Leptoxis compacta, is a narrow range endemic pleurocerid from the Cahaba River basin in central Alabama that has seen rapid range contraction in the last 100 years. Such a decline is expected to negatively affect genetic diversity in the species. However, precise patterns of genetic variation and gene flow across the restricted range of L. compacta are unknown. This lack of information limits our understanding of human impacts on the Cahaba River system and Pleuroceridae. Here, we show that L. compacta has likely seen a species-wide decline in genetic diversity, but remaining populations have relatively high genetic diversity. We also report a contemporary range extension compared to the last published survey. Our findings indicate that historical range contraction has resulted in the absence of common genetic patterns seen in many riverine taxa like isolation by distance as the small distribution of L. compacta allows for relatively unrestricted gene flow across its remaining range despite limited dispersal abilities. Two collection sites had higher genetic diversity than others, and broodstock sites for future captive propagation and reintroduction efforts should utilize sites identified here as having the highest genetic diversity. Broadly, our results support the hypothesis that range contraction will result in the reduction of species-wide genetic diversity, and common riverscape genetic patterns cannot be assumed to be present in species facing extinction risk.Many freshwater gastropod species face extinction, including 79% of species in the family Pleuroceridae. The Oblong Rocksnail,  Freshwater gastropods of the United States suffer one of the highest imperilment rates of any taxonomic group in North America . DespiteLeptoxis ampla  and little research attention . Pleurocis ampla . Many frdistance . HoweverLeptoxis compacta  can provide data that will enhance L. compacta management options (L. compacta will likely require reintroduction efforts to previously occupied habitat(s). Maintaining genetic diversity of imperiled species is important for mitigating extinction risk  Leptoxis compacta has undergone a severe genetic bottleneck and (2) genetic diversity of L. compacta is considerably lower than L. ampla, a sympatric and wider ranging species. We also examined how genetic diversity of L. compacta varies across its current range, specifically assessing whether broad patterns seen in many other riverine taxa like isolation by distance and strong genetic structure are seen in L. compacta.In this study, we used 2bRAD-seq  to generLeptoxis compacta was collected during two trips to the Cahaba River in June 2018 and June 2019. We collected L. compacta at four sites, and all sites except Cahaba River above Shades Creek were outside the previously documented contemporary range of L. compacta .A reduced representation genomic library was generated for genotyping using the AlfI enzyme and the 2bRAD library prep protocol of L. compacta samples pooled in equimolar concentrations and sequenced on a single lane. The other individuals were pooled in equimolar concentrations with samples from projects on conservation genomics of other pleurocerid species, and 87 individuals were sequenced per HiSeq 4000 lane. Although batch effects in RADseq data have been recently noted in studies that used different read lengths among sequencing runs  for any read that had five or more base pairs with Phred quality scores less than 20. Reads were processed with scripts from SHRiMP 2.23 (http://github.com/Eli-Meyer/2bRAD_utilities). As this step removes any sequence that is not part of the RAD-locus, adaptor sequences and non-target sequences are removed from the sequencing reads. RAD-loci, defined as the stretch of DNA cut by the AlfI enzyme, were assembled with the STACKS 1.48 pipeline denovo_map.pl as no reference genome is available for L. compacta. For denovo_map.pl parameters, we set minimum stack depth to five (-m 5), distance allowed between stacks to three (-M 3), and distance between catalog RAD-loci to two (-n 2). These parameters were determined to be most appropriate for our data following denovo_map.pl parameters were set to defaults.Raw Illumina reads were demultiplexed with the STACKS 1.48  module piMP 2.23  and subspopulations. In order to pass filtering steps, a RAD-locus had to be present in 75% of individuals from any given collection site and also present at three collection sites. RAD-loci that had a minimum minor allele frequency of less than 2.5% or heterozygosity higher than 50% were removed to limit the influence of paralogy and misassembly on final datasets. Sequencing coverage of RAD-loci with SNPs was measured with vcftools , expected heterozygosity (He), nucleotide diversity (Π), and FIS at each collection site were calculated by populations. The number of private alleles at each site was also reported by populations. Average allelic richness (Ar) was calculated with the R . We used the multiple SNPs per RAD-locus dataset and the R package adegenet  and the 2bRAD library prep protocol are available on FigShare (DOI: 10.6084/m9.figshare.12014619).All bash and R scripts used for processing and analyzing data are available at github.com/nathanwhelan. Demultiplexed raw Illumina reads have been uploaded to NCBI under BioProject L. compacta from Cahaba River at old Marvel slab upstream to Cahaba River at Booth’s Ford .DNA yields for two individuals were too low for library preparation so only 19 individuals were sequenced from Cahaba River at old Marvel Slab and Cahaba River at Booth’s Ford. The number of demultiplexed raw reads per individuals varied from 930,062-10,146,649 . Much of the variation in raw reads can be attributed to whether the individual was sequenced on a HiSeq 4000 lane with 48 or 87 samples. Aside from raw read number, we saw no evidence of batch affects like individuals from one sequencing run all clustering together in analyses (see below). After initial raw read filtering, the number of reads that passed quality filtering steps ranged from 865,314–9,838,187 . Assembly with the STACKS o at each collection site ranged from 0.0963–0.1568, and He ranged from 0.0980 to 0.1801 .The number of private alleles at each site ranged from 28–262 . Ho at eo 0.1801 . At each than He . The difectively . FIS val4–0.1934 , with thFST values among sites ranged from 0.0–0.055 . According to the AMOVA, significant genetic structure was present among collection sites (p = 0.004), but only 4.16% of variation was explained by collection site. In contrast, 81.8% of genetic variation was explained by within individual variation, further indicating high amounts of gene flow among collection sites.Pairwise .0–0.055 . We founK = 2). Most individuals from across the range of L. compacta had a genomic admixture profile that was dominated by a genomic background from a single ancestral population, likely indicating that overall genomic diversity has been lost across the range of L. compacta. Nevertheless, 14 individuals had varying levels of admixture with a second ancestral population  than all but two sites sampled for us study . Therefoon sites  were mucL. ampla . We thinL. compacta, a small number of individuals with a less common genomic background suggests that the species has lost genetic diversity through bottleneck and drift. Patterns seen in DAPC, ADMIXTURE, and fineRADstructure were not driven by geography as individuals with the less common genomic background were not found in adjacent sites (orange in DAPC and ADMIXTURE plots and upper right corner of fineRADstructure plot; L. compacta would have to exist elsewhere in the Cahaba River. The most likely hypothesis for explaining observed clustering and co-ancestry profiles (L. compacta was genetically diverse across its historical range prior to decline, but range contraction caused a considerable loss of genetic diversity. In turn, genetic drift resulted in the observed coancestry pattern of one ancestral population being more common in extant individuals (Given the well-documented decline of profiles –5 is a gividuals  and 5.L. compacta can be characterized by a single population with some subpopulation structure at Cahaba River above Shades Creek and Cahaba River at Booth’s Ford (o values compared to He values and the higher FIS values seen in collection sites with inferred subpopulation structure (FIS and He is null alleles. However, null alleles are unlikely as they would increase pairwise FST values (L. compacta to persist with greater genetic diversity as the species declined in the 20th century.Broadly, genetic structure across the current range of h’s Ford ; Fig. S1h’s Ford . That istructure ; Table 2T values  that areT values –5. TheseLeptoxis compacta suffered a massive decline during the 20th century, a period of intense mining, forestry, and urban development in the Cahaba River drainage (L. compacta was considered extinct less than a decade ago. Fortunately, L. compacta is not extinct. However, conservation efforts are needed to ensure the long-term survival of L. compacta as the species is at risk from both chronic habitat degradation and one-time catastrophic events. Two potential management strategies for L. compacta are habitat restoration and reintroduction with captively reared individuals.drainage . The decL. compacta. One site, Cahaba River at old Marvel Slab, was previously the focus of intense habitat restoration through the removal of a low-level dam (L. compacta was not found at this site by L. compacta could not survive or from a point of considerably lower carrying capacity. As the only undammed, major river in the southeastern United States, the Cahaba River is much less modified than most other systems in the southeast. Our findings suggest that imperiled gastropods will benefit from water quality and habitat improvements even in relatively “pristine” river systems. Improving habitat, or identifying suitable habitat, will be a necessary starting point for L. compacta reintroduction efforts.In this study, we report an 8.26 km known range extension for evel dam . The sitevel dam  as the sevel dam . ConsideL. compacta only exists along a single river path. This means that one catastrophic event such as a massive point source pollution event above Cahaba River at Booth’s Ford could result in extinction of L. compacta. Such an event is not merely a hypothetical. In 2016, a gasoline pipeline spill came perilously close to the Cahaba River (L. compacta. Once a suitable reintroduction site is chosen, managers will need to choose a broodstock site. This decision should be informed with genetic data. The absence of an isolation by distance effect across the current range of L. compacta indicates that managers do not need to prioritize potential broodstock sites based on whether they are geographically proximate to reintroduction sites. Rather, sites with high genetic diversity and ease of access should be prioritized for broodstock. Therefore, the Cahaba River above Shades Creek is likely an ideal broodstock location. Moreover, L. compacta is easy to sample and relatively easy to distinguish from other sympatric species at Cahaba River above Shades Creek, making it ideal from both a genetic and sampling standpoint.In addition to having a small range, ba River . To mitiL. compacta was considered extinct less than a decade ago, we now know more about this species than most other freshwater gastropods. This is helpful for conservation of L. compacta as the biggest barrier to effective management strategies for most freshwater gastropods is a lack of data. Future research efforts should focus on differences in dispersal dynamics among pleurocerids and causes of differences in riverscape genetic patterns seen between L. ampla and L. compacta. As more population genomic data becomes available for pleurocerids, we will be better suited to develop strategies to conserve these critically important components of many North American riverine ecosystems.Even though 10.7717/peerj.9789/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9789/supp-2Supplemental Information 2Tick marks represent individuals. Lcompop_01-20: Cahaba River at old Marvel slab; Lcompop_21-40: Cahaba River above Shades Creek; Lcompop_41-60: Cahaba River at Booth’s Ford; Lcompop_61-80: Cahaba River at canoe launch.Click here for additional data file.


Local cattle breeds need special attention, as they are valuable reservoirs of genetic diversity. Appropriate breeding decisions and adequate genomic management of numerically smaller populations are required for their conservation. At this point, the analysis of dense genome-wide marker arrays provides encompassing insights into the genomic constitution of livestock populations. We have analyzed the genetic characterization of ten cattle breeds originating from Germany, The Netherlands and Denmark belonging to the group of red dairy breeds in Northern Europe. The results are intended to provide initial evidence on whether joint genomic breeding strategies of these populations will be successful.Traditional Danish Red and Groningen White-Headed were the most genetically differentiated breeds and their populations showed the highest levels of inbreeding. In contrast, close genetic relationships and shared ancestry were observed for the populations of German Red and White Dual-Purpose, Dutch Meuse-Rhine-Yssel, and Dutch Deep Red breeds, reflecting their common histories. A considerable amount of gene flow from Red Holstein to German Angler and to German Red and White Dual-Purpose was revealed, which is consistent with frequent crossbreeding to improve productivity of these local breeds. In Red Holstein, marked genomic signatures of selection were reported on chromosome 18, suggesting directed selection for important breeding goal traits. Furthermore, tests for signatures of selection between Red Holstein, Red and White Dual-Purpose, and Meuse-Rhine-Yssel uncovered signals for all investigated pairs of populations. The corresponding genomic regions, which were putatively under different selection pressures, harboured various genes which are associated with traits such as milk and beef production, mastitis and female fertility.This study provides comprehensive knowledge on the genetic constitution and genomic connectedness of divergent red cattle populations in Northern Europe. The results will help to design and optimize breeding strategies. A joint genomic evaluation including some of the breeds studied here seems feasible. The genetic diversity of cattle breeds is shaped by evolutionary forces such as genetic drift, migration, selection and geographical separation. Livestock breeds differ greatly from natural populations since they have experienced strong human-mediated selection and directed mating decisions. As a result, a large number of highly specialized breeds has evolved to meet a variety of human needs . FurtherIn Northern Europe, various red cattle breeds with small to medium population sizes exist. While their occurrence is often confined to restricted geographical regions, such traditional breeds are known to be well adapted to their prevailing environmental conditions . HoweverThe breeding programs of the Northern red dairy cattle breeds are primarily organized at the national level, and especially for the smaller populations, their aim is to maintain genetic diversity in order to keep the breeds viable. However, running breeding programs for small-sized populations managed by commercial breeding organizations is often expensive, inefficient, and returns of investment are limited. Thus, from an economic and practical point of view, cooperation across breeding companies that aim at the establishment of a common reference population for genomic prediction is expected to be advantageous . InvestmIn large populations, genomic selection has accelerated genetic gain mainly by reducing the generation interval in dairy cattle breeding , 12. HowRecently, various studies have successfully shed light on the patterns of genetic diversity and the relatedness of cattle populations in Europe and worldwide –24. HoweBos taurus genome reference assembly ARS-UCD1.2 [Genomic data of ten red cattle breeds from Germany , The Netherlands  and Denmark  were available for the analyses in this study. Genomic data for German breeds were provided by the breeding organization Rinderzucht Schleswig–Holstein (RSH e.G.). The genotypes of Dutch cattle breeds were obtained from the Center for Genetic Resources (CGN) in The Netherlands, and genomic data of Traditional Danish Red were provided by Aarhus University. Animals had previously been genotyped with four SNP chips . Detailed information on the samples is in Additional file S-UCD1.2  availablS-UCD1.2 . Second,o) and average expected heterozygosity (He), were estimated with PLINK v1.09 [In order to assess within-breed genetic variation, the following parameters i.e., average minor allele frequency (MAF), average observed heterozygosity  between the alleles at two loci according to Hill and Robertson [r2 in each bin was plotted.To explore the overall levels of linkage disequilibrium (LD) in the breeds studied, the genome-wide pairwise LD was estimated for SNPs on the same chromosome and less than 2 Mb apart using PLINK v1.09 . LD was obertson . For theTo investigate the population structure and genomic variability within and across cattle breeds and to detect admixture, we applied three complementary approaches: principal component analysis (PCA), ADMIXTURE and TreeMix. In order to avoid oversampling of some breeds for the analyses of population structure , a datasBos taurus breed). Phylogenetic trees with migration events from 0 to 10 were constructed. The number of migration events that best fitted the data was identified using the fraction of the variance in the sample covariance matrix explained by the model covariance matrix [z score indicates that a population has arisen by admixture of the two source populations. For the calculation of the f3-statistics, for all possible combinations of populations, blocks of 1000 SNPs were used and a Bonferroni adjustment of the p-value was applied.A maximum likelihood based phylogenetic tree was constructed using TreeMix v1.13  to evalue matrix . Each ane matrix . Furthere matrix . This alST values were estimated using the estimator of Weir and Cockerham [To gain additional insight into the genetic relationships among the populations and to measure the degree of population differentiation, average genome-wide Fockerham  in PLINKockerham . Geneticrehh [Recurrent artificial selection can result in signatures of selection in the genome , which cage rehh . Informaa et al. . Accordia et al. , the staentclass1pt{minimaThe cross-population i et al. , was useAs for To visualize the detected signatures of selection, average a Viewer .o ranged from 0.303 ± 0.189  to 0.375 ± 0.129 (German Angler). Similarly, He was lowest for the Traditional Danish Red (0.296 ± 0.176) and Groningen White-Headed (0.302 ± 0.172) breeds, and highest for the German Angler (0.369 ± 0.122) and Red Holstein (0.359 ± 0.132) breeds indicating that Traditional Danish Red and Groningen White-Headed are less diverse populations.The estimated genetic diversity indices for the ten red breeds are in Table ROH were detected in all breeds, but their length and frequency differed among populations. Additional file mentclass2pt{minimThe LD decay as a function of inter-marker distance differed between breeds  explained 18.9% of the total variance in the given dataset. PC1 explained 5.27% of the total variance and clearly separated Traditional Danish Red from the other breeds  and from Red Holstein and Meuse-Rhine-Yssel to Red and White Dual-Purpose (z score = − 15.30). In addition, a migration event from either Holstein Friesian or Meuse-Rhine-Yssel to French Red Pied Lowland (z score = − 10.77) was confirmed by the f3-statistics  to 0.168  , French Red Pied Lowland and Deep Red (0.018), Red and White Dual-Purpose and French Red Pied Lowland (0.018), and Holstein Friesian and Red Holstein (0.018). In addition, the genetic differentiation between Red Holstein and German Angler was low (FST = 0.021) and that between Red Holstein and Red and White Dual-Purpose (FST = 0.053) was slightly lower than that between Red Holstein and Meuse-Rhine-Yssel (FST = 0.082).We estimated the genome-wide FT values  to measuBos taurus (BTA) chromosome 18 between 16.5 and 33.5 Mb. The annotated genes on BTA18 include ZNF423 (16.5–18.0 Mb), ADCY7, NKDI, BRD7 (18.5–19.0 Mb), FTO, IRX3, IRX6, and RPGRIP1L (21.5–26.0 Mb). In the German dual-purpose breed, the strongest evidence of selection was found on six chromosomes: BTA3, 9, 15, 20, 26 and 27. On BTA9, several genes are mapped within the identified genomic region (59.0–62.5 Mb), e.g., BACH2, RARS2, SLC35A1, and CFAP206. In the Red and White Dual-Purpose breed, putative signals of positive selection were detected on BTA15, which contained the KCNJ11 and NUCB2 genes (35.0–37.0 Mb) and the EIF4G2 and SBF2 genes (41.5–42.5 Mb). In addition, the results revealed variants in the genomic region from 39.0 to 41.0 Mb on BTA20, which is assumed to be under positive selection pressure and which includes the RAI14, SLC45A2, TTC23L and ADAMTS12 genes. A genomic region (21.5–23.5 Mb) under putative positive selection on BTA26 harbors the SLF2, LBX1 and BTRC genes. A signature of selection on BTA27 (30.5–34.5 Mb) contains the genes UNC5D and KCNUI. In the Dutch Meuse-Rhine-Yssel, the strongest signatures of selection were located on three chromosomes: BTA1 (148.5–150.0 Mb), a region that carries the PIPG gene, BTA3 (22.0–26.5 Mb), and BTA15 with the 34.0–37.0 Mb region containing the GRAMD1B gene and the 9.0–42.5 Mb region containing the EIF4G2 and ZBED5 genes.For the Red Holstein, Red and White Dual-Purpose, and Meuse-Rhine-Yssel breeds, putative signatures of selection are in Table MC1R gene. Furthermore, the region between 16.0 and 23.0 Mb contains several genes, e.g., ZNF423, ADCY7, NKD1, BRD7, FTO, IRX3 and RPGRIP1L. By comparing the Red Holstein and Meuse-Rhine-Yssel populations, we detected signatures of selection on BTA15 and 18. On BTA18, the signatures of selection between 15.0 and 15.5 Mb included the VPS35 gene and between 25.0 and 27.0 Mb the NDRG4 gene. Interestingly, the comparisons of Red Holstein with either Red and White Dual-Purpose or Meuse-Rhine-Yssel, showed that they shared some signatures of selection on BTA18 between 16.0 and 24.0 Mb. However, the ZNF215 in the 45.5–46.0 Mb region and P4HA3, KCNE3, CHRDL2 and NEU3 in the 53.5–56.5 Mb region, and on BTA21, two signatures of selection were found, i.e. one between 12.5 and 13.0 Mb with the candidate genes MCTP2 and one between 21.5 and 22.0 Mb with the candidate gene IQGAP1.As Fig. A continuous accumulation of inbreeding within a population is unavoidable if the number of breeding animals is finite , 59. TheIn this study, patterns of ROH and levels of genomic inbreeding differed both within and across the populations studied. The large amount of long homozygous DNA segments found in Traditional Danish Red and Groningen White-Headed represents recent inbreeding and might reflect a breeding population of limited size and consequently, mating between related animals. In Traditional Danish Red, it also reflects, at least in part, a strong reliance on semen collected long ago in the Danish national gene bank. The high inbreeding of Traditional Danish Red was also shown by Zhang et al.  and was The lowest amount of inbreeding was observed in German Angler. These results are in line with findings from Addo et al.  who repod et al. . ParametST value was lowest for this combination of breeds. It is assumed that these two dual-purpose populations originate from the same genetic source, but have been kept and managed in two distinct geographical regions. Moreover, since several years, the exchange of sires between these populations contributes to an increase in genetic similarity. Our study showed a larger influence of Red Holstein genes on Red and White Dual-Purpose than on Meuse-Rhine-Yssel. Red Holstein was frequently used in Red and White Dual-Purpose in order to improve milk yield, and Addo et al. [ST and gene flow between Deep Red and Meuse-Rhine-Yssel revealed by the phylogenetic tree. Genetic connectedness between these breeds was already described by Marjanovic et al. [Population structure and genetic connectedness of the cattle breeds analyzed in our study was assessed using three complementary approaches PCA, ADMIXTURE, and TreeMix. The results of the analyses agree well with each other. All the methodologies applied indicated close relationships and a similar genetic background for Red and White Dual-Purpose and Meuse-Rhine-Yssel. In the PCA plot, a considerable proportion of the variance was shared between these breeds. Furthermore, ADMIXTURE revealed a similar genetic background for these two breeds, and the phylogenetic tree indicated low genetic drift. In addition, the pairwise Fo et al.  identifio et al. . In the o et al. . Nowadayo et al. . In ordec et al.  and van c et al. . Dutch Ic et al. , 77. RecST values were highest between Traditional Danish Red and all other breeds, which indicates a high degree of genetic differentiation. The unique genetic background and a low level of admixture of Traditional Danish Red were also confirmed by Gautason et al. [Our study uncovered the highest degree of genetic differentiation for Traditional Danish Red compared to all other breeds. The PCA showed that the Traditional Danish Red breed was clearly separated from all other breeds. On the one hand, this is caused by a different geographical origin. On the other hand, the ADMIXTURE analysis confirmed a unique genetic background of Traditional Danish Red and a low level of admixture with other breeds. In addition, pairwise Fn et al. . In our n et al. , 79. Fron et al. . In locan et al. , 81. Hown et al. , 83. Thin et al. . The strn et al. , who repBRD7 gene and was previously described as associated with protein yield in Holstein cattle [FTO gene (22.0–22.5 Mb). Zielke et al. [BACH2 was identified as a key gene involved in the metabolism of milk fatty acids in dairy cattle [RARS2, SLC35A1 and CFAP206 genes. On BTA15, the EIF4G2 gene was located within a genomic region that influences lactation persistency in a genome-wide association study conducted by Do et al. [SBF2 was in close proximity to a SNP linked to growth traits in Charolais cattle [RAI14 and ADAMTS12 genes. RAI14 is located close to genomic regions associated with susceptibility of clinical mastitis in Holstein Friesian [ADAMTS12 gene has been reported in various genome-wide association studies for traits such as carcass weight and milk production [GRAMD1B gene that flanks the genomic region around the signature of selection at 34.3 Mb on BTA15 is associated with female fertility in Nordic Red cattle [In order to investigate whether directional selection occurred in the Red Holstein, Red and White Dual-Purpose, and Meuse-Rhine-Yssel breeds, genome-wide SNPs were used to detect signatures of selection within and across these breeds. Signatures of selection are assumed to occur in genomic regions that host essential genes with a role in the phenotypes that were selected for . For then cattle , 91. In e et al.  identifiy cattle . Recently cattle  reportedo et al. . Moreoves cattle . On BTA2Friesian  and milkFriesian . The ADAoduction –101. In d cattle . Furtherd cattle  detectedMC1R gene, which is responsible for variation in red coat color [BRD7 and FTO) are mainly associated with milk production traits and our results suggest directed selection for these traits in Red Holstein. The comparison of Red Holstein with Meuse-Rhine-Yssel revealed a high VPS35 gene. In Swedish Red cattle, Duchemin et al. [VPS35 gene that is associated with non-coagulating milk. Furthermore, the study of Pimentel et al. [NDRG4 gene (BTA18 at 26.3 Mb) that is significantly associated with fat and protein yield, interval from calving to first insemination, and days open in cows. XPEHH-analysis between Red and White Dual-Purpose and Meuse-Rhine-Yssel revealed a set of putative signals on BTA15 between 45.5 and 67.0 Mb. A genome-wide association study in Holstein cattle discovered a significant SNP for milk yield located in the P4HA3 gene at 54.4 Mb on BTA15 [CHRDL2 and NEU3 genes on BTA15. Beyond that, NEU3 was demonstrated to have functional effects on fertility and production traits in Holstein Friesian [MCTP2 gene, which was previously reported as a candidate gene for traits related to carcass merit and metabolic weight in different cattle breeds [IQGAP2 gene that is strongly associated with sole hemorrhage in Holstein cattle [The XPEHH analysis of Red Holstein and Red and White Dual-Purpose showed directional selection pressure in Red Holstein in the genomic region between 14.0 and 15.5 Mb on BTA18. Similarly, in Red Holstein, Rothammer et al.  reportedat color . Red Holn et al.  found anl et al.  showed aon BTA15 . Moreoveon BTA15  reportedFriesian . In our e breeds , 111. Thn cattle .In this study, different population genetic analyses were used to investigate the population structure and connectedness of certain red cattle breeds from Northern Europe at the genomic level. Ultimately, the objective is to create a multi-breed reference population in order to ensure a sustainable development and the conservation of these native breeds. Our results provide initial evidence that some of the red cattle populations studied  might benefit from a common prospective multi-breed genomic evaluation provided that the number of animals included in the training population is sufficiently large . HoweverThis study represents a detailed genetic characterization of ten cattle breeds belonging to the Red group in Northern Europe. The results indicate that some breeds are genomically distinct , whereas other populations show strong genetic similarity . Furthermore, we show that some breeds  have experienced intense gene flow in the past from higher yielding breeds such as Red Holstein. The results are relevant for application by breeding organizations and breed associations in order to guide and develop prospective breeding strategies.Additional file 1: Table S1. Information on breeds, sample size per breed, source and type of SNP chip.Additional file 2: Table S2. Details on data processing. For each analysis, steps of quality control and the size of used dataset are specified.Additional file 3: Table S3. Descriptive statistics of frequency and size of ROH per breed.Additional file 4: Table S4. Average level of genomic inbreeding (FROH) for different length categories with standard deviation in parenthesis per breed.Additional file 5: Figure S1. Linkage disequilibrium (LD) decay across the genome as a function of inter-marker distance for each breed. ANG: German Angler. DBE: Dutch Belted. DFR: Dutch Friesian Red. DR: Deep Red. GWH: Groningen White-Headed. IR: Improved Red. MRY: Meuse-Rhine-Yssel. RDM70: Traditional Danish Red. RDN: Red and White Dual-Purpose. RH: Red Holstein.Additional file 6: Figure S2. Genetic relatedness among the red cattle breeds from Germany, The Netherlands and Denmark (circles) and further European cattle breeds from the WIDDE database (triangles) using principle component analyses . ANG: German Angler, AYR: Finnish Ayrshire, BRV: Braunvieh, BSW: Brown Swiss, DBE: Dutch Belted, DFR: Dutch Friesian Red, DR: Deep Red, GNS: Guernsey, GWH: Groningen White-Headed, HOL: Holstein Friesian, IR: Improved Red, JER: Jersey, MON: Montbeliarde, MRY: Meuse-Rhine-Yssel, NRC: Norwegian Red Cattle, PRP: French Red Pied Lowland, RDM70: Traditional Danish Red, RDN: Red and White Dual-Purpose, RH: Red Holstein, SHO: Shorthorn, SIM: Simmental.Additional file 7: Figure S3. Plot of ADMIXTURE cross validation error from K = 2 to K = 40 revealed lowest cross validation error at K = 8.Additional file 8: Figure S4. Unsupervised model-based clustering results of 393 individuals using 19,717 SNPs. Presented is K = 2 to K = 12. ANG: German Angler. DBE: Dutch Belted. DFR: Dutch Friesian Red. DR: Deep Red. GWH: Groningen White-Headed. IR: Improved Red. MRY: Meuse-Rhine-Yssel. RDM70: Traditional Danish Red. RDN: Red and White Dual-Purpose. RH: Red Holstein.Additional file 9: Figure S5. Unsupervised model-based clustering results of 653 individuals from 21 breed. Presented is K = 10 to K = 18, with optimal number of ancestral populations K = 18. ANG: German Angler, AYR: Finnish Ayrshire, BRV: Braunvieh, BSW: Brown Swiss, DBE: Dutch Belted, DFR: Dutch Friesian Red, DR: Deep Red, GNS: Guernsey, GWH: Groningen White-Headed, HOL: Holstein Friesian, IR: Improved Red, JER: Jersey, MON: Montbeliarde, MRY: Meuse-Rhine-Yssel, NRC: Norwegian Red Cattle, PRP: French Red Pied Lowland, RDM70: Traditional Danish Red, RDN: Red and White Dual-Purpose, RH: Red Holstein, SHO: Shorthorn, SIM: Simmental.Additional file 10: Figure S6. Plot of f-index, representing the fraction of the variance in the sample covariance matrix explained by the model covariance matrix, as criteria for model fitting for number of migration events from 0 to 10.Additional file 11: Table S5. Negative z-scores obtained from the f3-statistics in THREEPOP.Additional file 12: Table S6. Pairwise genome-wide FST values (based on Weir and Cockerham [ockerham ) for allAdditional file 13: Table S7. Full list of annotated genes located in genomic regions detected as putative selection signatures.


Previous studies indicate that migrant integration is associated with migrants’ characteristics as well as restrictions and opportunities in receiving cities. However, the effect of receiving cities and the relationship between migrants and receiving cities have not been fully explored due to the lack of large samples from cities. The objective of this study is to examine the effects of receiving cities alone and their regulating role in the interaction with individual characteristics.Cross-city data on 154,044 Chinese domestic migrants above 15 years old in 289 cities from the 2017 China Migrants Dynamic Survey are used. Migrant integration is assessed by a four-dimensional model proposed by Esser, which is slightly adjusted according to the characteristics of Chinese migrants. A hierarchical linear model is used to measure the proportion of effects of city factors in migrant integration as well as the effects when city factors are considered alone and in interaction with individual factors.The individual-level and city-level factors are responsible for 69.81% and 30.19% of the effect on migrant integration, respectively. City political factors do not affect migrant integration directly, and cities with larger sizes and higher wages can directly and significantly improve integration, while higher housing prices will directly inhibit integration. From the cross-level interaction of city and individual, different social, economic and political factors at the city level have an indirect impact on migrant integration by inhibiting or strengthening the effect of individual-level factors on migrant integration.This study is one of the first to show the effect of cities and the relationship between receiving cities and migrants on migrant integration by keeping the national context constant. It is necessary to weaken the social and economic privileges associated with a city’s administrative level and reduce the negative impact of cities’ social and economic conditions by implementing city agglomeration, developing advantageous industries and optimizing the industrial structure. It is also essential to improve migrants’ socioeconomic capital through social support, occupation training and contiguous education. The large-scale integration of domestic migrants has become an important problem for Chinese urbanization in recent years. The rate of urbanization in China currently has exceeded 50%. However, urbanization is not the only process of population agglomeration in cities through migration from rural areas; the transformation of modes of production and life through citizenization has also contributed , 2. At pIn the context of the concern about migrant integration, literature and policy debates have often focused on the incompatibility of individual characteristics, including narrow social networks and human capital , limitedIn a context in which large-scale domestic migration has become widespread, the integration of migrants in receiving cities has become a problem that all cities need to pay close attention to. Specific corresponding countermeasures must be put forward by receiving cities according to their unique characteristics. Therefore, it is of great practical value to study the interaction between migrants and receiving cities. Specifically, this paper explores this issue from three aspects innovatively. (I) The proportion of the effects of individual-level and city-level factors is distinguished. (II) The effects of city-level factors and individual-level factors are examined separately, and the cross-level effects of migrants and cities are also measured. (III) The application of large-sample data can make the empirical analysis more stable and reliable and provide guidance for cities in China.The rest of this study is organized in five sections. Section 2 presents a brief literature review. Section 3 introduces the data sources and methodology. Section 4 presents the main findings. Section 5 discusses the findings and provides suggestions. Section 6 highlights the conclusions.The definition and concept of migrant integration were not particularly clear in early studies due to conceptual arbitrariness in the social science on the one hand and scientific controversies on the other hand. The adaptation, acculturation, assimilation and incorporation emerged . AdaptatThere are few detailed and empirical measurements of migrant integration in models whose subjects are immigrants before 1965, and migrant integration is described qualitatively , 15. ① RTo date, the multidimensional measurement of migrant integration has basically formed and guided contemporary models. The model of intergenerational integration distinguishes four dimensions of integration: the cultural dimension, which refers to the acquisition of skills and knowledge of language, customs and lifestyle; the structural dimension, which is the core of participation in the labor market and occupation; the social dimension, which refers to the interaction and contact with autochthonous people; and the emotional dimension, which concerns identity and belonging . The socThe research on the influence mechanisms of migrant integration has experienced a change from focusing on individual characteristics to focusing on both the individual and the context.The early models focus on the characteristics of the individual as well as the interaction between individuals. The differences in language, religion, race and culture between migrants and locals are the objective basis for the integration. These differences bring about inevitable conflict and potential integration for migrants and locals. Competition between individuals in valued economic and social conditions are first considered as the driving force of conflicts and assimilation , and theThe receiving society’s characteristics have begun to be mentioned with the deepening study of migrant integration. With the extension of the residence time of the migrants in the receiving locations, migrant integration differs in different places, and the role of the environment has gradually been recognized. However, as with individual factors, the emphasis is on the context’s various attitudes towards migrants, which can vary among “pressing, willing, indifferent, unwilling or blocking” and may cause different states of migrant integration: monistic, pluralistic, and interactionist . The focBoth individual and environmental factors and their interplay have been considered in depth with the important contribution and influence of immigrants to the receiving place. The interplay of migrants’ motives and skills and the receiving society’s opportunities and restrictions influences migrant integration , 27–30. Even if there are nuanced descriptions and explanations of migrant integration, this field remains fragmented, and the applicability of existing models in China remains to be examined. First, the study objectives are different. Existing models are generally based on transnational migrants with different ethnic and racial backgrounds. China has a large land area and a large number of domestic migrants, and they mostly share the same ethnic, racial and unified Chinese culture and official language among themselves and with the local people. This means that there will be no serious ethnic, racial or cultural discrimination and conflict. Second, the economic system is different. China has a market economy with socialist characteristics, while most other countries have a market economy . As a reReferring to foreign studies and characteristics of Chinese domestic migrants, Chinese scholars usually define migrant integration from the perspective of social equity. Migrant integration refers to migrants gradually gaining basic economic and social insurance, accepting or adapting to the subculture of receiving cities, and engaging positive interactions with the local population on the basis of equal treatment between migrants and the established population , 42. GenSummarizing previous research results, the important roles of both individual characteristics and city context in migrant integration have been proven. However, there are still two limitations that hinder further studies of Chinese domestic migrants. The first is the foundation of research, in which the utilization of survey data from a single city or several cities in one province has prevented scholars from examining city effects on migrant integration while keeping the national context constant . Second,The empirical analysis in this paper is based on the China Migrants Dynamic Survey (CMDS) conducted in 2017, which includes 289 cities and 154,044 Chinese domestic migrants over 15 years old who live in the receiving city for more than one month. The survey is conducted by the National Health Commission with the layered, multistage and probability proportionate to size (PPS) sampling method, and the total migrants in 2016 is the basic sampling frame. The sample cities account for 82.86% of all Chinese cities. Only those cities with poor natural conditions and very few migrants were not selected for the questionnaire survey. In this survey, a city is defined as a built-up area with urban functions, and Chinese domestic migrants are defined as people who have a Chinese household registration and migrate across the boundaries of county or higher administrative level in China. The data on city features are obtained from China’s City Statistical Yearbook 2018.A hierarchical linear model (HLM) is used to model how integration varies across individuals and cities and can be considered a generalization of the ordinary least squares regression when the dependent variable varies at more than one level , 49. UsiThe other model is a single second-level independent variable model with no variables at the first level (model 2). Model 2 is as follows:Yij is the degree of integration; βj0 is the intercept of city j; βnj is the slope of variable Xn in city j; rij is the residual of individual i in city j; γ00 and γn0 are intercepts; and γm0 and γnm are the slopes predicting βJ0 and βnj from variable Wm at the second level, respectively.If the variance of the intercept and slope ratios at the first level is significant at the second level, establishing that a model covering the variables at both levels (model 3) is necessary. Model 3 is as follows:Under the framework of a relatively mature and comprehensive four-dimensional measurement of migrant integration in the intergenerational model proposed by Esser, the selection and definition of some indicators are slightly adjusted according to the characteristics of Chinese domestic migrants. The four dimensions are cultural, structural, social and emotional integration . For theThe index system of migrant integration is standardized and used for the factor analysis. On this basis, the degree of migrant integration is calculated by taking the variance contribution rate after rotation of the three new factors whose cumulative contribution is over 86% as the weight  and convPrevious studies have proven that migrants’ characteristics, ability and capital are determinant factors. Therefore, migrants’ demographic and migration characteristics are first examined. Migrants’ demographic characteristics are measured by gender and age. Behind the age difference of migrants is the intergenerational difference of migrants, and to compare the differences in social integration among the young, the middle-aged and the elderly, we divide age into three categories according to the measurement of the United Nations: 14–45, 46–60 and > 60. In addition, the residence length is selected to describe migrants’ migration status. Moreover, based on the field-capital-habitus framework of sociology , variablThe cities’ contextual characteristics are proven to have fundamental effects on migrant integration. Therefore, cities’ political, social, economic and cultural statuses are examined to understand the unique background of receiving cities . First, The score of migrant integration is calculated through factor analysis. For the individual level, the average score of migrant interaction is 52.30, the range is 16.27–84.67, and the standard error is 13.44, showing that migrant integration is generally at a medium level and that there are substantial differences among migrants. For the city level, the values of the above three indicators are 51.19, 31.61–73.11, 9.34, indicating that the gap of migrant integration is also substantial among cities. From the region level, there are greater differences among the four regions of China. The central region has the highest score of 60.54 in migrant integration. The west and northeast regions follow, with scores of 57.80 and 54.55, respectively. The east region has the lowest score of migrant integration of 45.11. Although more Chinese domestic migrants migrate to the developed east region, their integration is not advanced.p = 0.000, df = 288), indicates that migrant integration is affected not only by personal characteristics but also by receiving cities’ characteristics. The individual-level and city-level factors are responsible for 60.81% and 30.19% of the effect on migrant integration, respectively, indicating that the effects of neither individual nor receiving city characteristics can be ignored. Therefore, a hierarchical model is needed to analyze the impacts of these two levels.Model 0, which passed the examination of the significance of intergroup correlation coefficients (ICC>0.059) and chi-square values Click here for additional data file.


The enzymatic synthesis of oligosaccharides depends on the availability of suitable enzymes, which remains a limitation. Without recourse to enzyme engineering or evolution approaches, herein we demonstrate the ability of wild‐type cellodextrin phosphorylase  and laminaridextrin phosphorylase  to tolerate a number of sugar‐1‐ phosphate substrates, albeit with reduced kinetic efficiency. In spite of catalytic efficiencies of <1 % of the natural reactions, we demonstrate the utility of given phosphorylase–sugar phosphate pairs to access new‐to‐nature fragments of human milk oligosaccharides, or analogues thereof, in multi‐milligram quantities. Milking phosphorylases: We demonstrate the ability of wild‐type cellodextrin phosphorylase  and laminaridextrin phosphorylase  to tolerate a number of sugar‐1‐phosphate substrates, thereby enabling the construction of new‐to‐nature analogues of human milk oligosaccharides. This is particularly well exemplified by the commercial, kilogram‐scale production of the cosmetic humectant α‐glucosyl glycerol by using sucrose phosphorylase.d‐galactose‐1‐phosphate , α‐d‐glucosamine‐1‐phosphate (GlcN1P) and α‐d‐mannose‐1‐phosphate (Man1P) as prospective donor substrates. As the latter two compounds are not commercially available at reasonable cost, we report the synthesis of these compounds in the Supporting Information. We have recently reported on structural rationalisation of the promiscuity of laminaribiose phosphorylase, which produces disaccharides Glc‐β‐1,3‐Glc and Man‐β‐1,3‐Glc when fed glucose and Glc1P or Man1P, respectively.In selecting sugar phosphates to assess with CDP and Pro_7066, we considered the synthesis of fragments of human milk oligosaccharides (HMOs), which have been the focus of recent enzymatic syntheses.4) or β‐1,4‐linked cellobiose (1) to generate new β‐1,4‐ and β‐1,3‐linkages, respectively. Turnover numbers were of the order of a few per second . However, active‐site alignments based on conserved catalytic aspartate and tryptophan residues, and phosphate/sulfate residues sitting in the pockets thought to be occupied by phosphate in sugar 1‐phosphates, leads to the conclusion that there is no obvious equivalent to the CDP R496 residue in the Pro_7066 active site ,1 and laminaribiose 4 for CDP and Pro_7066, respectively. Given the axial orientation of OH‐4 in Gal, once Gal is transferred onto an acceptor, further extension of oligosaccharide stops for CDP  due to the strict requirement for an equatorial OH‐4 in the acceptor. The Pro_7066 galactosylation reaction products, however, were more complex, with a series of oligomeric products being obtained (see section below).In order to maximise synthetic utility, the pH and temperature optima for CDP and Pro_7066 reactions were established . All further experiments were therefore conducted at 37 °C and pH 5.0 (CDP) or pH 7.0 (Pro_7066). We initially investigated Gal1P as a donor and natural disaccharide acceptors cellobiose 1 and 4 vs. 10–13 vs. 14–16), regardless of the acceptor intersugar linkages, whereas Pro_7066 (Table 1 and 4), it is less effective if the β‐1,4‐linkage is located at an internal position of the acceptor (compare acceptors 2 and 18).In general terms, CDP Table  showed aAfter careful analysis of glycan products by thin‐layer chromatography (TLC) and MALDI‐TOF MS, it became evident that Pro_7066 stops after the addition of a single galactose residue onto laminaribiose Figure  i, thus 19 (38 %) and 21 (77 %) for Pro_7066 reactions with acceptors possessing linkages that either match or do not match the enzyme specificity, respectively.This situation proved to be much less problematic in reactions with CDP than with Pro_7066 , the reaction mixtures were first treated with β‐1,3‐glucanase and then subjected to gel‐permeation chromatography. The yields of 19 (38 %) and 20  would yield single products, as reactions would not be complicated by the phosphorolysis of acceptors. Indeed, incubating reaction mixtures over three days with periodic product analysis by TLC and MALDI‐TOF MS, we were able to synthesise several further β‐galactosylated compounds 3) and Glc‐β‐1,3‐Glc‐β‐1,3‐Glc (5) resulted in 70 and 60 % yields of products 10 and 13, respectively. In contrast, β‐1,3‐galactosylation of mixed‐linked trisaccharides Glc‐β‐1,4‐Glc‐β‐1,3‐Glc (2), Glc‐β‐1,3‐Glc‐β‐1,4‐Glc (3) and Glc‐β‐1,4‐Glc‐β‐1,4‐Glc (18) by Pro_7066 gave 80, 22 and 47 % yields, respectively, thus indicating that Pro_7066 is more sensitive to acceptor oligosaccharide linkages than is CDP.The efficiencies of phosphorylase‐catalysed reactions with acceptors having both β‐1,4 and β‐1,3 linkages between glucose residues were variable. Yields of CDP‐catalysed β‐1,4‐galactosylation were not influenced by a mixed‐linked acceptor: reaction with both Glc‐β‐1,3‐Glc‐β‐1,4‐Glc , which has been used with α‐1,4‐glucan phosphorylase to synthesis α‐linked chitosan analogues,N‐acetyl‐glucosamine‐1‐P (GlcNAc1P), was tolerated to some degree by CDP and not at all by Pro_7066, as was evident from TLC and MALDI‐TOF MS analyses . We recently showed that a Paenibacillus laminaribiose phosphorylase has a relaxed substrate specificity and can accommodate mannose 1‐phosphate (Man1P) as a glycosyl donor.22 in near quantitative yield. This trisaccharide was previously identified as a component of the exopolysaccharide of cellulose‐producing bacterium Acetobacter xylinum.Changing GlcN1P to galactosamine‐1‐phosphate  had a profound negative effect on glycosylation efficiency: the latter compound was not accepted as a substrate by Pro_7066, and CDP showed only very low activity with this prospective donor substrate. Having the amino group acetylated, as in 1H and 13C NMR spectra of galactosylated oligosaccharides prevented detailed assignment of all resonances. However, it was possible to pinpoint characteristic signals of nonreducing terminal residues present in 9–17 and 19–25 (see the Supporting Information) but absent in the spectra of the corresponding oligosaccharide precursors 1–8. Mostly with the aid of 2D HSQC spectra, the β‐galactopyranosyl residue could be identified by the presence of resonances at δ=102–103 for C‐1β and δ=68.2–68.6 for C‐4 in 13C NMR spectra. In addition, a characteristic doublet of H‐4 with 3J<2 Hz at δ=3.7–3.9 and an anomeric signal at δ=4.3–4.5 supported the presence of β‐Gal residues in structures 9–16 and 19–23. The 1,4‐galactosylation of glucan acceptors was confirmed through observation of a characteristic signal at about δ=78.0 ppm, which appeared 2–3 ppm downfield from the often‐crowded region of pyranose ring carbons and belonged to C‐4 of the nonreducing termini of the corresponding acceptors. The regiochemistry of Prozo_7066‐catalysed 1,3‐galactosylation, leading to oligosaccharides 19–23, followed from the appearance of signals at δ=84–85 ppm, which are diagnostic of C‐3 of glucopyranose bearing a glycosyl residue at that position. In a similar manner, the above characteristic chemical shifts of C‐4 and C‐3 were used to confirm the presence of β‐GlcN‐1,4‐Glc and β‐GlcN‐1,3‐Glc linkages in trisaccharides 17 and 24, respectively, as wells as the β‐Man‐1,3‐Glc linkage in trisaccharide 25. The size of the oligosaccharide was confirmed by HRMS in all instances.The confirmation of the stereochemical and regiochemical outcomes of CDP and Pro_7066 reactions were confirmed by NMR spectroscopy, although considerable signal overlap in both the Escherichia coli. These studies highlight that the notion of one enzyme/one linkage does not hold for the phosphorylases, which, although stereo‐ and regiospecific, are not limited to use with only one donor substrate, if you push them hard enough. In turn, this restricts the range of enzymes that are required for the in vitro synthesis of β‐1,4 (CDP) and β‐1,3 (Pro_7066) linkages in the nonreducing terminal position of some classes of oligosaccharide.In conclusion, we have assessed the kinetic efficiency of wild‐type β‐1,4‐glucan (cellodextrin) phosphorylase CDP and β‐1,3‐glucan (laminaridextrin) phosphorylase Pro_7066 with a range of sugar‐1‐phoshates substrates; the outcomes of the reactions are tentatively explained on the basis of protein X‐ray crystal structures for both enzymes. These kinetic studies highlight the potential of the native enzymes to use sugar‐1‐phosphates other than Glc1P, and we have gone on to demonstrate the multi‐milligram‐scale production of novel oligosaccharides capped with either Gal, GlcN or Man in a series of examples that provide building blocks for the generation of analogues of human milk oligosaccharides. Although the kinetic efficiencies of these processes are often <1 % of that of the natural reaction—a feature that allows single turnover reactions even though these enzymes are naturally polymerases—the enzymes are easily produced in tens‐of‐milligram quantities from one litre cultures of transformed The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


We sampled extensively across the natural range of P. hallii in Mexico and the USA to generate double-digestion restriction-associated DNA (ddRAD) sequence data for 423 individuals from 118 localities. We used these individuals to study the divergence between the two varieties of P. hallii, P. hallii var. filipes and P. hallii var. hallii as well as the genetic diversity and structure within these groups. We also examined the possibility of admixture in the geographically sympatric zone shared by both varieties, and assessed distribution shifts related with past climatic fluctuations. There is strong genetic and morphological divergence between the varieties and consistent genetic structure defining seven genetic clusters that follow major ecoregions across the range. South Texas constitutes a hotspot of genetic diversity with the co-occurrence of all genetic clusters and admixture between the two varieties. It is likely a recolonization and convergence point of populations that previously diverged in isolation during fragmentation events following glaciation periods.Geographic patterns of within-species genomic diversity are shaped by evolutionary processes, life history and historical and contemporary factors. New genomic approaches can be used to infer the influence of such factors on the current distribution of infraspecific lineages. In this study, we evaluated the genomic and morphological diversity as well as the genetic structure of the C 4 grass Panicum hallii across its native range in North America. We observed strong genetic and morphological divergence across seven genetic clusters that follow major ecoregions. South Texas is a hotspot of genetic diversity with the co-occurrence of all genetic clusters and admixture between two described varieties. South Texas is likely a recolonization and convergence point of formerly diverged populations.Geographic patterns of diversity are shaped by historical and contemporary factors that drive evolutionary processes. Genomic approaches can be used to infer the influence of such factors on the current distribution of genetic variation. We studied diversity of the C Genetic variation is the raw material necessary to understand evolution and adaptation to diverse environmental conditions . A plantPanicum hallii at the population level. This species is a perennial C4 grass native to North America with a distribution that spans from the south-eastern part of Mexico through the South-Central and South-western regions of the USA , semi-tropical to temperate hardiness zones (south to north) and altitudinal . This species is also found in a variety of soil and ecological conditions, including nine ecoregions as defined by P. hallii var. hallii  and the more restricted P. hallii var. filipes (hereafter var. filipes). Both varieties occur sympatrically in a small part of their ranges. Variety hallii has a native distribution that extends from southern Colorado south into Mexico and from western Arizona to eastern Texas, and typically occurs on a variety of soil substrates, including sandy to shallow, dry, rocky and calcareous soils. In contrast, variety filipes is typically found in areas of transition between clay soils and mesic depressions along the Western Gulf Coastal Grassland ecoregion and the Pine Oak Forest ecoregion at the Rio Grande Valley  with a split that occurs in western Texas , a reduced representative genotyping method, and seven discriminant morphological markers to address three major questions: (i) What is the degree of divergence between the two varieties of P. hallii? (ii) How is the genomic and morphological diversity distributed within each variety? (iii) Is there evidence of admixture between individuals from these two varieties, especially when they occur in sympatry? Answering these evolutionary questions will help us to understand how evolutionary and ecological processes have shaped species diversity.Here we combine genomics, environmental niche models and common garden experiments using more than 400 samples to characterize the patterns and distribution of diversity in P. hallii seed collections derived from previous studies  absence locations, as well as pseudo-absence points sampled in the whole species distribution area [see  ]. Considering the wide geographic distribution of var. hallii covering extensive environmental heterogeneity, we also performed additional HSMs for the two largest infraspecific genetic clusters . As explanatory variables, we included eight (largely independent) bioclimatic variables from the WorldClim v.1.4 , Last Glacial Maximum  and Last Inter-Glacial  available in the WorldClim v.1.4 climatic database. For our distribution hindcast distribution analyses, we assessed the intrinsic uncertainty of the simulation of past climatic scenarios by projecting the HSMs using climatic data derived from three different global circulation models (GCMs). A complete description of the data and modelling methods can be found in the We implemented an ensemble modelling approach using the biomod2 package in R , we chose the combination of NspI and Mlucl restriction enzymes for the ddRAD method.In order to obtain a genome-wide representation of genetic diversity at low cost, we used ddRAD sequencing . Library preparation followed an in-house protocol. Briefly, for each sample, around 1 μg of genomic DNA was digested with the restriction enzymes NspI and Mlucl and then adaptor ligation fragments were selected in a range of 375–650 bp using Pippin Prep technology . Finally, the library samples were sequenced using the Illumina HiSeq 2500 and 4000 , producing around 2.6 billion of 2 × 100 or 2 × 125 pair-end raw reads.FastQC v. 0.11.3 . Raw fastq read quality was evaluated with . 0.11.3 . SequencP. hallii var. hallii v. 2.0 reference genome , using bbmap v. 37.50  to minimize the presence of loci that consist of repetitive regions that have been co-assembled  and found that the number of SNPs is very similar [see  ] and phylogenetic trees are concordant when mapping to either genome [see  ].To test for reference genome bias against var. Panicum section Diffusa is notoriously difficult  at The University of Texas at Austin were used as a control for species identification. A total of 31 samples were sequenced for the nuclear Internal Transcribed Spacer (ITS1) along with two new developed suitable chloroplast regions. The first marker, cp45676, spans the 3′ end of the intergenic region between ycf3 and trnS and includes the 5′ portion of trnS. The second marker, cp1204, spans the 3′ end of the intergenic region between psbA and trnK and includes the 5′ portion of trnK. These markers were specially designed to differentiate species from the section Diffusa of the genus Panicum. The ingroup contained 17 control samples belonging to 11 species of the genus Panicum, section Diffusa. Also, six samples acted as known controls for both varieties of P. hallii. A sample of Setaria viridis was used as an outgroup for phylogenetic inference.As the taxonomic identification of species from ifficult , we sequhttp://tree.bio.ed.ac.uk/software/figtree/). In addition, because the species in the Diffusa section also differ in ploidy, the ploidy level was estimated in these samples using flow cytometry analysis (BD LSRFostesca SORP).Approximately 570 bp of ITS1, 650 bp of cp1274 and 550 bp of cp45676 were amplified by polymerase chain reaction (PCR) using the same DNA samples that were sent for ddRAD. For ITS1, we used newly designed primers ddITS1F (5′-CCG TGA ACG TGT CAT CCA TG-3′) and ddITS1R (5′-GGT CCG AGC ACC AAG GCG-3′). For cp1204: 1204F (5′-GGC TTG TAC TTT CGC GTC TC-3′) and 1204R (5′-CGG TAC GAA CTT TTA TGC AAC G-3′). For cp45676: 45676F (5′-TAG GCA TAA TTC CCA ACC CA-3′) and 45676R (5′-CGA ACC CTC GGT AAA CAA AA-3′). Polymerase chain reaction mixtures were in 15 µL and were run on the MJR PTC-200 thermocycler. For ITS, we used the following conditions: 35 cycles of 95 °C denaturation for 60 s, 56 °C annealing for 45 s and 72 °C extension for 60 s. For the cp1204 and cp45676, we used a two-step PCR with 20 cycles of 95 °C for 60 s, 54 °C for 30 s, reduced by 0.1 °C every cycle, and 72 °C for 60 s, followed by 20 cycles of the same conditions but with a constant annealing temperature of 52 °C. Polymerase chain reaction amplicons were visualized with SYBRSafe  on 1 % agarose gels and prepared for cycle sequencing by treatment with a 10:1 mixture of Shrimp Alkaline Phosphatase and Exonuclease I . Cleaned PCR products were submitted to The University of Texas at Austin ICMB Core Facility for cycle sequencing and visualization. The chromatograms were inspected by eye, manually trimmed for quality and aligned, then, the sequences were manually assembled, using Geneious v. 7.1.9   implemented in the Adegenet v. 2.1.1 package (k-means clustering (find.clusters) to assess groups using the Bayesian information criterion (BIC); we used the cross-validation test  to retain the number of PCs with the lowest mean squared error using 100 replicates. We used three different SNP data matrices for several independent analyses including the full set of 16 397 SNPs for all 423 samples of P. hallii species, one set of 16 595 SNPs for var. hallii and one set of 13 167 for var. filipes.The high-quality SNP data set was used to identify  package  for the  package . Discrim package . We ran hallii and var. filipes, a common garden was established on 15 May 2014. Four hundred and eighty individuals from 76 different localities  were planted at Pickle Research Campus, The University of Texas at Austin, TX, USA , following the seedling method described at P. hallii varieties. During the months of July and August of 2014, one plant for each sampled locality was chosen to measure seed mass and panicle structure. For seed mass, 20 seeds per individual were weighed. Two representative mature panicles were collected and photographed to measure primary and secondary branch number and panicle length, using the Panicle Phenotyping tool P-trap  was performed on the seven phenotypic traits using R v3.6.3  to identTo test correlations between any two of these matrices, we performed a Mantel tests using the R package ‘vegan’ v2.5-6 , samples from var. hallii in sympatry with var. filipes  and a sample of var. hallii samples allopatric to var. filipes . We assigned the allopatric var. hallii samples to one population, HAL, and a random subset of var. filipes samples to another population, FIL. We then ran STRUCTURE , in contrast with the diploid condition of P. hallii  [see  ]. We limited our subsequent analyses to 600 samples which had a strong phylogenetic assignment to P. hallii.This study included a total of 649 individuals primarily representing counties ; Table 1 section . While treported , our samP. hallii samples. Almost all (99 %) of the raw reads passed our quality-filtering criteria. These high-quality reads were mapped against the P. hallii v. 2.1 reference genome assembly  and 13 167 for var. filipes . The high-confidence SNPs were fairly evenly distributed across the nine nuclear chromosomes of P. hallii  [see  ].We used the ddRAD approach to genotype the P. hallii has been described but for which contemporary samples are lacking. Several collection records show the presence of P. hallii samples in eastern Texas and Louisiana  independent models that showed true skill statistic (TSS) values above 0.75. The mean TSS over the full set of models was 0.83 and the area under the curve (AUC) score of 0.92 [see  ]. Among the selected climate predictors, the mean temperature of warmest quarter was estimated as the most important variable to predict the presence of var. filipes  [see  and  ]. The ensemble model for var. filipes shows high suitable areas from South Texas in the USA to the Nuevo Leon and Tamaulipas states in Mexico with decreasing suitability towards the south  independent models that showed TSS values above 0.75. The mean TSS over the full set of models was 0.78 and the AUC 0.94 [see  ]. Among the selected climate predictors, temperature seasonality was estimated as the most important variable to predict the presence of P. hallii  [see  and  ]. The projected suitable distribution for var. hallii shows a bigger continuous area with high suitability values encompassing the region of Texas and the states of Coahuila and Nuevo Leon in Mexico  than var. hallii, despite its narrow natural distribution  and had a positive Mantel statistic (r = 0.36) , hereafter called the ‘West’ cluster. In addition, there is a cluster including the Mexican samples and some Texas samples collected close to the Mexican border, identified as the ‘Tex-Mex’ cluster. In Central Texas, samples collected in the Edwards plateau and the Central Forest Grassland transition ecoregions form a group that we call the ‘Central Texas’ cluster. Finally, we identify a cluster occurring in South Texas where both varieties coexist under sympatric conditions that we name the ‘sympatric’ cluster. One hypothesis is that this population structure is the result of a northward and westward expansion of the species range following the LGM. To examine this hypothesis in var. hallii, we examined the correlation between expected heterozygosity and geographic distance to San Antonio. We expect a decreased genetic diversity as the distance from modern San Antonio (i.e. genetic diversity hotspot) increases. The West cluster shows a significant negative correlation between expected heterozygosity and distance from the hotspot. The other three clusters also show a negative, albeit non-significant, correlation between expected heterozygosity and distance to San Antonio  than var. hallii (filipes was more tolerant to cold stress (measured as a percentage of electrolyte leakage). Lastly, the lifespan (measured as senescence score across time) was longer for var. filipes than var. hallii.Principal component analysis using seven phenotypic characters showed a high divergence between var. . hallii . In gene. hallii . Also, vfilipes, where the samples collected in the coastal and inland form two different clusters. Interestingly, the var. hallii samples belonging to the sympatric group form a morphology-based cluster that is intermediate between the var. filipes and the rest of the var. hallii samples. Our Mantel test revealed a positive correlation between phenotypic distance of populations and their geographic distance   .filipes and var. hallii coexist in sympatry, some samples appear intermediate between the two varieties when genotypes and phenotypes are clustered  plus some var. hallii samples  collected in allopatric regions to serve as non-admixed controls.In South-west Texas, where var. lustered . Though  species , the twohallii collected from four localities in the sympatric zone were assigned membership to both var. filipes and var. hallii groups under four different arrangements of var. filipes learning samples  of the six samples with admixed compositions in the genotypic analysis [see  ].Morphological analysis of the samples of var. P. hallii individuals across the natural range of the species. We detected strong genomic differentiation between the two varieties of P. hallii, as indicated by the phylogenetic tree , extreme temperature conditions  are the better predictors of the P. hallii varieties distribution. We also corroborate annual precipitation as important predictor for var. hallii which almost occupies the geographic range of the entire species, but not for var. filipes that is more restricted to coastal and foothill environments and should be less tolerant to low precipitation conditions [see  and  ].We used ddRAD to genotype more than 400 tic tree  and the tic tree . More gehallii has heavier seeds than var. filipes  , which is a common pattern for plants that are typically exposed to drought conditions after germination in their natural habitat . Two samples from that site appear to be admixed, suggesting natural crosses of these two varieties. The Gonzales locality is particularly notable because the morphological and ecological characteristics are not typical for P. hallii. Morphologically, we were not able to definitively differentiate the two varieties in the field at this locality. Ecologically, this is the only locality in the Texas Blackland Prairie ecoregion where we collected both varieties of P. hallii, despite extensive collecting effort at other localities in the ecoregion. However, while there is detectable hybridization in the Gonzales locality, samples of both varieties with no evidence of admixture are also found in this locality, indicating that hybridization is not ubiquitous. Potential reproductive barriers between the two varieties have yet to be quantified, as they have been for other plant species  global biodiversity hotspot . Moreovehallii during Holocene might explain the lack of diversity in the recently colonized regions (i.e. West and Tex-Mex genetic clusters). In fact, there is a significant negative correlation between diversity (heterozygosity) and distance from southern Texas (San Antonio) for the var. hallii West cluster, consistent with a post-glacial expansion   , one of P. hallii is narrower than previously considered at the southern and eastern edges of the species distribution. Previous literature and collection records indicate that the natural distribution of P. hallii spans from the South-west USA  through Mexico to the southern Mexican state of Oaxaca . We also made at least 14 collection trips east of the Edwards Plateau ecoregion in Central Texas without finding P. hallii, suggesting that the eastern distribution of P. hallii is restricted to the Texas Blackland Prairies and East Central Texas forest ecoregions  efforts should focus on the verified samples. While the initial steps take time and may delay NGS efforts, they are a valuable investment as they improve the sequencing results and can save substantial time during bioinformatic analysis.P. hallii varieties. This evaluation made it possible to distinguish genetic structure at different biological levels. At the species level, divergence was found between both varieties using molecular and morphological data. Within varieties, it was possible to characterize population structure across the diverse geological and ecological native range of P. hallii. Within the sympatric zone, this set of data allowed us to identify putative admixed individuals between var. filipes and var. hallii. Taken together, these data illustrate the geographical distribution of genetic diversity in P. hallii. A practical application of these results could be the creation of a core collection of these samples to streamline screening for particular traits of interest. Future work could include increased sampling efforts for var. filipes, especially in the Rio Grande Valley and Mexico to explore the genetic diversity in the Western and South boundary of the distribution. In addition, to confirm admixture between the varieties, the plastome from samples in the sympatric area can be examined to confirm admixture between the varieties. Furthermore, studies of local adaptation could be conducted by crossing members of different populations  Short Read Archive (SRA) under project number SRP110713.


Importation of foreign genetics is a widely used genetic improvement strategy. However, even if the foreign genetic merit is currently greater than the domestic genetic merit, differences in foreign and domestic trends mean that the long-term competitiveness of an importation strategy cannot be guaranteed. Gene flow models are used to quantify the impact that a specific subpopulation, such as foreign genetics, can have over time on the genetic or economic benefit of a domestic industry.We used a deterministic recursive gene flow model to predict the commercial performance of lambs born across various subpopulations. Numerous breeding strategies were evaluated by varying market share, proportions of rams selected for mating, genetic trend, superiority of foreign genetics over domestic genetics and frequency of importation. Specifically, an Ireland-New Zealand case study was simulated to quantify the potential gain that could be made by using foreign sire contributions  in a domestic sheep industry (Ireland).Genetic and economic gains were generated from alternative breeding strategies. The ‘base scenario’ (i.e. representing the current industry) predicted an average genetic merit value of €2.51 for lambs born and an annualised cumulative benefit of €45 million (m) after 20 years. Maximum genetic (€9.45 for lambs born) and economic  benefits were achieved by implementing the ‘PRO-intense-market scenario’ which involved shifting market share away from conservative domestic breeders and reducing the proportion of rams that were selected for mating by progressive domestic breeders from the top 40% to the top 20%, without the use of any foreign genetics. The ‘PROFOR scenario’, which considered the use of foreign and progressive domestic genetics, predicted an average genetic merit value of €7.37 for lambs born and an annualised cumulative benefit of €144 m, after 20 years.Our results demonstrate that there is opportunity for a domestic industry to increase industry benefits without the use of foreign genetics but through an attempt to shift the market share away from conservative domestic breeders towards progressive domestic breeders. However, the importation and use of progressive foreign genetics may be an effective method to trigger a change in behaviour of conservative domestic breeders towards the use of progressive genetics. Within a breeding pyramid, subpopulations can be stratified into various tiers across multiple species such as sheep, beef and dairy cattle –3, by usGene flow models are also useful to account for the time-lag involved when transitioning from one breeding strategy to another –3. UnlikImportation and the use of superior foreign genetics, as an alternative to domestic genetics, is a strategy that has been used to accelerate the domestic genetic gain that can be achieved , 11. HowA gene flow model with multiple flows of predicted genetic merit across subpopulations of a sheep industry was created using Microsoft Excel. The model predicted the genetic improvement in overall economic merit of future generations of commercial sheep as a consequence of the incorporation or exclusion of foreign genetics in a proportion of ram breeder flocks. The model also assessed the impact of the implementation of various breeding strategies using multiple scenarios (described below). Depending on the scenario under investigation, the market share of each subpopulation varied on an annual basis. Similarly, for each subpopulation, the age structure of the ram and ewe population, genetic trends, genetic merit,  the proportion of rams selected for mating and selection differentials could also be varied. Discount factors and the level of superiority of foreign sires over domestic sires, as well as the size of the national ewe population and weaning rate, which ultimately determined the number of lambs born alive per year, could be adjusted within each modelled scenario.Annualised cumulative benefits were calculated to quantify and compare the impact of each alternative breeding strategy investigated. The final output of the gene flow model was the predicted average genetic merit value of commercial lambs born during a given year, which were then aggregated to industry-level benefits and expressed as a cumulative value. The model could be refined to suit any species or any country. To start, the model generated predictions for the genetic merit of animals within a number of subcategories including: ewes, rams born, rams mated and lambs born during a historic phase from the year −10 to year 0. This historic phase was identical for every strategy and reflected the current genetic merit of subpopulations and their recent genetic trends. Year 0 was described as the base year for the model. Thereafter, a recursive predictive phase was generated from year 1 onwards. This predictive phase modelled future gene flow and genetic trends in accordance with each breeding strategy scenario. Standard partial budgeting techniques were used to aggregate the benefits into present values, with a focus on the overall industry impact after a certain time horizon.The historic phase was classified as year −10 up to and including year 0. The genetic merit of animals in each subpopulation at year 0  generated by the industry in year The total annual income (€ millions) generated by the industry in year The discount factor The total income generated over a specific period of time was evaluated, while accounting for the lag between selection decisions and the flow of genetic contributions. The discounted cumulative benefit, denoted as , and a group representing those not engaged , with the national performance recording system operated by Sheep Ireland . The genThe subpopulations modelled represent the Irish sheep industry and are described in Table The average genetic merit value of rams born within the FOR subpopulation in year 0 of each scenario  and commercial (COM) subpopulations were established based on industry data supplied by Sheep Ireland and were calculated as €0.38 and €0.01 per year, respectively. The progressive breeders using foreign genetics subpopulation (PROFOR) were assumed to have the same genetic trend as the PRO subpopulation (i.e. €0.38 per year). The conservative subpopulation (CON) was assumed to make no genetic progress (i.e. €0.00 per year) in the Irish €uro-star Replacement Index, during either the historic or recursive phases of the model.The relationship between the domestic maternal index  and the foreign maternal index , was calculated using genetic evaluations of animals imported into Ireland from progressive New Zealand flocks as part of a production validation study . These aThe FOR genetic trend was calculated to be €0.18 per year for ewes mated and rams born during the historic phase, using incremental changes in genetic trend for the aforementioned mentclasspt{minimaTable The proportions of ewes and rams mated within each age group and subpopulation are outlined in Tables The accuracy values associated with the DOM maternal index (i.e. the Irish €uro-star Replacement Index) were retrieved from the Sheep Ireland database for each subpopulation. The accuracy values associated with year 0 were 0.39, 0.45, 0.45, 0.45 and 0.38 for CON, PRO, PROFOR, FOR and COM, respectively. The accuracy values were incorporated into selection differential calculations for rams born within the recursive phase of the model.The current number of breeding ewes in the Irish sheep population was assumed to be 2,560,180 with a weaning rate of 1.3 lambs per ewe [Several scenarios were investigated to reflect various possible changes within each of the subpopulations and included shifts in market share and/or the proportion of rams selected for mating, as well as variation in the frequency of importation of FOR sires into the domestic industry. A schematic view of possible scenarios for implementation within the industry is in Fig. The ‘base scenario’ replicated the current Irish industry and predicted future trends where no changes were implemented, with the COM subpopulation continuing to source 87% and 13% of rams from CON and PRO subpopulations, respectively.A series of scenarios were then undertaken to evaluate the impact of a shift in the market share away from the CON subpopulation towards more progressive domestic or foreign subpopulations .In the ‘PRO-intense scenario’ a reduction in the proportion of rams selected for mating was investigated, whereby the PRO subpopulation only retained rams for mating that were ranked in the top 20% of rams (instead of from the top 40% as in the base scenario) for genetic merit on the domestic market index. No shift in market share was associated with this scenario.In the ‘PRO-market scenario’, the impact of increasing the market share of the PRO subpopulation was investigated whereby the market share of the CON subpopulation was reduced by 5% per year of the market share value in the previous year, from year 1 to 20. This market share was transferred to the PRO subpopulation, which thereby increased by 5% of the value in the previous year, per year. The proportion of rams selected for mating was  the same as in the base scenario.In the ‘PRO-intense-market scenario’, a combination of a 5% shift per year of the market share value in the previous year towards the PRO subpopulation and a reduction in the proportion of rams selected for use at mating was investigated, where only rams that ranked in the top 20% for genetic merit on the domestic market index were retained.In the ‘PROFOR scenario’, FOR genetics were introduced into the domestic market. From year 1 onwards, market share of the CON subpopulation declined by 5% of the market share value in the previous year, per year; in the first 5 years (i.e. years 1 to 5) this market share was evenly divided between the PRO and FOR subpopulations (i.e. 2.5% increase in market share for both PRO and FOR per year). From year 6 onwards, no FOR subpopulation existed but rather the market share of both the PRO and PROFOR subpopulations grew by 2.5% annually. The PROFOR subpopulation was generated based on sire matings from the PRO and FOR subpopulations within the first 5 years of this scenario.In the ‘FOR scenario’, the COM subpopulation sourced FOR genetics directly from New Zealand through a single shipment of live rams and/or frozen semen in year 0, with the objective of widespread use via laparoscopic artificial insemination. From year 1 onwards, the market share of the CON subpopulation declined by 5% of the market share value in the previous year, per year. This market share was evenly divided between the PRO and the FOR subpopulations. Two sub-scenarios within the FOR scenario were also investigated, i.e. a ‘FOR-5 scenario’ and a ‘FOR-10 scenario’ that followed similar trends for market share shifts as described in the FOR scenario, however new shipments of FOR genetics were imported every 5 or 10 years, respectively.The mean age of rams and ewes mated within each subpopulation, based on data sourced from the Sheep Ireland database are in Table Genetic trends for rams, ewes and lambs, adjusted for the mean age of the subpopulation, were greatest for the PRO and PROFOR subpopulations. The genetic trend of rams in the FOR subpopulation, was equal to that of the PRO and PROFOR subpopulations. However, the genetic trend of ewes in the FOR subpopulation was inferior to that of PRO and PROFOR subpopulations (− €0.78), and therefore resulted in a lower overall genetic trend for lambs born − €1.58) compared to the PRO or PROFOR subpopulations. The genetic trend for the COM subpopulation was positive for all groups of animals, albeit making little genetic progress  and the ACB generated, over and above the base scenario, after 20 years of implementation. The PRO-intense-market scenario contributed the greatest genetic and economic benefits to the industry after 20 years and resulted in a 3.7- and 4.0-fold increase in the rate of genetic and economic gain above the base scenario, respectively Table . The incWith increasing interest in the incorporation of superior genetics within the agricultural industry, research must investigate the potential benefit of the use of foreign sires in terms of genetic gain and consequent economic benefit within domestic industries. Previous studies in the dairy and beef cattle industries , 3, usinModelling genetic gain within an industry is not a new concept , however. [Although a deterministic modelling approach was undertaken in the current study, alternative modelling approaches such as stochastic simulation models could also have been considered . However. , stochas. –3, 5, asWithin the present and other studies , 3, a brReducing the proportion of rams selected for mating within a subpopulation was of key importance in maximising the genetic gain that can be achieved by the Irish sheep industry. The PRO-intense-market scenario, by incorporating a reduction in the proportion of rams selected for mating from 40% to 20% and an annual shift in market share, achieved the greatest increase in genetic and economic benefits, at 377 and 397% above that generated by the base scenario, respectively. The importance of selection intensity on maximising the rates of genetic gain in the Australian dairy industry was highlighted by the study of Pryce et al. , where t. [As previously discussed and confirmed by Santos et al. , foreignHowever, the additional superiority of the FOR genetics  did not prove to be as beneficial as predicted. This could be explained by the frequency of importation of the FOR genetics and the modest annual shift in market share of 5% of the value in the previous year, away from conservative breeders towards progressive breeders modelled in the current study. In the FOR, FOR-5 and FOR-10 scenarios, the average genetic merit value of lambs born within the PRO and FOR subpopulations equalised during years 12, 15 and 17, respectively. In addition, the breeds of New Zealand sheep imported as part of [ Ireland . The imp Ireland . The potFurthermore, the authors carried out a sensitivity analysis on the FOR-5 and FOR-10 scenarios to examine the potential impact of the importation of foreign genetics with a greater foreign genetic trend than that achieved by the New Zealand population within this particular case study. Results were expressed relative to the current genetic trend within the PRO subpopulation (i.e. €0.38), in order to demonstrate the difference in foreign genetic trend relative to the domestic trend. As previously mentioned, the PRO-intense-market scenario generated an additional €179.93 m for the industry (PVB), at an ACB of €14.44 m per year and increased the genetic merit of lambs born to €9.45 after 20 years of implementation. The genetic and economic benefits achieved within the adjusted FOR-5 and FOR-10 scenarios reached similar levels to those achieved by the PRO-intense-market scenario when the foreign genetic trend was increased to 2.5 and 3.5 times that of the domestic genetic trend, respectively. The adjusted FOR-5 and FOR-10 scenarios generated a PVB and an ACB of €188.21 m and €15.10 m, and of €184.62 m and €14.84 m, respectively. A significant increase in foreign genetic trend was required in order to generate gains equal to the PRO-intense-market scenario. This was attributed to the modest shift of 5% per year of the market share value in the previous year. An increased shift in market share away from conservative breeders at a rate of 10% each year as part of the FOR-5 scenario increased the PVB by €50.24 m, the ACB by €4.03 m, and the genetic merit of lambs born by €1.88 m after 20 years (results not presented). However, in a volatile industry where tradition is at the forefront within the domestic Irish industry and substantial genetic progress is only in recent years being achieved, 5% was a realistic but changeable parameter within the model.. [Although the genetic trends within Ireland and New Zealand differ, breeding objectives within both countries are similar, as industries attempt to breed a robust ewe that is suitable in any grass-based production system. Previous work completed by Santos et al.  demonstr.  highlighIn practice, realisation of this opportunity for industry benefits would depend on considerable change in the farmer ram buying behaviour. Experience in the beef industry in Ireland would suggest that a national development programme with quite rigorous genetic improvement requirements has been shown to be effective at changing farmer behaviour. For this reason, the authors recommend that the Department of Agriculture, Food and the Marine, in collaboration with Sheep Ireland  (i.e. thThe findings of the current study demonstrate that it is possible to substantially increase the genetic and economic benefits to the domestic industry, without foreign sire contributions, but through the strategic use of domestic genetics. Essential to its success is a shift in the market share from conservative breeders towards progressive breeders. The use of foreign genetics may play a key role in triggering this shift. Deterministic modelling, which incorporated age proportions, genetic time-lags, the proportion of rams that are selected for mating, superiority of foreign genetics compared to domestic genetics, selection differentials and proportion of market share proved to generate realistic methods that could be incorporated into the national breeding programme in an attempt to increase genetic and economic benefits to the domestic industry. This model now provides a template for other industries to quantify their genetic and economic benefits as a result of foreign sire contributions, regardless of country or species.Additional file 1. Derivation of Eq.