAS panel is therefore composed of 319 wheat varieties, mainly elite UK winter wheat varieties, but other European countries are also represented.Puccinia striiformis (Pst) isolate 08/21  virulent on Avalon and avirulent on Cadenza was obtained from Amelia Hubbard at the UK Cereal Pathogen Virulence Survey at NIAB. This isolate was used to screen both the AxC DH population and the YR-GWAS panel. The full virulence profile of Pst ‘08/21 Solstice’ is as reported in S4 Table of [Table of . Seeds wTable of . Lines wThe wheat 90k Illumina iSelect SNP array  was usedth edition . Default settings of single trait linkage analysis, i.e. 10 cM step size, minimum cofactor proximity of 50 cM, 30 cM minimum separation of selected QTL, and a threshold calculated using Li and Ji option [P) values of 3.9, were used. The QTL was mapped based on interval mapping followed by two rounds of composite interval mapping using cofactors. Finally, to estimate the allelic effect and the explained phenotypic variance, a final QTL model was run.Only SNPs on linkage group 2B, i.e. 353 SNPs, which had been scored and were polymorphic in both the AxC and YR panel were used for QTL mapping of seedling test using QTL library in GenStat 17i option  with alpKinship matrix and principal components were calculated using Genomic Association and Prediction Integrated Tool (GAPIT) statistical package in R software , 22. Assyr7 bulk) and 54 individuals scoring < = 2 (referred to as the Yr7 bulk). At cost of a potential small loss in genetic resolution, lines with intermediate scores (2.1–6.9) were omitted from the bulks to ensure that phenotyping errors did not dilute the allelic discrimination potential of the bulks.Resistant and susceptible bulks were created by pooling equal amounts of DNA of 51 individuals with an infection score of > = 7 (referred to as the We used the NimbleGen SeqCap EZ in solution custom capture probe set (~110 Mb) to enrich genomic DNA from the two bulk segregated pools along with the purebred Avalon and Cadenza parental lines. The design and targets of the capture probe set that is used here is as previously reported . The finThe pipeline and algorithm for the processing of this sequencing data is summarized below and in SNP calling was carried out on the mapped datasets using Samtools mpileup  and thenYr7 and yr7 bulks  = 52.6) that explains 70.6% of the variance with a peak at position 110.9 cM on chromosome 2B. Yr7.A QTL scan for loci conferring resistance to Pst 08/21 showed a single major QTL  in the GWAS scan is located at 112.9 cM, close to the peak of the QTL interval shown in yr7 susceptible haplotype represented by ‘Avalon’  predominates (251/329) over the resistant ‘Cadenza’ (red shaded) haplotype, several minor haplotypes, some represented by single cultivars, show evidence of recombination between susceptible-like and resistant-like haplotypes. Vertical lines delineating seven recombination ‘blocks’ have been inserted to highlight where multiple distinct and independent haplotypes show blocks of SNPs, presumably in a state of identity-by-descent, have undergone historical recombination. This historic recombination is useful as it allows us both to partly resolve map order, as in the sub-division of SNPs co-segregating in AxC at position 109.24cM into Blocks 2–4, and to tentatively rule out all but Block 6 as potentially carrying the causal gene, since each of the other Blocks have both fully ‘Cadenza’ and fully ‘Avalon’ partial haplotypes in association with susceptibility to Pst 08/21. The notion that graphical genotypes as displayed in Yr7 in terms of vertical transmission of haplotypes 25 and 26 through known pedigrees. Haplotype 25 is possessed by varieties Brock, Tara and Vault belonging to the same lineage and Haplotype 26 is shared by three separate lineages . In each case, the founding Yr7 donor in the lineage is highlighted in bold.In order to gain information on how widespread Yr7 and yr7) were sequenced and mapped along with the purebred Avalon and Cadenza parental lines; over 3.6Gbp of the RefSeqv1 reference was mapped in each of the four datasets with an average depth of coverage of approximately 3.3X. To focus on the targeted regions for enrichment only, reads were aligned to the sequences that were used for the capture probe set design, known as the capture probe design-space. It was observed that ~95% of this reference was mapped across the four datasets with an average depth of coverage of approximately 45X in Avalon and Cadenza and 80X in the pools Yr7 and yr7  which for Yr7 contains 1,262 ‘Cadenza’-specific SNPs and for yr7 823 ‘Avalon’-specific SNPs. Yr7 Cadenza represent the intervals 633,430,001–663,920,001bp, 681,790,001–693,030,001bp and 702,660,001–721,190,001bp . There are 589 high confidence genes observed in these regions.n Avalon . The homYr7 interval were used in a BLAST search against RefSeqv1 to find their relative positions . 277 iSelect SNPs had hits (93.6%) and of these SNPs; 246 (88.8%) were anchored to chromosomal pseudomolecule 2B. The relative homozygosity scores  for these iSelect SNP positions were extracted for each of the Yr7/yr7 datasets and plotted in Yr7-linked iSelect SNP positions on chromosome 2B (216 or 87.8%), fall within the peak region 633,430,001–734,910,001bp that we identified using mapping-by-sequencing. We noted that the 5 SNPs that we previously found in our candidate genetically defined interval ‘Block 6’ (Yr7 Cadenza specific (>1E+36) and yr7 Avalon specific homozygosity scores (>1E+16) but also low Yr7 Avalon specific (<6) and yr7 Cadenza specific homozygosity scores (<1) that are characteristic of the Yr7 locus. Furthermore, the 5 SNPs were found in the 10.3 Mbp interval 683,029,160–693,314,916bp on chromosome 2B.BLAST analysis was used to infer common features with the sequence (SNP)-based AxC genetic map and the Chinese Spring based RefSeqv1. 296 iSelect SNP sequences from the Block 6’ , all shoYr7-linked polymorphisms could be examined in more detail. The candidate window encompassed 589 high confidence genes, 10 of which are annotated as disease resistance associated and are therefore candidate genes for Yr7 resistance . This gene is also seen in the peak interval that was defined here and is one of only 10 of the gene candidates that we report in Yr7 gene against a Chinese Spring background. Interestingly, there were no homozygous Avalon specific alleles in the yr7 bulk in this gene, although there was sufficient sequencing coverage in this region to define SNPs. Therefore, it appears that Avalon matches Chinese Spring closely in this region.Once we had established that the combination of gene enrichment, mapping-by-sequencing and the homozygosity haplotyping algorithm, was specifically and robustly detecting an interval which overlapped the genetically defined interval, the number and nature of sistance . The 10 reported . We usedThe 5 SNPs that we previously found in our candidate genetically defined interval ‘Block 6’ that showed the highest–log(P) scores in the GWA scan , were foYr7 genetic locus. Yr7 was named by Macer in 1963 [Yr7 in French varieties ‘Camp Remy’ and UK varieties ‘Tonic’, ‘Cadenza’ and ‘Brock’ [Yr7 has been tentatively mapped by association with QTL in several previous studies. [Yr7. Furthermore, the Yr7 gene was mapped to the long arm of chromosome 2B [A strong body of evidence links yellow rust resistance present in the cultivars ‘Lee’ and ‘Thatcher’  and their derivatives with the  in 1963 . Pathotystudies.  interpreYr7 with great precision, identifying a set of only 10 gene candidates that matched both positional and functional annotation criteria, based only on our own results. Comparison of this list with the published identity of Yr7 by [Yr7, thus giving retrospective validation to our method. We highlight the benefit of combining our mapping-by-sequencing approach with the near complete wheat reference genome sequence (RefSeqv1) in refining our candidate interval. Furthermore, this was possible even though the reference sequence is based on the variety Chinese Spring while our trait was found in the variety Cadenza. The identification of Yr7 by [In this work, we are able to re-map f Yr7 by  which emf Yr7 by  exploiteYr7 locus. The candidate window encompassed 589 high confidence genes, including 10 disease resistance associated genes that included the closest Chinese Spring homolog (TraesCS2B01G488000) of the candidate Yr7 gene reported previously [Here, we have taken yellow rust resistant and susceptible bulked doubled-haploid populations, performed gene enrichment and, using mapping-by-sequencing, identified a 60Mbp region on chromosome 2B that contains the T. monococcum identifying a region on chromosome 3 that was likely to contain the Eps-3Am deletion that results in early flowering [This analysis has taken the principles demonstrated by SHOREmap  and appllowering  and latelowering . Here, tYr7 gene. The identification of a small genetic region containing a candidate gene of interest in a hexaploid demonstrates the power of this analysis and its broad applicability.Within the defined candidate gene, we were able to select homozygous alleles at positions that were conserved with the parental lines. This revealed a small subset of 9 Cadenza specific alleles in the Yr7 bulk, of which, 4 are predicted to be non-synonymous and are potential markers for the Yr7 locus. The line-by-line SNP genotyping of the biparental population provided an ordered genome scan that treated homoeologues as distinct. The GWAS approach gave further context as to the population history of Yr7 deployment and the historical recombination evident in the variety panel provided more genetic discrimination than many hundreds more AxC lines could possibly have provided, a fact that was particularly important given the unfavorable physical: genetic distance ratio found in the Yr7 interval. The nature of this particular YR panel i.e. the fact that it is composed purely of commercial UK winter wheat varieties and important historic founders provides breeders with knowledge of those lineages in which Yr7 is found and those where it is not, allowing them to pick the most appropriate Yr7 donor and flanking markers to suit the objectives of the cross and the constraints of their marker assisted selection schemes. Finally, the mapping-by-sequencing defined a list of only 10 genes of which one was the candidate gene itself and also generated additional markers for this gene. We noted that the SNPs that we found in our candidate genetically defined interval closely overlapped the interval defined by mapping-by-sequencing.Each of the approaches taken has contributed something to the discovery of potential causative SNPs underlying the Yr7.Although the technique of mapping is by now an established method in the genetic toolbox of species which boast a fully assembled genome, polyploid large-genome crop species whose genomes have continued to evade gold standard sequencing and are expensive to re-sequence are still problematic to work with.  showed tShort lists of candidate genes or polymorphisms produced by the hybrid mapping-by-sequencing/GWAS approach demonstrated here can be subjected to validation of their function in wheat. Current methods of choice include in silico TILLING (if the wild type background possesses the trait of interest)  or gene S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.