Celiac disease (CD) affects ~1 in 141 individuals in the United States, requiring adherence to a strict gluten-free diet. The Codex Standard and the European Commission states that gluten level of gluten-free foods must not exceed 20 ppm. The FDA requires food bearing the labeling claim “gluten-free” to contain <20 ppm gluten. Accurate quantitation of gluten in fermented-hydrolyzed foods by antibody-based methods is a challenge due to the lack of appropriate reference materials and variable proteolysis. The recent uses of proteases  to hydrolyze immunopathogenic sequences of gluten proteins further complicates the quantitation of immunopathogenic gluten. The commercially available antibody-based methods routinely used to detect and quantitate gluten are not able to distinguish between different hydrolytic patterns arising from differences in fermentation processes. This is a severe limitation that makes accurate quantitation and, ultimately, a detailed evaluation of any potential health risk associated with consuming the food difficult. Utilizing gluten-specific antibodies, a recently developed multiplex-competitive ELISA along with western blot analysis provides a potential path forward in this direction. These complimentary antibody-based technologies provide insight into the extent of proteolysis resulting from various fermentation processes and have the potential to aid in the selection of appropriate hydrolytic calibration standards, leading to accurate gluten quantitation in fermented-hydrolyzed foods. Celiac disease (CD) is an immune mediated enteropathy triggered by the interaction of the prolamin and glutelin fractions of proteins from wheat, barley, and rye with the intestinal mucosa of sensitive individuals . Upon inApproximately 1 in 141 people in the US are affected by CD and adherence to a strict gluten-free diet is the only option to prevent inflammatory symptoms in sensitive individuals , 9. In 2Several qualitative and quantitative analytical methods are used for the detection and quantitation of gluten in foods. The strengths and limitations of each method have been summarized in The reliable detection and accurate quantitation of gluten in fermented-hydrolyzed foods is another challenge that warrant further discussion. This review will discuss the challenges involved in the detection and quantitation of fermented-hydrolyzed gluten by antibody-based methods and a potential path forward in overcoming the challenges. Although significant progresses have been made by using mass spectrometry-based methods in this direction, this review will only discuss mass spectrometry-based methods briefly and will particularly focus on antibody-based methods.Cereal-based fermented-hydrolyzed foods can be classified into different categories depending on the grain source, type of fermenting organism, and differences in the fermentation process. Wheat, rye and barley are commonly used in fermented-hydrolyzed foods such as beers, soy sauces, vinegars, and sourdough breads.Beer is the most widely consumed alcoholic beverage made from celiac-toxic cereals, mainly barley, and wheat. During mashing, malting, and fermentation, the gluten is proteolyzed by enzymes, resulting in the formation of peptides. Gluten-derived peptides tend to remain in the final beer product and often contain immunopathogenic sequences . StudiesSoy sauces are popular fermented foods that are commonly used to impart flavor. Soy sauce is produced in a two-stage fermentation process of soybean and wheat. First, koji (a mold-covered mixture of soybeans and wheat) is generated, which is mixed with salt water to form moromi. The moromi is allowed to age for several months, during which fermentation is catalyzed by lactic acid bacteria and yeast . SeveralMalt vinegars are produced by fermentation of cereals containing gluten, mostly barley and wheat. Vinegars made from distilled ethanol, are generally produced from non-gluten-containing raw material such as corn, beet, or sugar cane, but in some cases also gluten-containing cereals. The raw materials are typically processed in a manner that avoids the presence of any non-volatile compounds  from the finished product. However, exceptions to this occur when the distillation process is poorly performed. Thus, it is not uncommon to observe gluten peptides in some vinegars . Gluten Sourdough is a mixture of flour , water, and other ingredients that are fermented by naturally occurring lactic acid bacteria and yeasts. The potential of sourdough lactic acid bacteria as a source of proteolytic enzymes has also been investigated recently. Although primary proteolysis during sourdough fermentation is exerted by wheat or rye endogenous enzymes that are activated by the low pH, studies have shown that certain strains of lactic acid bacteria used in sourdough fermentation can produce peptidases that can proteolytically cleave the gliadin fraction of wheat gluten under certain conditions –54. HoweQuantitation of gluten in fermented-hydrolyzed foods poses a challenge due to lack of methods that can recognize the highly variable proteolytic peptide patterns that vary between fermentation processes, as well as due to the lack of suitable hydrolytic calibrants. This is further complicated by the lack of clinical information correlating peptide content with biological activity. Further, it is unknown how to interpret the immunopathogenicity based on the amount or profile of gluten protein/peptides being detected in several different fermented-hydrolyzed foods. The regulatory threshold of 20 ppm intact gluten was based on studies examining the immunopathogenicity of intact gluten. Whether the biological activity is the same for gluten peptides that are produced during fermentation is unknown , 56–59.The protein/peptide profile generated during the fermentation of different foods is dependent on numerous parameters. These include the ingredients used, time, temperature, and fermenting organisms. A slight change in these parameters can lead to wide variations in the protein/peptide profile. As such, it is impossible to generalize the profile for the different fermentation processes. The protein/peptide profiles of different fermented foods were examined using a recently developed multiplex-competitive ELISA. The ELISA utilized HRP (Horseradish peroxidase)-conjugated gluten specific antibodies  from nine gluten ELISA test kits. The antibodies were utilized in a competitive ELISA format by multiplexing the nine gluten specific antibodies into a single assay plate as described previously . Figure Aspergillus niger (AN-PEP), Sphingomonas capsulate, EP-B2 (cysteine endoprotease from germinating barley), ALV003 (mixture of cysteine endoprotease and PEP), and Pseudolysin (lasB)] have been recently used to enzymatically hydrolyze gluten proteins in an attempt to prevent proliferative responses in gluten specific T cells (Aspergillus niger derived PEP (AN-PEP) and the ALV003 have been evaluated in clinical trials for their effectiveness in mitigating gluten-induced immune responses in celiac patients , indicating that beers treated with PEP are still immunogenic  using the multiplex-competitive ELISA and western blots , 56. TheIn the western blot, 3 gluten-reduced beers, RA (Lane 5), RC (Lane 9), and RD (Lane 11), resulted in bands with the Veratox for Gliadin, cat # 8,480 detector antibody . Bands aThe RIDASCREEN® R5 competitive ELISA utilizes a mixture of pepsin-trypsin digested prolamin fractions from wheat, rye, and barley as the calibrator for quantitation purposes. Though awarded First Action by the Association of Official Analytic Chemists Official Methods of Analysis (AOAC OMA) , 85, theIn a mass spectrometry study, we evaluated the potential of the hydrolyzed wheat prolamin  as a calibrant for the detection of gluten in a model sorghum beer containing 200 mg/L added gluten, brewed with or without the addition of PEP . By massAnother limitation of the R5 competitive ELISAs is the use of gliadin as the calibrant without the inclusion of the glutenin fraction of gluten. Though the gliadin fraction of gluten is mainly responsible for exacerbating celiac disease, glutenin proteins have also been shown to stimulate celiac small intestinal T cells and can induce a toxic response in patients , 84. In A G12 antibody based competitive ELISA is also available for the detection of gluten. Although, the G12 antibody was raised against a prominent immunogenic gluten peptide, it may not recognize all known potential immunogenic-sequence-containing gluten peptides. This is complicated by the fact that not all immunogenic sequences associated with CD is known due to incomplete understanding of the pathogenesis of CD , 88, 89.Competitive ELISAs based on the G12 and R5 antibodies cannot distinguish between the protein/peptide profile pattern of different fermented foods, and while they target gliadins, they do not accurately detect glutenins, which also contain immunopathogenic sequences , 83, 84.Recognition of protein/peptide profile differences among different fermentation processes is the first step toward selection of appropriate calibration standards and, eventually, to the development of a method that can accurately quantitate gluten in fermented-hydrolyzed foods. Accurate quantitation requires that the calibration standard be identical to the protein/peptide profile of the fermented-hydrolyzed foods. This means encompassing all the gluten components  present. A single calibration standard will not be suitable for all fermented-hydrolyzed foods. As such, any analytical method that is used to analyze multiple fermentation products must be able to distinguish between the different protein/peptide profiles so the appropriate calibrant can be selected to ensure accurate quantitation.The novel multiplex-competitive ELISA included gluten specific antibodies from nine different commercial ELISA test kits. Utilizing antibodies that target different gluten epitopes, it was possible to distinguish between the protein/peptide characteristics of several different fermentation processes [Using the multiplex-competitive ELISA, it was possible to distinguish between the wheat beers, barley beers, sourdough breads, and the soy-based sauces using cluster analysis. Of the 26 barley beers analyzed, 25 clustered separately from wheat beers and 24 clustered separately from sourdough breads. Only one barley beer clustered with the majority of soy-based sauces. It was also possible to distinguish samples with similar composition or processing within a particular category of fermented-hydrolyzed food by this method  . The varWestern blot analysis utilizing the same gluten specific antibodies used in the multiplex-competitive ELISA confirmed the cluster analysis by the multiplex-competitive ELISA , 56. AltIt is obvious that further research is needed before accurate quantitation of gluten content in fermented-hydrolyzed foods can be achieved. The multiplex-competitive ELISA provides a first step by making it possible to determine the suitability of different hydrolysates as calibration standards for different fermentation-hydrolysis processes. This method also helps rule out false negative results. For examples, in It is currently impossible to accurately quantitate gluten in fermented/hydrolyzed foods and assess its potential immunopathogenicity using antibody-based methods. This is complicated by the fact that no current commercial antibody-based assay targets all the components of gluten. Further complicating the quantitative analysis of hydrolyzed gluten is the lack of appropriate calibrants that reflect the protein/peptide profiles characteristic of the various forms of fermentation. It is therefore necessary to first distinguish between the protein/peptide profiles to ensure the use of appropriate calibration standards for accurate quantitation. Mass spectrometry has potential by virtue of its ability to directly detect the peptides and proteins; however, its use as a routine analytical method is still in its infancy. In the meantime, the multiplex-competitive ELISA along with the western blot analysis make it possible to distinguish between the different protein/peptide profiles resulting from different fermentation processes and, ultimately, select appropriate standards for calibration.EG oversaw the project. RP carried out the experiments, wrote the manuscript. All authors discussed the results and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Senecio squalidus (Oxford ragwort), originated following the introduction of hybrid plants to the UK approximately 320 years ago. The source of the hybrid plants was from a naturally occurring hybrid zone between S. aethnensis and S. chrysanthemifolius on Mount Etna, Sicily. Previous studies of the parent species found evidence for multiple incompatibility loci causing transmission ratio distortion of genetic markers in their hybrid progeny. This study closes the hybridization triangle by reporting a genetic mapping analysis of the remaining two paired cross combinations between S. squalidus and its parents. Genetic maps produced from F2 mapping families were generally collinear but with half of the linkage groups showing evidence of genomic reorganization between genetic maps. The new maps produced from crosses between S. squalidus and each parent showed multiple incompatibility loci distributed across the genome, some of which co-locate with previously reported incompatibility loci between the parents. These findings suggest that this young homoploid hybrid species has inherited a unique combination of genomic rearrangements and incompatibilities from its parents that contribute to its reproductive isolation.A new homoploid hybrid lineage needs to establish a degree of reproductive isolation from its parent species if it is to persist as an independent entity, but the role hybridization plays in this process is known in only a handful of cases. The homoploid hybrid ragwort species,  Senecio squalidus, and its two progenitor species. Multiple genomic regions, including genomic rearrangements, were found to contribute to hybrid incompatibility between these species. In particular, the new hybrid species had inherited a mixture of genetic incompatibilities against both parents. These results supported the hypothesis that re-assortment of pre-existing parental genetic incompatibilities in hybrids can contribute to reproductive isolation between hybrids and their progenitors, potentially leading to further divergent evolution.A genetic mapping approach was taken to study the inheritance of genetic incompatibilities between a new homoploid hybrid species,  Heliconius butterflies  meiotic problems caused by possession of different parental chromosomal rearrangements and (2) the negative interaction of new combinations of alleles inherited from both parents. However, fitness can be recovered through re-assortment of both chromosomal rearrangements and genetic incompatibilities in later generation hybrids  from its parental species, S. aethnensis and S. chrysanthemifolius. The human-aided translocation of hybrids to the UK from a natural hybrid zone on Mount Etna, Sicily, approximately 320 years ago was crucial to the origin of S. squalidus as a new stabilized hybrid species, and its subsequent invasive spread , as well as local adaptation associated with latitude within the UK and between the UK and Sicily (3 generation (2 families derived from crosses between S. aethnensis and S. chrysanthemifolius have characterized genetic incompatibilities in the form of transmission ratio distortion (TRD), breakdown of fitness at the F2 generation, and associations between transmission ratio distortion loci (TRDL) and quantitative traits  Hybrid speciation has been accompanied by little major genomic restructuring as suggested by similar genome structures of the parent species; (2) intrinsic genetic incompatibilities are present between S. squalidus and its parents; and (3) they are likely to have been inherited in S. squalidus from its parent species. We discuss our findings in terms of their wider implications for understanding hybridization and homoploid hybrid speciation.The genetic mapping studies reported here complete the hybridization triangle of all paired cross combinations between the two parents and their hybrid derivative, and thus increase our understanding of hybrid evolution in this system. Specifically, we test the following hypotheses based on prior knowledge about this hybridizing 2 mapping families were founded from each of three paired cross combinations between three glasshouse grown (F0) individuals representing each of S. aethnensis, S. chrysanthemifolius, and S. squalidus. Senecio aethnensis and S. chrysanthemifolius F0 parental individuals were originally sampled as seed from populations VB and C1 on Mount Etna as described in S. squalidus F0 individual was sampled as seed from the Oxford (Ox), UK, population as described in 1 families where the maternal and paternal species of each individual were known. Floral emasculations have been shown to be unnecessary for these typically strongly self-incompatible species . From each of the three originally paired species combinations, one reciprocally crossed pair of F1’s was chosen to found each of three F2 mapping families, maintaining approximately equal frequencies of maternal cytoplasm per family. The family hereafter referred to as F2AC was derived from the original cross between S. aethnensis and S. chrysanthemifolius, F2AS was derived from the cross between S. aethnensis and S. squalidus, and F2CS was derived from the cross between S. chrysanthemifolius and S. squalidus. The F2 individuals were labelled in a way to keep track of their maternal origin. The analysis of the F2AC family was previously described in F species . Seeds r0, F1 and F2 plants. The number of F2 offspring from which DNA was extracted was 100, 100 and 107 for the F2AC, F2AS and F2CS mapping families, respectively. Approximately 10 % of samples were extracted twice to serve as quality controls.Genomic DNA was extracted from leaf tissue samples as described in 2 mapping families were genotyped for a total of 75 codominant genetic markers that were found to be polymorphic in the F0 and F1 parents. These comprised 61 expressed sequence tag simple sequence repeats (EST SSRs) and EST indels that were developed from the Senecio expressed sequence tag database  according to 2 mapping family was derived from two outbred F0 parents, between two and four alleles segregated at each polymorphic genetic locus. Genotype data for AFLPs and other genetic markers were first formatted according to JoinMap outcrossed mapping family (Crossed Parent or CP type) that allows markers with different segregation patterns, phases and dominance expression to be combined for genetic map construction. Linkage groups (LGs) were identified as sets of markers sharing at least one logarithm of odds (LOD) linkage score >3 and genetic distances <20 Kosambi centiMorgans (cM) following LG regression mapping. Marker order for each LG was determined by iterative rounds of regression mapping, excluding markers that had a large influence on marker order or goodness-of-fit statistics. Each genetic map was summarized for total length according to two different estimators . The relative orientation of equivalent LGs on different genetic maps with five or more shared markers was identified by comparing the results of Kendall’s tau correlation tests of shared genetic marker rank order in R v3.1.2 . The rel2 tests using Microsoft Excel 2010 (Microsoft Corp). Genetic markers were considered to show TRD if χ2 tests were significant at a 95 % confidence level before examining the effect of using a 100-fold stricter confidence level to account for multiple marker tests per map.To identify genomic regions containing genetic incompatibilities between species, genotype data for each marker in each mapping family were initially tested (Test 1) for TRD against the null hypothesis of Mendelian segregation with χAll markers linked to a genetic incompatibility locus are expected to show similar patterns of TRD. Therefore, clusters of genetic markers showing similar patterns of TRD and linked by less than 10 cM map distance were identified as TRDLs. Single genetic markers showing independent patterns of TRD and located more than 10 cM from other markers with TRD were considered as additional TRDLs, albeit with less supporting evidence. The extent of a genomic region affected by TRD was estimated as halfway to the next marker not showing TRD.0 parent, the influence of pre-zygotic incompatibilities acting at the haploid gametophyte stage was assessed by performing χ2 tests of allelic frequencies against null expectations. Dominantly scored markers or markers where parental genotypes shared some alleles could not be tested in this way. Test 4: Also, for those TRDLs containing genetic markers for which all alleles could be assigned to each F0 parent, deficits or excesses of heterozygotes in terms of F0 parental origin were tested with χ2 tests against null expectations. Test 5: Two-locus negative epistatic interactions, also known as BDMs were tested by building contingency tables of genotype combinations for the most distorted marker within each pair of TRDLs and performing Fisher’s exact tests for biases in genotype frequency combinations.Genotype data were further examined to determine the possible causes of TRD by means of the following tests. Test 2: Cytonuclear incompatibilities dependent on cross direction were examined by testing for genotypic TRD in the subsets of each mapping family in each reciprocal cross direction (i.e. sharing the same species cytoplasm). Test 3: For those TRDLs containing genetic markers for which all alleles could be assigned to each FThe locations of observed TRDLs and genomic rearrangements were compared between the different genetic maps by transposing them onto the F2AC map according to common genetic markers. For this, the location of the genetic marker in the F2AC map that showed most TRD in each TRDL and the midpoint of each rearranged genomic region were used for subsequent analyses of co-location. Co-location between TRDLs identified by genotype tests in each pair of genetic maps after transposition onto the F2AC genetic map was tested using sampling without replacement tests as described in Senecio EST database, primer sequences, F0 genotypes and genetic mapping information are provided in 2 genotype data used to construct the F2AS and F2CS genetic maps are available at .Details of all genetic markers used to genotype individuals for the construction of genetic maps including: type , EST match in the n = 10 chromosomes expected for these Senecio species , indicating that some regions of each map show better marker coverage than others.Each map comprised 13 to 15 LGs based on our minimum linkage criteria of greater than 3 LOD linkage between pairs of markers and <20 Kosambi cM distance between markers. However, some pairs of LGs showed weaker evidence of linkage or were part of the same equivalent LG in another genetic map leading to the conclusion that these maps correspond to the  species . The F2A[see]. The maps showed high synteny overall as indicated by high Kendall’s tau correlation coefficients for genetic marker rank order (P < 10–05 for each map) in accordance with expectations of reduced recombination within rearranged regions.Paired comparisons between the three genetic maps showed that almost half (62 to 70) of the genetic markers were shared between each pair of maps allowing identification and orientation of equivalent LGs see. The mapnk order . Based onk order . Nonethenk order , Table 22 mapping families differed significantly in the frequency of genetic markers showing genotypic TRD . Unmapped genetic markers were more likely to show genotypic TRD in each mapping population.Results of all TRD tests for genetic markers analysed in the F2AS and F2CS families are presented in After combined markers with genotypic TRD into TRDLs, it was apparent that fewer TRDLs were detected in the F2AS and F2CS maps  , Fig. 3 Further tests indicated the possible causes of TRD at a locus. Thus, Test 2 indicated examples of asymmetric TRD (i.e. dependent on cross direction) at three of four TRDLs resolved in the F2AS mapping family, but at none of the six loci resolved in the F2CS family . Rather S. squalidus-like genotypes. Two of these TRDLs, EC296B and E8M5_110, while on separate LGs in the F2CS map, form part of a large TRDL located on the AC1 LG of the F2AC map  TRD (Test 3) were significant for one F2AS and four F2CS TRDLs , whereas[see]. Co-location tests at larger map interval lengths were probably more conservative as the random chance of multiple TRDLs occurring within the same large interval increased. Visual inspection of the maps identified these co-located TRDLs on LGs 1, 4, 7 and 10. No significant co-location of TRDLs was evident from comparisons of the F2AC and F2AS or the F2AS and F2CS maps. Co-location tests involving TRDLs that remained significant after correction for multiple testing found significant co-location of TRDLs on LGs 1 and 4 of the F2AC and F2CS maps [see[see].Comparison of the genomic distribution of TRDLs indicated that all LGs contained TRDLs across the three genetic maps with between one and five TRDLs observed per LG , Table 32 families generated from pairwise species crosses indicate that the homoploid hybrid species, Senecio squalidus, has inherited genetic incompatibilities from both of its parental species, S. aethnensis and S. chrysanthemifolius. These findings lend support to a model of how reproductive isolation of a homoploid hybrid species can be initiated by inheritance of pre-existing genetic incompatibilities between the parental species . It seems, therefore, that genome size increase caused by retrotransposon proliferation activated by hybridization as reported in Helianthus  and F2AS (LG4) maps and three on LGs 3, 9 and 10 of the F2CS map . These rMimulus guttatus . Other results can be found in the supporting information.Mapping family genotype data and genetic map information has been deposited with the DRYAD Digital Repository  to RJA as Principal Investigator.A.C.B., S.J.H. and R.J.A. conceived the study. A.C.B. performed the experiments and analysed the results. A.C.B., S.J.H. and R.J.A. contributed to writing the paper.None declared.Supplementary Table S1Click here for additional data file.Supplementary Table S2Click here for additional data file.Supplementary Table S3Click here for additional data file.Supplementary Table S4Click here for additional data file.Supplementary Table S5Click here for additional data file.Supplementary Figures S1 and S2Click here for additional data file.


Ambystoma annulatum) across its distribution in the Central Interior Highlands (CIH) of North America, an area of high species endemism, to infer the ecological and evolutionary history of the species.For many endemic species with limited dispersal capacities, the relationship between landscape changes and species distributions is still unclear. We characterized the population structure of the endemic ringed salamander  markers.Ambystoma annulatum exist in two strongly supported nuclear genetic clusters across the CIH that correspond to a northern cluster that includes the Missouri Ozark populations and a southern cluster that includes the Arkansas and Oklahoma Ozarks and the Ouachita Mountains. Our demographic models estimated that these populations diverged approximately 2,700 years ago. Pairwise estimates of genetic differentiation at microsatellite and mtDNA markers indicated limited contemporary gene flow and suggest that genetic differentiation was primarily influenced by changes in the post‐Pleistocene landscape of the CIH.A. annulatum. The low mtDNA diversity suggests a retraction into and expansion out of refugial areas in the south‐central Ozarks, during temperature fluctuations of the Pleistocene and Holocene epochs. Similarly, the estimated divergence time for the two nuclear clusters corresponds to changes in the post‐Pleistocene landscape. More recently, decreased A. annulatum gene flow may be a result of increased habitat fragmentation and alteration post‐European settlement.Both the geologic history and post‐European settlement history of the CIH have influenced the population genetic structure of  Ambystoma annulatum) across its distribution in the Central Interior Highlands of North America, an area of high species endemism, to infer the ecological and evolutionary history of the species. Both the geologic history and post‐European settlement history of the CIH have influenced the population genetic structure of A. annulatum.We characterized the population structure of the endemic ringed salamander ( For aquatic organisms, gene flow is higher within subdrainage basins than among subdrainage basins as movement is constrained within aquatic ecosystems  which is a species of conservation concern across its distribution  restriction fragment length polymorphisms (RFLPs) on samples collected across the Missouri portion of the species range, Phillips, Suau, and Templeton  overall genetic diversity across the range will be low as a result of the range contraction during the Pleistocene; (b) the southern Ozark Highlands or Ouachita Mountains will have higher genetic diversity as would be expected given a northern recolonization of the CIH following the Pleistocene glacial cycles; and (c) gene flow will be limited as a result of land use changes post‐European settlement  given that A. annulatum is a forest‐dependent species and is sensitive to landscape perturbations.Herein, we employ nuclear microsatellite genotypes and mitochondrial DNA sequences to estimate the genetic diversity and differentiation across ecological and evolutionary time scales for 22.1A. annulatum are known or presumed to occur across the Ozark Highlands and Ouachita Mountains in Missouri, Arkansas, and Oklahoma  following the protocols outlined by Peterman, Connette, Spatola, Eggert, and Semlitsch . Forward primers were fluorescently labeled and amplified in two multiplexed polymerase chain reactions  and deviations from Hardy–Weinberg Equilibrium (HWE) with genepop on the web  and GenoDive v2.0 , pairwise fixation index values , and inbreeding coefficients (FIS). We tested for isolation by distance (IBD) using the “vegan” package  in arlequin v.3.5 , and tRNAPro to the 5′ end of the D‐loop (control region) using the THR and DL1 primers , and ~15 ng of DNA using thermocycler conditions set by Shaffer and McKnight , nucleotide diversity (π), and ΦST in arlequin.We edited and aligned mitochondrial sequences in 2.3A. annulatum demographic history in the CIH using approximate Bayesian computation (ABC) implemented in diyabc v2.1.0  of each cluster to vary as amphibian population sizes are highly variable in both time and space 2  and average HE was 0.650 ± 0.078 (SD) (Table SD), and average rarefied allelic richness was 1.96 ± 0.89 (SD) across all populations (Table FST was 0.211 ± 0.037 (SD) and average FIS was 0.058 ± 0.054 (SD)  in our structure analyses  and two well‐supported clusters within the southern cluster  and HE was 0.625 ± 0.082 (SD), whereas in the southern cluster diversity average HO was 0.623 ± 0.075 (SD) and average HE was 0.686 ± 0.069 (SD) (Table SD) and average rarefied allelic richness was 2.16 ± 1.18 (SD), whereas the average allelic richness was 5.64 ± 1.03 (SD) and average rarefied allelic richness was 1.68 ± 0.07 (SD) in the southern cluster (Table FST was 0.191 ± 0.074 (SD) and FIS was 0.034 ± 0.044 (SD) whereas average FST was 0.190 ± 0.065 (SD) and FIS was 0.091 ± 0.057 (SD) in the southern cluster (Table p > .05). The AMOVA results indicated significant subdivision among groups  Table . Within ps Table .A. annulatum, all but one pairwise comparison of sampling locations revealed significant genetic differentiation . As in our pairwise FST comparisons, the highest inferred migration rates were between the St. Louis #1 and St. Charles sites in northeast Missouri; 15.1% of individuals in the St. Louis #1 site were identified as immigrants from St. Charles  ranged from 0.000 to 0.694 and nucleotide diversity (π) ranged from 0.000 to 0.002 . Tests for IBD indicate that distance was a significant predictor of genetic differentiation based on mtDNA haplotypes .Across all populations, haplotype diversity  than in the southern CIH cluster . Our model was well estimated as none of the simulated summary statistics were significantly different from the observed data (p = .135–.811).We estimated that the divergence time between the northern and the southern CIH clusters was approximately 1,381 generations , corresponding to the early to middle Holocene  than the northern CIH cluster . We acknowledge that the effective population sizes may be overestimated since we observed substructure within A. annulatum; simulation studies have shown that estimates of effective population size are higher in populations with genetic substructure than in randomly mating populations of similar size , genetic differentiation between watersheds is pronounced enough to merit subspecific status , show similar phylogeographic breaks between the northern and southern Ozarks that were attributed to the palaeodrainages, contemporary drainages, and subplateaus in the Ozarks , one of which was found within most Missouri sampling sites , global climate change, and increased prevalence of disease . Locality data are only provided to the county level as exact coordinates are sensitive due to the conservation status of this species; however, further information is available from the authors upon request. Click here for additional data file.


Oncorhynchus tshawytscha) and GreenshellTM mussel . These programs have provided significant benefit to the industry in which we are now developing genomic resources based on genotyping-by-sequencing to complement the breeding programs, enable evaluation of the genetic diversity and identify the potential benefits of genomic selection. This represents an opportunity to increase genetic gain and more effectively utilize the potential for within family selection.Over the past 40 years New Zealand (NZ) aquaculture has grown into a significant primary industry. Tonnage is small on a global scale, but the industry has built an international reputation for the supply of high quality seafood to many overseas markets. Since the early 1990s the industry has recognized the potential gains from selective breeding and the challenge has been to develop programs that can overcome biological obstacles  and operate cost-effectively on a relatively small scale while still providing significant gains in multiple traits of economic value. This paper provides an overview of the current status, and a perspective on genomic technology implementation, for the family based genetic improvement programs established for the two main species farmed in NZ: Chinook (king) salmon ( TM mussels (GSM)  and Chinook (king) salmon (Oncorhynchus tshawytscha), produce over NZD $400 million (m) p.a. in export revenue; in 2017 revenue was $308 m for GSM and $97 m for king salmon 1. Although the NZ tonnage is small on a global scale, the industry has built an international reputation for the supply of high quality seafood to many overseas markets. Demand for NZ farmed seafood currently outstrips production and the industry has a goal to produce higher value branded products to meet domestic and export market demands. In addition to the potential of aquaculture to contribute significantly as adequate nutrition for a growing global population, there is an increasing demand for high quality seafood from an expanding middle class, as countries like China continue to develop.The New Zealand (NZ) aquaculture industry began more than half a century ago, but it was not until the 1990s that selective breeding programs were implemented for stock improvement  and operate cost-effectively on a relatively small scale while still providing significant gains. The first king salmon family breeding program was established in 1994 by commercial salmon farming company Southern Ocean Seafood Ltd. In 2002, the Cawthron Institute set up the first GSM families using wild parents and have since established a family based breeding program, now operated and managed by Breedco Ltd. In 2007, the second largest salmon farming company, Sanford Ltd., moved away from mass selection and has since developed a combined between- and within-family selection program. The breeding programs for both these species are designed so that the families are evaluated on one or more commercial farms, either in mixed family groups or as separate replicated families. To complement these well-established breeding programs and enhance emerging programs, we are now developing genomic solutions for the NZ aquaculture industry. The reduction in DNA sequencing costs coupled with multiplex assays has enabled restriction enzyme based sequencing techniques to be explored as a simultaneous SNP discovery and genotyping method for the industry. This genotyping strategy, together with advanced statistical analysis, allow genetic diversity to be assessed, pedigree established/verified and genomic selection to be implemented.This paper provides an overview and describes the benefits to the industry from the genetic improvement programs. In addition, a perspective of the use of genotyping-by-sequencing (GBS) as a tool to deliver genomic solutions to established and emerging breeding programs is discussed. To ensure NZ aquaculture meets the desired revenue target, progress in all sectors will be required, including the implementation of practical methods of genetic improvement.2. This Pacific salmon species was imported to NZ in the early 1900’s from the McLeod River, a tributary of the Sacramento River in California , sexual maturation and filet color. By 1997, the first genetic parameters for harvest traits were estimated and included harvest weight, filet color, tail fade (color variation in the tail region of the filet), body fat, filet texture and gaping (slits between the muscle blocks) and 2 year sexual maturation . ModeratIndices for selection of parents maximized growth rate while maintaining quality and minimizing losses from early maturity. For some breeding programs, selection for body conformation, improved performance and survival over summer are also included. Early maturation at 2 years is no longer a focus as it is controlled mainly through husbandry and the use of lights in the sea pens. Additional traits have been analyzed in the last 6 years including spinal curvature using digital X-radiography  and feedTM mussel is the trade-marked name of farmed green-lipped mussels . A local delicacy for centuries, this endemic species is widely distributed along the coasts of the three main islands of NZ (3.Greenshellds of NZ . These m4.Farming of GSM (grown to market size on suspended ropes) commenced in the 1970’s to better manage the production of this species which in turn results in better quality than those harvested from the wild. Today, following years of innovation, the industry produces 84,000 to 104,000 metric tons p.a. and is the largest aquaculture industry in NZ based on tonnage and export value. Marketed primarily in the form of frozen on the half shell, the industry exports to over 70 countriesAlthough the industry still relies predominantly on wild-caught juveniles (spat), a Primary Growth Partnership (PGP) program operated by SPATNZ is developing commercial scale hatchery protocols. This has enabled the production of selectively bred GSM spat for commercial production where previously farming wild spat precluded genetic improvement. This hatchery is designed to supply ∼30% of current NZ production. The wild spat that supports the industry is sourced from two main areas; attached to seaweed washed onto Ninety Mile Beach (North Island) or collected using specially designed ropes in the northern part of the South Island. However, these spat supplies are both limited and unpredictable, and the retention of spat on seaweed after translocation to mussel farms is typically less than 5 % . HatcherDuring the early 2000’s, a novel rearing system for producing large numbers of bivalve families was developed  to overcBreeding has focused on producing attractive, fast growing, resilient and high-yielding mussels suited to the predominant half-shell product. Reviewed in The current selection methods utilized by the industries discussed are pedigree-based mixed-model BLUP (best linear unbiased prediction) to estimate genetic parameters and breeding values (EBVs) using quantitative measurements on individuals , positioning for use in genomic selection whereby trait and genomic information are simultaneously collected, to assessing the genetic diversity of broodstock that have never been DNA profiled previously. All of these applications are making use of the KGD method from n = 469) as described in 5,6, the variants, together with the recorded allelic depth, were utilized for the KGD software7 to establish relatedness within (self) and between the samples. Using the following filtering parameters applied to the data , ∼46 K SNPs were observed. Processing 120–380 samples/lane (1 × 101 nt sequencing read and 27 Gb data/lane), the mean sample depth and call rate ranged from 2.17 to 4.10 and 0.66 to 0.80, respectively, with a mean self-relatedness of 0.98 observed.The RE-GBS was performed on a subset of males and females from 2 year classes  that would give the same rate of increase in inbreeding as the population under study. The Ne estimation was based on the r2 measure of linkage disequilibrium between SNPs on different chromosomes . Sample size corrections were not used, as medians of ratios were used instead of means.Utilizing allelic data, levels of genetic variation within the broodstock were determined by three different estimates of diversity: number of variable SNPs detected within the group, Minor Allele Frequency (MAF), and effective population size, Nomosomes , with r2g errors . The fire, rare alleles  are easily lost (resulting in a MAF of zero) so that many of the remaining SNP are represented as having common alleles . In a frequency distribution (see The number of informative SNPs (>0) ranged from ∼38 to ∼46 K, with the MAF distributions from combined and per group shown in tion see , a highee increases. Males from each year class have the least diversity  with the 2014 females exhibiting the most diversity. In males the estimated effective population sizes are lower than desirable whereby a total Ne of 50 per year class is recommended in breeding programs to avoid loss of genetic variation and restrict inbreeding (e is expected to increase.The diversity measures are consistent in that the number of polymorphic SNPs increases as the proportion of polymorphic SNPs with MAF > 0.2 decreases and as Nbreeding . HoweverIn addition to utilizing the allelic data to assess the levels of genetic variation within the broodstock, the allelic sampling from the GBS data was used to calculate the relatedness between all the possible combinations of the 469 broodstock using the KGD method  and is de. Distinct groups by year class were also detected in the data subset (This example of utilizing RE-GBS coupled with KGD analysis indicates that there can be low levels of genetic variation within some king salmon broodstock groups based on number of SNPs observed, their MAF distribution, and the low Na subset . The 201The above example highlights the utility of RE-GBS to provide a genetic diversity summary, over and above DNA parentage for a cost similar to many oligonucleotide low-plex SNP methods for parentage assignment. The full potential will be realized when “DNA parentage” genotyping is replaced with genotyping methods that offer greater efficacy, such as RE-GBS. This simultaneous SNP discovery and genotyping platform enables genome wide analysis and development of genomic selection in aquaculture systems without set-up costs associated with targeted GBS and fixed SNP array technologies, where on-going genotyping costs can be high, particularly in smaller industries where large volume discounts may not be achieved. This paper has shown that selective breeding of NZ aquaculture species is well established and the benefits from breeding have been clearly demonstrated. Within the next decade we expect those benefits to grow through the application of modern genomic techniques and RE-GBS provides a cost-effective method, not only within the established family programs, but also for smaller companies who previously have had no genetic management.All authors contributed to the development of the aquaculture breeding programs in NZ, on which this perspective is based and provided information that was included in this paper. JS, SC, and KD wrote the first draft of the manuscript with input from SW, NK, and RR. JS, SW, BB, and DS organized the sampling of the salmon broodstock. RR, NK, JS, SC, and KD provided the genomics information. SC and KD completed the salmon GBS and analysis. All authors contributed to manuscript revision, read and approved the submitted version.BB, DS, PB, MP, RR, and MT are employed, or sub-contracted directly, by commercial aquaculture companies in New Zealand. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Python bivittatus) has been reproducing in the Florida Everglades since the 1980s. These giant constrictor snakes have caused a precipitous decline in small mammal populations in southern Florida following escapes or releases from the commercial pet trade. To better understand the invasion pathway and genetic composition of the population, two mitochondrial (mtDNA) loci across 1,398 base pairs were sequenced on 426 snakes and 22 microsatellites were assessed on 389 snakes. Concatenated mtDNA sequences produced six haplotypes with an average nucleotide and haplotype diversity of π = 0.002 and h = 0.097, respectively. Samples collected in Florida from morphologically identified P. bivittatus snakes were similar to published cytochrome oxidase 1 and cytochrome b sequences from both P. bivittatus and Python molurus and were highly divergent . The average number of microsatellite alleles and expected heterozygosity were NA = 5.50 and HE = 0.60, respectively. Nuclear Bayesian assignment tests supported two genetically distinct groups and an admixed group, not geographically differentiated. The effective population size (NE = 315.1) was lower than expected for a population this large, but reflected the low genetic diversity overall. The patterns of genetic diversity between mtDNA and microsatellites were disparate, indicating nuclear introgression of separate mtDNA lineages corresponding to cytonuclear discordance. The introgression likely occurred prior to the invasion, but genetic information on the native range and commercial trade is needed for verification. Our finding that the Florida python population is comprised of distinct lineages suggests greater standing variation for adaptation and the potential for broader areas of suitable habitat in the invaded range.The invasive Burmese python ( Advancements in molecular tools and techniques have allowed for the delimitation of taxonomic units and genetic diversity, and identification of nonnative animals and plants in the absence of reliable morphological data , intrinsic ecological constraints, and climatic suitability  population in the Greater Everglades Ecosystem (GEE) in Florida, USA. This giant constrictor snake has been reproducing in southern Florida since approximately the mid‐1980s  and possibly even syntopically  and Indian python (P. molurus) as distinct species. To date, the GEE population has been morphologically identified as Python bivittatus throughout the invasive range.b (Cyt b) and two in the control region and used 10 cross‐species microsatellites developed by Jordan, Goodman, and Donnellan (P. bivittatus were subsequently isolated (N = 18) and combined with six cross‐species markers to identify 61% average expected heterozygosity (HE) and 2–6 alleles per locus  were identified for P. bivittatus in the native range using eight microsatellites  genes with population‐specific nuclear microsatellite markers to investigate diversity, relatedness, effective population size, population structure, and introduction dynamics of P. bivittatus captured in Florida , southeastern Miami‐Dade County, and the Florida Keys  did not produce consistent scores and were excluded here. To optimize previously published annealing multiplex (MP) temperatures, Pmb‐U21 was reassigned to MP1 and MS09 was reassigned to MP9. Annealing temperatures and PCR parameters followed Hunter and Hart . The majority of individual genotypes (N = 389) included all 22 loci, with a small percentage of samples missing ≤ seven loci.To address fine‐scale genetic diversity and population structure in the invasive population, 18 population‐specific microsatellites were developed through next‐generation sequencing and incorporated with six cross‐species loci ), which is the probability that two individuals drawn at random from a population will have the same genotype at the assessed loci sib), a related, more conservative statistic for calculating P(ID) among siblings  of clusters (presumably populations). In determining the number of clusters, the algorithm attempts to minimize deviations from Hardy–Weinberg equilibrium (HWE).The program q) from each of K clusters. Multiple Markov chains can delineate differences within populations; therefore, 20 parallel chains were analyzed for K = 1–11, with a run length of 200,000 Markov chain Monte Carlo repetitions, following a burn‐in period of 50,000 iterations. The most probable number of groups, K, was assessed using the mean log likelihood (Ln P(D)) and by calculating ∆K, an ad hoc quantity related to the change in posterior probabilities between runs of different K values , and the percentage of individuals in a cluster with q ≥ 0.90 was calculated. geneclass was used to detect first‐generation migrants born in a population other than the one in which they were sampled without a priori population categorization , information index (I), NA, average effective number of alleles (EA), and private alleles (PA) using genalex 6.501  within and among clusters and individuals with 9,999 permutations. The statistical significance of the correlation between genetic and geographic distance matrices, or isolation by distance, was assessed with a Mantel randomization test performed with genalex 6.5 with 999 permutations comparing pairwise genetic distance  using the linkage disequilibrium (LD) method at the three lowest allele frequency levels , with 95% confidence intervals (CI) following the bias‐corrected method of Waples  were computed for all pairwise relationships via ML‐RELATE  were estimated for all pairwise relationships via COANCESTRY  was calculated following Lynch and Ritland  to test for multiple paternity. Where not specified previously, the default parameters were used in the previous analyses.RY Wang, . From me Ritland , where r2.32.3.1b , 0.8 mm dNTP, 3 mm MgCl2, 0.24 μm of each primer, 0.04 units of Sigma Jump Start Taq DNA polymerase. PCR cycling profile: 5 min at 94°C; then 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C; then 10 min at 72°C. Amplified products were purified using ExoSap‐IT  for PCR cleanup. DNA sequencing was accomplished with the BigDye terminator protocol .Mitochondrial DNA variation was assayed at two protein‐coding loci: Cyt 2.3.2geneious 5.4.6 , haplotype diversity (h), sequence diversity (k), and the standard neutrality test, Tajima's D, using DnaSP v5.0 (Table N = 293), while the concatenated mtDNA matrix only included those individuals sequenced at both loci , we calculated pairwise ΦST  using Arlequin 3.5 . The second sequence (ISDB016‐11.COI‐5P) was from a snake housed in a zoo in the same state and differed by four base pairs (bps) from ISDB081‐13. Python regius  system  analyses.Pairwise genetic distances were calculated using Tajima‐Nei between the invasive population samples and structure clusters. The number of base pair discrepancies between haplotypes is provided in parenthesis.We created haplotype networks using PopART  were identified with high haplotype diversity in the invasive python samples corresponding to both 3.1MS13 locus in cluster 1 indicated the evidence of null alleles due to homozygote excess (>0.05), but there was no evidence of stuttering, large allele dropout, or linkage disequilibrium. The loci produced an unbiased P(ID) estimate of 5.63 E−15 and a P(ID)sib estimate of 2.99 E‐07, indicating that unique individuals can be confidently identified across the region. The Bayesian structure Ln P(D) estimates indicated similar values and generally plateaued at K = 4 clusters, while the structure harvester analysis according to Evanno et al.  were also investigated; however, the majority of the genotypes were “roughly symmetrically” assigned across the four or six populations, respectively, indicating that these values of K are not identifying real population structure , cluster 2 (N = 42), and a third, admixed group (N = 55), containing q ≤ 90% assignment to the two clusters . After the sequential Bonferroni adjustments, linkage disequilibrium was found for 39 of 231 (16.9%) comparisons, likely due to population substructure tested below. Separate analyses of the three groups identified in structure resulted in HWE for all loci and linkage equilibrium for cluster 1 and admixed. However, linkage disequilibrium was found in cluster 2 for two pairs of loci . This deviation may be due to inbreeding or cryptic subpopulation structure . geneclass detected 16 samples with a probability <0.01. These samples were all members of cluster 2 defined by structure.Across the 389 samples, Hardy–Weinberg disequilibrium was found for FST values among the three structure‐defined clusters were low, but significant (p ≤ 0.017): cluster 1 versus 2 (0.029), cluster 1 versus admixed (0.004), and cluster 2 versus admixed (0.012). RST values were not significant, likely owing to the minimal time for mutations to occur. Low levels of nuclear diversity were found for all samples assessed together and grouped by clusters . In cluster 2, 19 private alleles were found in 33 samples, while the admixed group contained 10 private alleles in 14 samples distributed across the q‐values. Cluster 1 had a single private allele in five samples. Effective population sizes using the linkage disequilibrium method were similar for the three allele frequencies tested; therefore, the 0.01 frequencies are reported (Table p = 0.27).t p ≤ 0.07: clustep = 0.03). However, a normal “L”‐shaped allele distribution curve was obtained, indicating a larger proportion of alleles in the low‐frequency allele classes . Cluster 2 indicated a reduction of effective population size (M = 0.766), but was not below the critical value threshold. The admixed group was significant under the Wilcoxon's TPM (p = 0.015). Nonsignificant bottleneck test values may have been due to smaller sample sizes.Assessing the 389 samples together, the stepwise mutational model (SMM) of the sign test was significant  over the three groups, which indicates inbreeding and/or a founding effect on the population. Overall, the average number of alleles was 5.50 and HE was 0.60 (Table rxy = 0.091) were between first (rxy = 0.125) and second (rxy = 0.0625) cousins on average, and inbreeding coefficients were also indicative of a cousin relationship. Across the structure cluster and all estimators, cluster 1 had the highest level of relatedness, cluster 2 had moderate levels, and admixed had the lowest level. Simulations using population allele frequencies estimated that 20% of the population was related, while observed values estimated that 24% of the samples were related. Analysis of the small number of collected hatchlings indicated that they were likely related at either the half‐sibling or full‐sibling levels.The inbreeding coefficient, 3.2b produced six novel haplotypes in 419 sequences across 799 bps with relatively high genetic distance (range 0.13%–4.30%) and number of polymorphic sites , with the five other haplotypes represented in lower proportions  and CO1 . The AMOVA identified variation levels of 48.60% and 51.40% within and among clusters 1 and 2, respectively.The es Table . The majes Table . Interes3.4b haplotype that we found in the invasive range matched all but one nucleotide to one of the published P. bivittatus mitochondrial genome sequences  was larger than the distance found in the most recent taxonomic assessment that separated P. bivittatus and P. molurus into species .The invasive Burmese python population in Florida appears to be derived from multiple genetic sources with strongly divergent mitotypes corresponding to species‐level differentiation. The Cyt cytotype in an admixed clade until, over time, only either the dominant (parent) or introgressed cytotype is retained . In natural systems, the sigmoid shape of the structure plot  in the native range through sympatric associations or secondary contact , although assessment of mitochondrial DNA is needed to determine whether divergent mitotypes are also present (Duan et al., The taxonomic uncertainty regarding species boundaries in the genus Python bivittatus prefers riverine forests and flooded grasslands, while P. molurus occupies dry, sandy, and woodland areas (Schleip & O'Shea, Field observations in the native range indicate that the two species utilize distinct habitats with some overlapping ranges. NE generations ago with a ≥90% reduction in population size (Williamson‐Natesan, A bottleneck and/or founding event was indicated 0.2–4 NA = 5; Duan et al., In comparison with our findings, on average, native range Burmese pythons had nearly twice the number of alleles and higher average heterozygosities, with the exception of the Yunnan population, which had similar allelic values (While the genetic diversity in the invasive Burmese python population is lower than that found in the native range, it is likely to increase in the large, rapidly growing invasive population, especially if additional animals are released. Multiple paternity was identified in the invasive population which could also contribute to accelerated increases in diversity. Of note, the genetic confirmation of multiple breeding events by different sires lends support to the Judas control technique in which radio‐tagged snakes are used to reveal the location of conspecifics during breeding (Smith et al., Recently, eDNA has become an important tool to estimate occurrence and detection probabilities and track the invasion front of the Burmese python populations (Hunter et al., P. bivittatus and P. molurus species boundaries. Morphological voucher specimens and broader phylogeographic sampling throughout the native range, including sympatric areas, could improve taxonomic uncertainty. Further, genomic‐level assessment and transcriptomic studies could address fine‐scale population structure and the Burmese python's adaptation to the novel environment (Castoe et al., The limited number of well‐documented, high‐quality published sequences hinders our ability to investigate None declared.MEH designed the research, performed the research, analyzed the data, and wrote the paper. KMH and RWS designed the research, collected samples, and wrote the paper. BJS collected samples and wrote the paper. MCD performed the research, analyzed the data, and wrote the paper. NAJ and JSSB analyzed the data and wrote the paper.https://doi.org/10.5066/f7hh6hkj.The data presented in this manuscript are available from  Click here for additional data file.


Bletilla striata is a well-known traditional Chinese herb with varieties of functions. In China, the natural resources of Bletilla striata have been severely damaged because of the excessive exploitation and destruction of natural habitats. The aim of present study was to provide a reference for fully exploiting and utilizing the germplasm resources of Bletilla striata.Bletilla striata from different area in China was analyzed by SCoT and IRAP molecular marker technique. A total of 209 bands were amplified by 20 groups of SCoT primers, of which 201 (96.17%) were polymorphic, and 47 polymorphic bands (94%) were observed in 50 bands amplified by 8 groups of IRAP primers. The 50 populations of Bletilla striata were divided into two major groups by SCoT and IRAP at the genetic similarity coefficient value of 0.60 and 0.68 individually. The partition of clusters in the unweighted pair group method with arithmetic mean dendrogram and principal coordinate analysis plot based on the SCoT and IRAP markers was similar.The genetic diversity of 50 varieties of Bletilla striata among different areas. Our results will provide useful information for resource protection.Results indicated the abundant genetic diversity of  Bletilla striata is a well-known traditional Chinese herb, which was first described in Shennong BenCao Jing (Shennong’s Materia Medica) 2000 years ago. In China, there are four unique populations: Bletilla striata, Bletilla formosana, Bletilla ochracea and Bletilla sinensis [Bletilla striata is capable of restraining blood leakage, stopping bleeding, dispersing swelling and promoting tissue regeneration. Thus it could be effectively applied in the treatment of hematemesis, hemoptysis, traumatic bleeding, chapped kin, and ulcerative carbuncle [Bletilla striata can be added in medicated diets or drinks when stewed together with chicken or duck and extracted by boiling water or brewed as wine material [Bletilla striata include rubbing its mucilaginous roots in inkstones with vermilion for writing [Bletilla striata results in demand exceeding supply.arbuncle , 17. Besmaterial . Additio writing . In indu writing . Moreove writing . The widBletilla striata have been severely damaged because of the excessive exploitation and destruction of natural habitats [. More than 10 years ago, Bletilla striata was listed as one of the key protected wild medicinal plants (http://rep.iplant.cn/). The price of Bletilla striata has soared 20-fold in the past 10 years [Bletilla striata resources, the artificial cultivation of Bletilla striata and its related species has been developed in the majority of regions in China [Bletilla striata also has been launched [Bletilla until now.In China, the natural resources of habitats . More th10 years . In ordein China . More imlaunched . As partBletilla striata.Molecular markers are one of the most important methods to allow cultivar identification. They are widespread used on account of their simple operation and high detection efficiency . The difBletilla striata species in China and construct a molecular phylogenetic tree, which provides a theoretical basis for protection, selection and development.The objectives of this study are to investigate the genetic relationships among some The oligo nucleotide sequences of SCoT and IRAP primers are summarized in Table r = 0.752, indicating that the clustering results are good).Twenty groups of primers were used to detect genetic polymorphisms in the samples. The primers produced 209 clear SCoT bands, including 201 polymorphism bands. The total number of scored bands varied from 6 (for SC8 + SC11) to 14  with an average of 10.5 bands per primer .Eight groups of primers were used to detect genetic polymorphisms in the samples. The primers produced 50 clear IRAP bands, including 47 polymorphism bands. The total number of scored bands varied from 4 (for LIRE-Tar-F + LIRE-del-F1) to 9 (for DO-F + LIRE-del-F1) with an average of 6.3 bands per primer Table . The numPCoA analysis was done to see the displacement of the accessions and to further confirm the clustering pattern obtained from the dendrogram. The result of the three-dimensional (3D) plots of PCoA analysis for SCoT marker analysis was also similar to that of clustering in the dendrogram Fig. . PCoA anBletilla striata  of the SCoT data revealed that out of the total recorded variation, 79.38% was recorded within the species level whereas 20.62% was compartmentalized amongst the species. Similar in distribution to that of SCoT, IRAP marker data accounted for 77.36% variability at inter-specific level and 22.64% at intra-specific level. Which indicated high genetic variation within populations, and using SCoT marker reveled higher variation within and among populations of Bletilla striata becomes complex because of prolonged cross pollination and natural selection. In this study, we collected a variety of Bletilla striata from China. The genetic diversity and phylogenetic relationships of the samples were analyzed using SCoT and IRAP markers. The SCoT marker systems are proved to be efficient and inexpensive ways to provide molecular data to assess genetic diversity, and it has been used successfully to determine genetic relationships for many plants ([H. vulgare) cultivars. IRAP markers involve the PCR amplification of DNA sequences between two nearby retrotransposons, using a primer designed from the LTR sequence of a retrotransposon, which were recently used extensively in studying genetic diversity, genetic relationship and germplasm management [In general, the genetic characteristics of a species are affected primarily by various ecological and biological traits, modes of reproduction and breeding and also by the various human mediated anthropogenic activities . The genetic background of wild  plants . Specific information is in Table The A modified cetyltrimethyl ammonium bromide (CTAB) method was used for DNA extraction . DNA samPistacia chinensis [Karim Sorkheh et al. used IRAP for germplasm analysis of wild hinensis , and Ahmhinensis . We usedPCR amplification was performed in a final reaction volume of 20 μL containing 10× buffer, 0.1 mM dNTPs, 0.3 μM of each primer, 1 U Taq polymerase, and 20 ng of template DNA. The amplification was performed with the following thermal program: 94 °C for 4 min; followed by 36 cycles of denaturation at 95 °C for 50s, annealing at 50 °C for 40 s, elongation at 72 °C for 2 min; and extension at 72 °C for 5 min. All PCR reactions were performed twice. The products were kept at 4 °C.PCR amplification was performed in a final reaction volume of 20 μL containing 10× buffer, 800 mM dNTPs, 0.3 nM of each primer, 1.25 U Taq polymerase, and 20 ng of template DNA. The amplification was performed with the following thermal program: 94 °C for 4 min, 36 °C for 1 min, 72 °C for2 min; followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 36 °C for 1 min, elongation at 72 °C for 2 min; and extension at 94 °C for 4 min, 36 °C for 1 min, 72 °C for2 min. All PCR reactions were performed twice. The products were kept at 4 °C.DNA amplification products for SCoT and IRAP markers were analyzed by electrophoresis in 1.80% agarose gels. The electrophoretic patterns of the products were recorded using a gel imaging system . Genotypes were built based on different DNA band patterns on gels.ί was calculated using PIC = 1 − ΣPί2 according to Botstein et al. [Pί is the allele frequency at locus ί. Nei’s genetic diversity index (ℎ) was calculated using POPGENE 1.31[Data matrices of Inter-simple sequence repeat and start codon targeted polymorphic marker profiles were generated by scoring (1) for presence and (0) for absence of individual allele. Polymorphism information content (PIC) is a property value of a marker based on its allelic number and distribution frequency in a population. PIC for marker n et al. , where PGENE 1.31. The res


Platycladus orientalis  is famous for its lifespan spanning hundreds, and even thousands, of years. Most ancient P. orientalis populations are widely distributed in China, with accessible historical records, making them valuable genetic resources. In this study, the distribution pattern of ancient P. orientalis in China was analyzed based on 13 bioclimatic factors. Additionally, a specific-locus amplified fragment (SLAF) sequencing method was applied to detect single nucleotide polymorphisms (SNPs) in the genomes of 100 accessions from 13 populations. The resulting data revealed that the suitable areas for the distribution of ancient P. orientalis populations were accurately predicted with four main climatic factors. A total of 81,722 SNPs were identified from 461,867 SLAFs for 100 individuals, with an average sequencing depth of 10.11-fold and a Q30 value of 82.75%. The pair-wise genetic distance and genetic differentiation of 13 populations indicated that the BT-T population exhibited the largest divergence from the other populations. A neighbor-joining phylogenetic tree suggested the relationship between many individuals was inconsistent with the geographical location, possibly indicative of a history of transplantation and cultivation. All individuals were clustered into nine genotypes according to a structural analysis and the relationships between individuals were clarified in phylogenetic trees. This study highlights the importance of the de novo genome sequencing of ancient P. orientalis and may provide the basis for the conservation of P. orientalis genetic resources, the identification of supergene families, and the evaluation of related genetic resources. Platycladus orientalis (L.) Franco (common name: Chinese thuja) is an evergreen tree species of the family Cupressaceae that is endemic to China, with the common name of Chinese thuja. This species is one of the most widely used trees for landscaping in China, and it was planted in some temples, mausoleums, and gardens thousands of years ago. Additionally, P. orientalis is highly resistant to drought and cold conditions, as well as diseases, and is widely distributed throughout China, with the exception of Qinghai and Xinjiang provinces , T4 DNA ligase (NEB), ATP (NEB), and an ScaI adapter. The restriction-ligation reactions were heat-inactivated at 65 °C, and the samples were digested with ScaI at 37 °C. The restriction-ligation samples (diluted) were used as the template for a PCR amplification with Taq DNA polymerase (NEB), dNTPs, and an EcoRV-primer containing a barcode. The E.Z.N.A.® Cycle Pure Kit  was used to purify the PCR products. Pooled samples were incubated at 37 °C with EcoRV, T4 DNA ligase, ATP, and a Solexa™ adapter , after which they were purified with a Quick Spin column  and separated on a 2% (w/v) agarose gel. Fragments that were 400–500 bp were isolated with the Gel Extraction Kit (Qiagen). These fragments were then used as the template for a PCR amplification with the Phusion™ Master Mix (NEB) and the Solexa Amplification primer mix to add the barcode. The amplified fragments that were 450–500 bp long were excised and purified as previously described and diluted for the paired-end sequencing, which was completed with the Genome Analyzer II high-throughput sequencing platform .For each accession, the CTAB method was used to isolate DNA from the dried leaves of a single plant for a subsequent SLAF-seq analysis . Because 38.51%)  based on 38.51%) . The resOryza sativa indica) data as a control to prevent false positive results [The sequence error rate was estimated using rice ( results . The Q v results . The Gen results  and SAMt results  were usePhylogenetic relationships were deduced from the SLAF-seq data. The SNPs were used to construct phylogenetic trees for all accessions according to the neighbor-joining method  in the MP. orientalis at the population and species level. Considerable genetic differentiation was observed among populations. A phylogenetic tree suggested the relationship between many individuals was inconsistent with the geographical location, possibly indicative of a history of transplantation and cultivation. All individuals were clustered into nine genotypes according to a structural analysis. On the basis of these findings, strategies have been proposed for conserving ancient P. orientalis populations.In summary, we herein reveal the genetic diversity of ancient


Pterois volitans through the Western Caribbean and the Gulf of Mexico. Here, we present a genetic analysis of lionfishes collected along Mexican coasts, examining their connectivity with other Caribbean localities  and the role of ocean currents on population structure. We collected 213 lionfish samples from seven locations comprising four countries. To evaluate genetic structure, mitochondrial control region and nuclear inter-simple sequence repeat markers were used. We found that lionfish collected along Mexican coasts show a similar haplotype composition (H02 followed by H01 and H04) to other Caribbean locations, and the H03 rare haplotype was not found. Haplotype composition in the southwest Gulf of Mexico suggests a discontinuity between the southern and northern areas of the Gulf of Mexico. The southern area clustered more strongly to the Caribbean region, and this is supported by the complexity of water circulation in the semi-enclosed region of the Gulf of Mexico. Mitochondrial genetic diversity parameters show small values, whereas nuclear markers produce medium to high values. Only nuclear markers highlighted significant genetic differentiation between the southwest Gulf of Mexico and Caribbean region, confirming a phylogeographic break between both regions. Separate analysis of Caribbean locations indicates restricted larval exchange between southern and northern regions of the Mesoamerican Barrier Reef System, potentially in response to regional oceanographic circulation.The recent expansion of the invasive lionfish throughout the Western Hemisphere is one of the most extensively studied aquatic invasions. Molecular studies have improved our understanding of larval dispersal, connectivity, and biogeographical barriers among lionfish populations, but none have included Mexican localities, an important area for the larval dispersal of  Pterois volitans L. 1758; Scorpaeniformes, Scorpaenidae) is an important and well-documented case of an aquatic invasive species. This species has recently invaded the Western Atlantic, and has rapidly dispersed throughout the Caribbean and Gulf of Mexico  has been demonstrated; however, the species has not exhibited genetic structure among the Gulf of Mexico and Caribbean region  what goes into the average reported in others studies (range 0.2%-15% see [PCR amplification and electrophoresis conditions of the anchored at 3’ end nuclear gene (ISSR) were conducted for 213 individuals, following Labastida et al.  using th markers –38 which markers . The mea-15% see ) indicatSequencher 4.1 , and sequences were subsequently aligned with Muscle [Mega 6.0 [The forward and reverse sequences were assembled and edited using h Muscle  in Mega Mega 6.0 .Pterois volitans in the invaded area [DnaSP 5 [h, also called genetic diversity) and nucleotide diversity (π) with Arlequin 3.5 [To identify haplotypes in the studied region, our dataset was compiled and compared with the nine haplotypes reported for ded area  using Dn[DnaSP 5 . Geneticquin 3.5 .ΦST values based on Tamura and Nei distances  using Arlequin 3.5, and the sequential Bonferroni correction, as implemented by Rice [JModelTest 2.1.9 [Arlequin 3.5 . To identify the most plausible phylogeographic breaks in the study region, different AMOVA scenarios were tested to maximize the percentage of variance among regions based on previous knowledge and hypothesis of phylogeographic breaks for lionfish , Nei’s gene diversity (h), total genetic diversity in the pooled samples (HT), mean diversity within each locality (HS), proportion of genetic diversity distributed among all localities (GST) using Popgene 1.31 [h values at each loci as variables. Those statistics were processed by Statistica 7.0.Candidate loci under natural selection were assessed using scan 2.1 , and theene 1.31 . To testPhiPT statistic, an analogue of Wright’s FST, determined for all pairs of populations with 9999 permutations using GenAlEx 6.4 [GenAlEx 6.4.Differentiation between localities was tested with the population pairwise AlEx 6.4 –51, and AlEx 6.4 . Followih: 0.393–0.480 and π: 0.00177–0.00230; High quality mtDNA control region sequences were obtained for a total of 176 lionfish individuals in the studied area, producing a final alignment length of 679 bp. The number of polymorphic sites varies from three to nine, determining a haplotype number of two or three, depending on the locality . As prevΦST values were very low and not significant  for 210 individuals among the seven localities. Analysis for candidate loci under natural selection identified one locus under “very strong” evidence for selection, and the alpha value (α = 1.47) suggests diversifying selection. Consequently, the locus under selection was excluded from our dataset. Private bands  obtained by ISSR were commonly used to distinguished related species –54; we dP = 93.6% and h = 0.261±0.170) even for Mexican localities (P = 87.2% and h = 0.253±0.174). At the locality level, genetic diversity parameters (P = 56% and h = 0.188) and higher values for the Belize locality (P = 86% and h = 0.271). The Kruskal-Wallis test indicated a significant difference among Nei’s gen diversity (h) , and the multiple comparison test detected significant difference between the Veracruz and Belize localities. Total variation (HT) was 0.263 (±0.028) and mean genetic variation within samples (HS) was 0.233 (±0.024). Coefficient of genetic differentiation (GST) was 0.113.Considering the whole dataset, genetic diversity parameters were high  between the southwest Gulf of Mexico and the Mexican Caribbean region as indicated by the most probable scenario (number 2) of phylogeographic break  for Diplodus species , whereas Machkour-M’Rabet et al. [Strombus gigas L. 1758 . It should be noted that the maximum value of expected heterozygosity using dominant genetic markers is 0.5 because only two alleles are produced at each locus [The low genetic diversity of lionfish observed in our study using mtDNA was expected due to the founder effect associated with the rapid colonization by an invasive species. Our results were slightly lower but generally consistent with those previously reported –9, 36. Ht et al.  revealedch locus . The lowch locus , as democh locus . The firch locus  above thch locus . Based och locus , reinforΦST and AMOVA results), whereas nuclear ISSR presented a high and significant separation between the southwest Gulf of Mexico and the Caribbean region . This kind of discrepancy between mitochondrial and nuclear genetic markers in population structure studies has already been demonstrated [S. gigas; [Eretmochelys imbricata L. 1766; Testudines, Cheloniideae; [Montastraea annularis Ellis & Solander 1786 , the foniideae; ), and folinidae; , using mlinidae; ).The very recent invasion of the southern area of the Gulf of Mexico, probably remained undetected by mtDNA, while the ISSR genetic markers that are capable of highlighting contemporary events , 67–68 cNassau grouper and Mutton snapper) [An analysis of the Caribbean region using ISSR genetic markers has shown a restricted connectivity between Caribbean locations considered in this study such as the Xcalak and Belize sites, suggesting new breaks in the dispersal capacity of lionfish. Considering the larval period of the lionfish , this ssnapper) .A previous study  suggestsS1 FigEach circle represents a haplotype, the size corresponds to haplotype frequency, and the colors correspond to the localities sampled. Brown circles represent other haplotypes present in the Atlantic region, but that were not encountered in this study. Lines connecting haplotypes represent one mutational step and small grey circles denote missing intermediate haplotypes. Localities abbreviation: Puerto Rico (PR), Belize (BZ), Xcalak (XC), Banco Chinchorro (BC), Puerto Morelos (PM), Cuba (CU), Veracruz (VC).(TIF)Click here for additional data file.S2 FigA) IBD considering all localities for the Caribbean region, (B) IBD at small spatial scale (without Puerto Rico data).((PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(A) including all individuals, (B) without individuals from Veracruz locality. Best scenarios are in grey. For details about scenarios tested see (PDF)Click here for additional data file.S4 TableNumber of individuals (n), haplotype designated (H01-H02-H03-H04).(PDF)Click here for additional data file.