An Act to prohibit and prevent genetic discrimination, has recently been enacted but may be subject to constitutional challenges, information about potential risks to insurability may raise issues in the clinical context. We conducted a survey with women in Quebec who have never been diagnosed with breast cancer to document their perspectives. We complemented the research with data from 14 semi-structured interviews with decision-makers in Quebec to discuss institutional issues raised by the use of genetic information by insurers. Our results provide findings on five main issues: (1) the reluctance to undergo genetic screening test due to insurability concerns, (2) insurers' interest in genetic information, (3) the duty to disclose genetic information to insurers, (4) the disclosure of potential impacts on insurability before genetic testing, and (5) the status of genetic information compared to other health data. Overall, both groups of participants (the women surveyed and the decision-makers interviewed) acknowledged having concerns about GD and reported a need for better communication tools discussing insurability risk. Our conclusions regarding concerns about GD and the need for better communication tools in the clinical setting may be transferable to the broader Canadian context.Genetic stratification approaches in personalized medicine may considerably improve our ability to predict breast cancer risk for women at higher risk of developing breast cancer. Notwithstanding these advantages, concerns have been raised about the use of the genetic information derived in these processes, outside of the research and medical health care settings, by third parties such as insurers. Indeed, insurance applicants are asked to consent to insurers accessing their medical information (implicitly including genetic) to verify or determine their insurability level, or eligibility to certain insurance products. This use of genetic information may result in the differential treatment of individuals based on their genetic information, which could lead to higher premium, exclusionary clauses or even the denial of coverage. This phenomenon has been commonly referred to as “Genetic Discrimination” (GD). In the Canadian context, where federal Bill S-201,  Breast cancer is the most common cancer in women worldwide, with nearly 1.7 million new cases diagnosed in 2012 (World Cancer Research Fund)Beyond expected benefits and hopes from genomic medicine, individuals' risk stratification could also interest third parties outside the medical context, such as insurers. For life insurance underwriting, an individual's insurability and payable premium are established on the basis of one's probability of dying prematurely  defined as the differential treatment of a group or an individual based on their genetic data Human Rights Act (Canada)Genetic Non-discrimination ActIndustry Code: Genetic Testing Information for Insurance Underwriting of the Canadian Life and Health Insurance Association (CLHIA) states that its members will not require any insurance applicant to undertake genetic testing but that members will require access to the ensuing genetic test results if they were made available to the applicant or her physician  a survey with women and (B) interviews with decision makers:The survey with women aimed to capture the knowledge, perspectives, experiences and needs of a sample of Quebec women regarding genetic information and insurance. This survey was administrated on a paper questionnaire at the end of a discussion group centered on the development of an information toolkit for the PERSPECTIVE risk estimation approach. Inclusion criteria for participation in the discussion groups were: being a woman between 35 and 55 years of age, not having received a breast cancer diagnosis, and being able to discuss in French. Each of the four discussion groups were comprised of 8–12 women from different regions of Quebec. Participants were invited to answer our 20-min paper questionnaire consisting of closed-ended questions and the opportunity to comment on their answers. A total of 36 women completed the questionnaire after discussions. This paper discusses only results from the paper questionnaire on insurance concerns (not from discussion groups).14 semi-structured interviews were also conducted with decision-makers involved in the management of breast cancer screening programs and policies in Quebec (Canada). Decision-makers were identified through our network of collaborators and from publicly available sources of information. They were invited to take part in a 1 h-long interview in French, either in person or over the phone, with a researcher trained in sociology and in interview-based research (JH). Our group of decision-makers comprised two clinicians with an active involvement in public health, five experts in public health involved in the evaluation or the implementation of the current breast cancer screening program, four regional managers and three national managers involved in the administration of programs and policies with regards to breast cancer in Quebec (Canada). Interviewees were deliberately selected for maximizing the representation of the following factors: social geography, administrative hierarchy , professional experience  and gender balance , collaboratively with the Quebec City team , eight closed-ended questions followed by open-ended questions (blank space to provide an opportunity to explain their answer). The Quebec City Team  hosted discussion groups and distributed the paper questionnaire. Then, the McGill team  conducted a content analysis of open-ended answers from participants . Initial results were organized into thematic codes. Responses that could not be categorized were excluded from the analysis. Discrepancies were resolved through majority vote among the researchers. Some participants did not answer each closed-ended and open-ended questions.Interviews with decision-makers were conducted (JH) using questions designed by the McGill research team (JH and EL) to guide discussions. The analysis was thematic and the content of each interview was annotated in English. Annotations were discussed among researchers and lead to the development of a themed framework. Triangulation of data, investigators and methodologies was used to derive a more complete understanding of the many dimensions of insurability issues in the context of breast cancer risk stratification assessment  and Quebec City (n = 12). The other half of respondents lives in smaller cities, specifically Rimouski (n = 12), Baie-Saint-Paul (n = 4), Mont-Joly (n = 1), and Saint-Georges (n = 1).Respondents provided basic data including their location, age, level of education, occupation/income source and family history of cancer. This information is presented in Table n = 15) and senior high school diploma (including CEGEPn = 13). The other respondents only completed secondary (n = 5) or primary (n = 1) school degrees while a minority did not receive any formal education (n = 2).The majority of respondents (78%) completed post-secondary education: university degree (n = 17) or part-time (n = 5). The second half of respondents received other forms of income including unemployment (n = 1), welfare (n = 7), invalidity/disability (n = 5) or pension payments. We note that one participant did not answer this question (n = 1).In terms of occupation/source of income, almost half of the respondents were employed, working full-time (n = 17)Nearly half of the participants shared having a family history of breast cancer (n = 22) indicated that they would be reluctant to undergo breast cancer genetic screening test knowing results could become accessible to insurers (Table n = 13), followed by losing current insurance coverage (n = 8), being the victim of a data confidentiality breach (n = 7), and having to pay increased insurance premiums (n = 4).Most respondents n = 2 indicatn = 13). In this group, some respondents explained their choice by indicating that such tests should be used for the purpose of disease prevention only (n = 4).A minority of respondents expressed not being reluctant to undergo breast cancer genetic screening test knowing that results could be accessible to insurers (n = 28) assumed that breast cancer genetic screening test results would negatively impact their ability (or that of a family member) to obtain personal insurance (Table n = 19) or their relatives (n = 2).The majority of respondents  did not think they would experience a negative impact on their ability to obtain insurance. Among them, two respondents nevertheless recognized the risk associated with genetic testing as a general factor used in underwriting by insurers.n = 29) said “no,” while seven answered positively  thought that insurers have an interest in knowing their genetic information for the purpose of determining their insurability (Table n = 12), and it can have a negative impact on insurability (n = 5).A majority of respondents  thought they did not have a duty to disclose genetic test results in order to obtain a life insurance policy (Table n = 10) indicated that they lack legal knowledge on this subject, and two respondents stated that genetic information should be treated as an exception to the general requirement of disclosure.The majority of respondents  considered having a duty to disclose genetic test results to prospective life insurers.A minority of respondents (n = 16) of the 36 women answered, but all of these respondents answered yes. Respondents who agreed that women should be informed were invited to answer a sub-question about communication tools. They were asked if information should be included in the “communication tools previously mentioned” or in a “communication tool designed especially” for insurance issues. The “communication tools previously mentioned” could have been understood by women as the website explaining breast cancer risk estimation presented during the group discussions. Some respondents indicated that information on insurability should be included in communication tools previously mentioned (n = 7) while others preferred a communication tool designed especially on insurance issues (n = 3)  considered genetic information to be different from other types of medical information (Table n = 11); associated with a risk to develop a future health condition (n = 7); and constituted confidential information (n = 4).The majority of respondents (on Table . Respondn = 12) of respondents did not consider genetic information to be different from other types of medical data.A minority  by insurers was discussed, five interviewees mentioned being familiar with insurers' use of such information in their underwriting processes Table : line 1.n = 5) discussed the interest and means that insurers have to access the genetic information of their applicants  recognized that other factors aside from genetic test results can impact applicants' insurability but are uncertain on their use in underwriting. For instance, two interviewees specified that family history might play a significant role. Moreover, three interviewees thought that insurers could decide to investigate further the possibility of a familial genetic predisposition to a disease based on non-medical data they obtained . Furthermore, three interviewees recognized their lack of knowledge about the extent of use of both genetic and non-genetic information by insurers for underwriting purposes, which as one interviewee stated, may change depending on different types of insurance products. Likewise, two interviewees were uncertain about the impact of genetic test results on the insurability of asymptomatic women, women who have or maintain a low breast cancer risk, or those having taken preventive actions to reduce a high risk.A majority of interviewees  believed that women should be informed about the potential impact on insurability before undergoing genetic testing for risk assessment . Six of them thought that the anxiety induced by the possibility of being treated differently in the context of personal insurance could undermine women's participation in genetic screening. One regional manager considered that merely rumors of insurers discriminating based on genetics would be enough to lower participation. One public health expert argued that a lack of participation derived from fear of GD in private insurance might impact the rationale behind public genetic screening programs. Only one of the respondents who mentioned the potential impact on insurance considered that the fear of GD would not, alone, be sufficient to affect women's participation in breast cancer screening.Without being prompted, many decision makers raised concerns about whether public fears of GD in the insurance context could affect the uptake of breast cancer genetic screening offer. The impact that insurers' access to genetic test results may have on women's participation in said screening was the dominant theme about insurance discussed by a majority of interviewees  stated that a public debate should be initiated about the issue of access to genetic information by insurers. One remarked that the confidentiality of patient data is a concern that should be discussed among ethicists. Likewise, one suggested that the use of genetic data from multiple sources  for underwriting purposes should be reviewed. One recognized the legitimacy of the principle of individual underwriting but would like its application to be constrained in the case of genetic information.Some interviewees  discussed the nature of genetic information in the context of insurance underwriting. One thought that the tangible quantitative nature of genetic risk-stratification makes it valuable for insurers when assessing individuals' risk. Two decision-makers reasoned that the use of predictive genetic test results should be limited in underwriting since it conveys only a probability of developing a disease or symptoms.Other interviewees  also proposed potential avenues to explore on the issue of insurers' access to genetic test results. One public health expert put forward the idea to limit insurers' access to patients/participants' genetic information in medical records. Another public health expert suggested that access to genetic information by insurers should be prevented. Likewise, two other decision-makers mentioned the potential use of a moratorium on the access and use of genetic information in insurance.Some interviewees  the reluctance to undergo genetic screening test due to insurability concerns, (2) insurers' interest in genetic information, (3) the duty to disclose genetic information to insurers, (4) disclosure of potential impacts on insurability before genetic testing, and (5) the status of genetic information compared to other health data.n = 22) expressed being reluctant to undergo breast cancer genetic screening test if results were accessible to insurers. Similar concerns have been documented in previous studies involving BRCA1/2 testing such as by Armstrong et al. in 2003 where 55% of the 574 women rated their fear of GD in life insurance as moderate or very important  also shared that it would affect their willingness to undergo genetic testing  was concerned that breast cancer genetic test results demonstrating a predisposition to develop the disease might have a negative impact on their capability, or that of their relatives, to obtain or maintain insurance. In addition, a majority of survey respondents and decision-makers interviewed in our study felt that genetic test results would be part of factors that insurers take into consideration in the underwriting process. Similarly, a survey from Parkman et al. showed that respondents from United States are either somewhat or very concerned that life insurers might use genetic test results as a factor for determining coverage and costs  considered that insurers have an interest in their genetic information for the purpose of assessing their insurability and establishing their premium. Indeed, in the revised Industry Code on Genetic Testing, the CLHIA has reiterated that “insurers will not initiate or require any applicant to undergo a genetic test as part of the process of applying for insurance.”  mentioned being unsure how insurers interpret and use hereditary disease information, genetic and, non-genetic data, in their risk assessment. Two interviewees pointed out that in the case of a screening program aimed at asymptomatic women, the effect of breast cancer genetic test results in underwriting is more problematic because some women—even at low risk—may never develop the disease and yet may still be perceived to be at higher risk than the average population by insurers. Such concerns about insurers' interest in genetic data have been reported in other studies where patients have been reluctant or refused to undergo breast cancer genetic testing or, expressed a preference for paying higher out-of-pocket costs if doing so could potentially prevent insurers from having access to their test results Until recently, Canada had not yet officially adopted specific policies or laws against GD, such that standard insurance law disclosure requirements would apply. For example, art. 2408 of the n = 20) thought that they were not required to disclose genetic test results to insurers when applying for insurance. Although this has not been explored by our survey, these respondents may also not be aware that insurance contracts usually require that an authorization  be granted for the insurer to have access to their medical records. This is important since insurers have stated that, with the consent of the applicant, they will seek access to genetic test results that have been made available to the applicant or her physician, as they do for all other types of health-related informationOur study found that most women were not aware of this duty to disclose since more than half of survey respondents  agreed that potential impact on insurability should be disclosed. Decision-makers interviewed in our study agreed that informed consent should include the disclosure of the impact of genetic tests on insurability and that health professionals should be trained to disclose on it. This touches a question underlined by previous researchers about informing patients and research participants about potential impacts of genetic tests results on insurability   consider genetic information to be different from other health data. This finding is not surprising given previous results of respondents' reluctance to undergo genetic testing and their concerns that associated test results may negatively impact their insurability  have mentioned supporting the adoption of a moratorium on the use of genetic information by insurers while others (n = 2) have mentioned that it should be at least regulated. A few decision-makers (n = 3) explicitly added that there is a need for a public debate involving ethicists, jurists and law/policy makers on the use of genetic information in insurance. According to them, this debate has to consider: all types of genetic information and not just breast cancer, the confidentiality of genetic data, and whether this information should benefit from an exception in the context of insurance underwriting.Two decision makers pointed out that in the context of risk-stratification, where personalized risk is measured in percentages, genetic information is probabilistic, which brings up concerns about how it is being used by insurers in their underwriting process. In view of current concerns about GD, uncertainties related to the nature of genetic information, and its possible impact on participation in genetic screening test, some decision-makers (n = 20). Furthermore, even if most participants answered all closed-ended questions Q1-7, some chose not to share reasons underlying their responses through the optional open-ended format.The survey with women included 36 respondents from Quebec, which is a small sample. However, making statistical inference was not planned in our methods, and the composition of the survey group met our goals of diversification of age, occupation, location and education. Another limitation of our study is item nonresponse in the survey, which is significant for some close-ended question such as Q8, the last question on the questionnaire, who has a non-response rate of 56% . All authors made a substantial contribution to the analysis and interpretation of data, provided a critical revision and agreed with the final version of the manuscript and all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In contrast, cities in regional clusters were more likely to have high rates of minorities and high natural growth rates than would be expected by chance . Our findings suggest that multiple highly connected foci of measles transmission coexist in China and that migrant workers likely facilitate the transmission of measles across regions. This complex connection renders eradication of measles challenging in China despite its high overall vaccination coverage. Future immunization programs should therefore target these transmission foci simultaneously.Measles is a highly contagious and severe disease. Despite mass vaccination, it remains a leading cause of death in children in developing regions, killing 114,900 globally in 2014. In 2006, China committed to eliminating measles by 2012; to this end, the country enhanced its mandatory vaccination programs and achieved vaccination rates reported above 95% by 2008. However, in spite of these efforts, during the last 3 years (2013–2015) China documented 27,695, 52,656, and 42,874 confirmed measles cases. How measles manages to spread in China—the world’s largest population—in the mass vaccination era remains poorly understood. To address this conundrum and provide insights for future public health efforts, we analyze the geospatial pattern of measles transmission across China during 2005–2014. We map measles incidence and incidence rates for each of the 344 cities in mainland China, identify the key socioeconomic and demographic features associated with high disease burden, and identify transmission clusters based on the synchrony of outbreak cycles. Using hierarchical cluster analysis, we identify 21 epidemic clusters, of which 12 were cross-regional. The cross-regional clusters included more underdeveloped cities with large numbers of emigrants than would be expected by chance ( Measles is a highly infectious disease. Eradication of measles can nevertheless be achieved with vaccination of 90–95% of a population, as shown in theory and practice. In China, however, measles continues to infect thousands of people each year despite vaccination coverage above 95%. This conundrum challenges measles elimination in China and worldwide. Here we characterize the geospatial distribution of measles and epidemic connections among cities across China. Using incidence data reported during 2005–2014 for all 344 cities in China, we show that the municipal burden of measles differed substantially and some cities were highly connected and experienced synchronous outbreaks. We identify 14 cities that experienced endemic transmission during 2005–2010, and 21 transmission clusters, including 6 cross-regional clusters that link the less developed inland regions and the industrial east. We find that three transmission foci coexist in China—cities with large minority populations, inland cities with more emigrants, and mega industrial cities hosting more immigrants—and that migrant workers, connecting the latter two foci, likely facilitate measles transmission across regions. This complex connection, along with the differing disease burden among cities, renders measles elimination challenging in China despite the high overall vaccination rate. Future immunization programs should therefore target these three foci. Morbillivirus. Before vaccine licensure in 1963, the disease infected virtually all children. Infection typically causes fever, runny nose, cough, red eyes, and sore throat, followed by a rash spreading over the body. While most cases recover, complications range from diarrhea, otitis media, pneumonia, encephalitis, seizures to death . Even when evaluated on a per capita basis, these cities still had incidence rates significantly higher than the national average . Such asynchrony in endemic cities also creates challenges for eliminating measles in China.Remarkably, the identified clusters did not include most of the industrial cities that experienced endemic measles prior to the 2010 nationwide SIA. Mega-industrial cities tend to attract migrant workers from a larger number of locations across China and as a result, the measles epidemic cycle in each city is likely influenced by multiple sources and does not resemble that of one particular source. In addition, those endemic cities did not have synchronous epidemics with one another, and were likely each a transmission hub. For instance, the three endemic cities in Guangdong—Guangzhou, Shenzhen and Huizhou—had asynchronous epidemic cycles despite their close vicinity and high connectivity  nor among those in cross-regional clusters . Birth rate is an important factor in measles epidemiology, as it affects the recruitment rate of susceptible individuals and in turn epidemic frequency [On a related note, high birth rates were only found to be associated with high measles incidence rates among cities in regional clusters  were conducted prior to 2010 , which mSecond, while the CISDCP surveillance system includes nearly all medical institutions in China ,51, undeIn summary, our analysis of the geography of measles demonstrates that multiple endemic cities co-exist in China, and that these cities are predominantly industrial cities with large numbers of migrant workers, whose movement likely facilitates the spread of measles across regions in China. In addition, cities with large minority communities and inland, underdeveloped cities likely have lower vaccination rates and are more vulnerable to the resurgence of measles. Measles has reappeared in many cities in China since 2013. Our findings suggest that elimination of measles remains challenging in China due to the heterogeneity in vaccination implementation and complex connections among regions.S1 Tabler≥0.85 were identified as within the same cluster. The 1st column shows the cluster id number, the 2nd shows the total number of cities included in each cluster, the 3rd lists the cities in each cluster, the 4th lists the province(s) in each cluster, and the 5th lists the region(s) in each cluster. The numbers in the parentheses in the 4th and 5th columns indicate the numbers of cities located in each province or region.Cities with a Pearson correlation coefficient (DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 FigThere are six geographical regions in China; provinces in the same region are labeled and outlined in the same color: North in red, Northeast in navy blue, East in blue, South Central in purple, Southwest in brown, and Northwest in black. Prefecture city level population density is shown by color as indicated in the legend.(TIF)Click here for additional data file.S2 FigCities labeled in blue and shown by an ‘x’ experienced endemic transmission during this phase.(TIF)Click here for additional data file.S3 FigNone of the 344 cities experienced endemic transmission during this phase.(TIF)Click here for additional data file.S4 FigCities labeled in green and shown by an ‘o’ experienced endemic transmission during this phase.(TIF)Click here for additional data file.S5 FigOnly those with three or more cities are included here; a complete list of clusters is shown in (PDF)Click here for additional data file.S6 FigOnly those with three or more cities are included here; a complete list of clusters is shown in (PDF)Click here for additional data file.


In Asia cattle were domesticated around 10,000 years ago and in Korea cattle are being raised since 2000 B.C. There are three native breeds of cattle in Korea viz. Brown Hanwoo, Brindle Hanwoo and Jeju Black. While one of these breeds, Brown Hanwoo, is a part of a Food and Agricultural Organization and national genetic evaluation plans, others get little attention. This study is an effort to understand and provide a detailed insight into the population structure and genetic variability of the Korean cattle breeds along with other Asian breeds using various methods. In this study we report the genetic variation and structure of the Korean cattle breeds and their comparison with five other Asian cattle breeds along with a panel of animals from European indicine breed, had the lowest gene diversity while Yanbian had the highest followed by Mongolian and Korean cattle. Amongst the Korean breeds Brown Hanwoo had the highest diversity followed by Brindle Hanwoo and Jeju Black. The genetic diversity in Asian cattle breeds was found comparable to the European taurines and more than the African taurines and Zebu cattle. Korean cattle breed, Brown Hanwoo was consistently found to be closer to Yanbian, a Chinese cattle breed. We found low divergence and moderate levels of genetic diversity among the native Korean breeds. Indicine introgression from Hainan was seen in other Asian breeds. From Europe, Limousin, Holstein and Hereford introgression was found in Asian breeds.Asian cattle were found to be least differentiated which reflects their recent history. Amongst the Asian breeds Hainan, which is an In this study we provide a genome-wide insight into the genetic history of the native cattle breeds of Korea. The outcomes of this study will help in prioritization and designing of the conservation plans.The online version of this article (doi:10.1186/s12863-016-0444-8) contains supplementary material, which is available to authorized users. Modern day breeds are a result of natural and artificial selection and adaptation to the local climate. Due to various reasons about 16 % of the cattle breeds are already extinct and 30 % face the risk of extinction. Preserving the local cattle breeds thus becomes necessary in order to prevent the depletion of the genetic resources of the country. Accessing the genetic diversity of a population provides an insight into the history, genetic structure and current status of the population which form the very basis for genetic improvement.Cattle is known to be domesticated approximately 10,000 years ago near present day Turkey and Pakistan however, the earliest remains of domestic cattle found in north-east Asia dates back to only 5000 years . Two maitaurine type but China also has some indicine/zebu type cattle breeds and hybrid breeds , Korean Brindle cattle  and Jeju Black . These breeds differ from each other in the coat color and levels of nose darkness [taurine, African taurine and Indicine (Zebu) cattle and five Asian breeds from data published by Decker et al. [In North-East Asia , most of the cattle breeds are ds Table . Asian cl origin . Asian cdarkness . Geograpdarkness . Like modarkness . It was darkness . Hanwoo darkness , 9. In tr et al. .Table 1BIn our study we present a detailed insight into the genetic diversity and structure of Korean cattle breeds in comparison with Chinese, Mongolian and Japanese breeds. The outcomes of this study would shed light on the genomic structure of Korean breeds as well as other Asian breeds and this information could be used as a primer to design conservation strategies and breeding programs.taurine, African taurine and Zebu data was used from the Bovine Hapmap project. All the genotype data was then merged to make one final dataset. In the final dataset there were 576 samples and 35,598 SNPs.Blood samples were collected from Brown Hanwoo (BH), Brindle Hanwoo (BNH) and Jeju Black (JB). All three breeds are found in their native track in three different regions of the country. Breeds and number of samples used for the study are described in detail in Table Genomic DNA for genotyping assays was extracted from the blood sample using DNeasy 96 Blood and Tissue Kit . DNA quantification was performed using a NanoDrop 1000 . DNA samples were submitted for genotyping with total DNA of 900 ng, 260/280 ratio >1.8, and DNA concentration of 20 ng/ul. The genotyping for animals was done by the Animal Genome & Bioinformatics Division of the National Institute of Animal Science, RDA, Korea, using a BovineSNP50 BeadChip Ver.1. .http://pngu.mgh.harvard.edu/purcell/plink/) [Genotype data was imputed using Beagle program . Plink v/plink/) ) was useTo understand the genetic diversity of the cattle populations we used Hierfstat R package . GeneticPopulation structure of the Korean cattle breeds was studied using multivariate approach and model based methods. Multi-dimensional scaling (MDS) was used to capture the preliminary glimpse of the genetic structure of the Korean cattle populations and also to remove outliers, if any. MDS  and higher than the zebu cattle breeds (0.162). YB cattle breed had Hs = 0.271 while the Korean native breeds including BH, BNH and JB had Hs value of 0.257  and we found less inbreeding than expected as Fis values were negative for all the breeds ranging from −0.16 in BRM to −0.23in HN. Among all the three native breeds BNH had the lowest Fis value. The negative Fis values averaged over all the loci indicated a lack of population structure in the populations. Given the small population size of native Korean breeds (BNH = 4000 and JB = 1000) considerable diversity still exists in the native populations.The genetic diversity within populations was accessed as a measure of heterozygosity i.e., expected heterozygosity (Hs) and observed heterozygosity (Ho). Gene diversity in three Korean cattle breeds was found to be slightly less 0.257) than the European 57 than ttaurine and zebu breeds. Amongst the three native breeds the Fst estimates of BH with other European taurine breeds were the least. The island breeds BNH and JB were found to be more differentiated from the taurine and zebu breeds. On an average, the genetic differentiation between the Asian breeds was found to be less than the other breeds in the study.Genetic differentiation between the 20 populations was studied using pairwise Fst estimates , followed by BNH (0.143) and JB (0.142). Amongst the three native breeds, BNH and JB were found to be most distantly related than others (0.176). Genetic distances of Korean breeds with that of other breeds were the least with LMS (0.22) and the highest with HN (0.32). Compared to other Asian breeds genetic distance of BH from WAGY was found to be the smallest (0.202).Provesti’s genetic distances between populations were calculated using adegenet R package and were plotted as a neighbor joining tree , LX, ND and QC placed themselves center to all the major clusters i.e. zebu, European taurine and African taurine cluster. This shows the influence of European and indicine cattle breeds on the formation of these Asian and African breeds.Population structure of the Korean cattle breeds was studied using multivariate approach and model based methods. Principal component Analysis (PCA) was used to place the Korean Cattle breeds with respect to the European ers Fig. . The firthst population used in the study was a local Korean cattle breed called “Chosun”. But based on DAPC, PCA and MDS results we understood that these animals were only crossbreds so we removed the entire population from the analysis and used only 20 populations in this study  formed one group together. The optimum value of K was found to be K = 15  of ancestry with BH and ~10 % each with BNH and JB. Both island breeds BNH and JB were found to have BH admixture in them. Admixture of some fraction of African cattle was observed in Asian cattle breeds.We performed unsupervised hierarchal clustering of our data as implemented in Admixture software . Admixture estimates the ancestry in a model-based manner from the autosomal SNP panel from a set of unrelated individuals. The data was then plotted in order of fraction ancestry that they shared with other breeds Fig. . In our Taurine and Indicine. Within taurine, Korean cattle breeds along with Japanese WAGY and Chinese YB formed a separate group. HN and LX formed a group with indicine cattle breeds. As we added migration events we found influence of European cattle, LMS on both Asian and African cattle. When adding more migration evnts than 8 we found introgression from HOL and HFD into the Asian cattle breeds. Introgression of Indicine genetic component from HN was seen in the Asian breeds. We also found influence of MG on the Chinese LX and QC breeds.Treemix analysis was used to study the population splits and gene flow Fig. . We firsTaurine and less than European Taurine. This could be attributed to the recent genetic history of Korean cattle breeds. However, genetic diversity of Korean cattle breeds in our study was considerably lower than that reported by Kim et al. [In our analysis, diversity was assessed as a measure of expected heterozygosity (Hs) and observed heterozygosity (Ho). Diversity in Korean cattle breeds was found to be more than Zebu and African m et al. . This mim et al. , based om et al. . Within m et al. . Howevertaurine cattle breeds. Zebu breeds were found to be least inbred amongst these 15 populations.In Korea only BH has a dedicated breeding program and the number of animals is ~3 million while number of animals for JB and BNH is only a few thousands. We used Fis as a measure to study inbreeding within these populations. Fis values indicated an excess of heterozygotes in BNH (−0.230) and JB (−0.216) which are the island populations. Compared to BH (−0.210), YB −0.205) was found to be less inbred. The Fis values in our study were different from that reported by Choi et al. [ was fountaurine and Zebu cattle.Pairwise Fst was used to study the population differentiation between the twenty breeds in the study. The Fst values for Korean cattle breeds in our study were lower than the European breeds ranging from 0.02 and 0.06. YB and BH were found to be least differentiated from each other (Fst = 0.01). JB and BNH were found to be most differentiated from one another (Fst = 0.06) while they were found to be less differentiated from BH which is a mainland breed. The divergence of JB and BNH in two different directions from the mainland breed makes a good example of the classical island model. Lower Fst values in the Korean populations suggest that they had not yet differentiated well into completely separate/independent breeds. While BH was found to be closer to MG, LX, HN and QC and WAGY (0.02), BNH and JB were found to be more differentiated from other Asian breeds. Based on Fst values, we found that Asian breeds were still genetically closer to each other than the European, African taurine and Indicine cattle in varying proportions were observed in the Korean cattle breeds. Genetic influence of Korean cattle breeds from LMS and BS was nicely captured by PCA and DAPC plots. Admixture analysis (Fig. taurines.We also found evidence of an admixed lineage of Korean cattle breeds. Influence from European sis Fig.  at all tsis Fig.  there waModern day Asian cattle are a result of introgression from various European and Indicine breeds. Mongolian, Qinchuan and Yanbian cattle still contain a high level of admixture from various other breeds while present day Korean cattle were seen to be less admixed. While Korean cattle were found to be less admixed with the breeds outside Korea, they still were found to be admixed amongst themselves. Based on all the population metrics used to study genetic diversity we conclude that the Korean populations are still very closely related and have not yet differentiated enough to be considered as separate breeds. These breeds could rather be referred to as the subpopulations of BH. YB was found to be closest to BH and thus it could be developed as an alternate meat breed of the country. Since YB is an unselected population it can also serve as a model to study the effect of selection and breeding on BH. BNH and JB are two valuable genetic resources of the country and we suggest relevant measures to be taken to increase the number of individuals in the two island breeds and thus prevent the loss of diversity that may occur due to small population size.


Caenorhabditis elegans, upon exposure to environmental extremes. Transcriptional control of the metazoan heat shock response is mediated by the heat shock transcription factor HSF-1. In addition to regulating global stress-responsive genes to promote stress-resistance and survival, HSF-1 has recently been shown to regulate stress-independent functions in controlling development, metabolism, and longevity. However, the indirect role of HSF-1 in coordinating stress-dependent and -independent processes through post-transcriptional regulation is largely unknown. MicroRNAs (miRNAs) have emerged as a class of post-transcriptional regulators that control gene expression through translational repression or mRNA degradation. To determine the role of HSF-1 in regulating miRNA expression, we have performed high-throughput small RNA-sequencing in C. elegans grown in the presence and absence of hsf-1 RNAi followed by treatment with or without heat shock. This has allowed us to uncover the miRNAs regulated by HSF-1 via heat-dependent and -independent mechanisms. Integrated miRNA/mRNA target-prediction analyses suggest HSF-1 as a post-transcriptional regulator of development, metabolism, and longevity through regulating miRNA expression. This provides new insight into the possible mechanism by which HSF-1 controls these processes. We have also uncovered oxidative stress response factors and insulin-like signaling factors as a common link between processes affected by HSF-1-regulated miRNAs in stress-dependent and -independent mechanisms, respectively. This may provide a role for miRNAs in regulating cross-talk between various stress responses. Our work therefore uncovers an interesting potential role for HSF-1 in post-transcriptionally controlling gene expression in C. elegans, and suggests a mechanism for cross-talk between stress responses.The ability of an organism to sense and adapt to environmental stressors is essential for proteome maintenance and survival. The highly conserved heat shock response is a survival mechanism employed by all organisms, including the nematode  A complhsf-1 upon HS ;+HS and hsf-1(-);+HS treatment conditions as determined by the Benjamini-Hochberg correction for multiple testing. The complete output from the Benjamini-Hochberg correction test can be found in miR-784, miR-231, miR-86, miR-53, miR-47, and miR-34 are normally upregulated by HSF-1 during HS.We were next interested in further examining the miRNAs normally regulated by HSF-1 during HS . By sepa2-fold change of each miRNA, is listed  to uncover the biological processes predicted to be suppressed by the miRNAs upregulated by HSF-1 during HS . The cat2-fold change of these 12 miRNAs are plotted in the presence and absence of HSF-1 during HS (hsf-1(+);+HS and hsf-1(-);+HS treatment conditions as determined by the Benjamini-Hochberg correction for multiple testing. The complete output from the Benjamini-Hochberg correction test can be found in miR-48 and miR-228 are normally downregulated by HSF-1 upon HS.We were next interested in examining the miRNAs normally downregulated by HSF-1 during HS . By sepa2-fold change of each miRNA, is listed ;+HS and hsf-1(-);-HS treatment conditions using the Benjamini-Hochberg correction for multiple testing. The complete results from the Benjamini-Hochberg correction test can be found in miR-72 is normally upregulated by HSF-1 independently of HS.To uncover a HS-independent role for HSF-1 in regulating miRNA expression, we next focused on the miRNAs normally regulated by HSF-1 independently of HS . By sepa2-fold change of each miRNA, is listed (hsf-1(-);-HS] in order to represent the normal degree of HSF-1 regulation on miRNA expression. However, the function of miR-72 is unknown, thus warranting future studies regarding miR-72.Next, we determined the known functions of the miRNA we found to normally be upregulated by HSF-1 independently of HS. The description according to WormBase, along with the logTo uncover potential mRNA targets of the miRNAs regulated by HSF-1 independently of HS, we performed integrated prediction-based analysis by comparing predicted mRNA targets to genes we previously determined to be regulated by HSF-1 independently of HS via mRNA-seq  7]. Due. Due7]. We next used DAVID to uncover the biological processes predicted to be suppressed by HSF-1-regulated miRNAs independently of HS . The cathsf-1 RNAi independently of HS, thus these miRNAs would normally be downregulated by HSF-1 (2-fold change of these 5 miRNAs are plotted in the absence of HSF-1 and in the presence and absence of HS (hsf-1(-);+HS and hsf-1(-);-HS treatment conditions using the Benjamini-Hochberg correction for multiple testing. The complete results from the Benjamini-Hochberg correction test can be found in miR-239b, miR-1820, and miR-228 are normally downregulated by HSF-1 independently of HS.We were next interested in determining a HS-independent role for HSF-1 in downregulating miRNA expression . By sepa2-fold change of each miRNA, is listed (hsf-1(-);-HS] in order to represent the normal degree of HSF-1 regulation. miR-239b is known to be highly upregulated upon aging, and mutants lacking miR-239a/b exhibit an increased lifespan and resistance to stress  or hsf-1 RNAi [hsf-1(-)] from the L1 larval stage to the L4 larval stage prior to treatment with a 33°C 30 minute heat shock (+HS) followed by a 12 hour recovery. (B) Quantification of fluorescence intensity confirms that hsf-1 RNAi blunts hsp-70 promoter activity upon HS. Quantification of the fluorescent images in (A) demonstrate hsf-1 RNAi decreases hsp-70 promoter activity by ~80%. (C) hsf-1 RNAi blunts hsp-16.2 promoter activity upon HS. Fluorescent images are shown of synchronous pY46H3A.3(hsp-16.2)::GFP worms fed control RNAi [hsf-1(+)] or hsf-1 RNAi [hsf-1(-)] from the L1 larval stage to the L4 larval stage prior to treatment with a 33°C 30 minute heat shock (+HS) followed by a 12 hour recovery. (D) Quantification of fluorescence intensity confirms that hsf-1 RNAi blunts hsp-16.2 promoter activity upon HS. Quantification of the fluorescent images in (C) demonstrate hsf-1 RNAi decreases hsp-16.2 promoter activity ~60%. (E) Heat shock protein genes are the top genes induced during a 30 minute 33°C HS. mRNA-seq performed in parallel to miRNA-seq [hsp genes are the highest induced group of genes in response to HS, and shows these genes are dependent on HSF-1, further validating our experimental conditions. For , error bars represent standard deviation and significance was determined with the Bonferroni post-test where ‘***’;q-value<0.001.((TIF)Click here for additional data file.S2 FigA) Dendogram correlation between biological duplicates. Clustering of the biological duplicates for each miRNA-seq condition reveals conserved alignment between replicates. The dendogram was generated by the program CummeRbund to provide insight into the relationships between different conditions. (B-E) Differential expression analysis shows little deviation between biological duplicates. Scatter plots of the miRNA-seq reads for each biological replicate, for each condition, shows similarities between biological duplicates. The x-axis is representative of the reads from the first biological replicate, and the y-axis represents reads from the second biological replicate. The closer the R2 value is to 1, and the closer each point is to the line, is representative of similarity between replicates.((TIF)Click here for additional data file.S3 Fighsf-1(+);-HS control. The q-value is the FDR-adjusted p-value of the test statistic as determined by the Benjamini-Hochberg correction for multiple testing.Volcano plots show all unchanged  and significantly altered  miRNAs, relative to the (TIF)Click here for additional data file.S4 FigA) Venn diagram analysis of the miRNAs significantly upregulated as compared to the control. The Venn diagram shows the overlap among miRNAs that were found to be significantly upregulated  as compared to the hsf-1(+);-HS control for each of the indicated comparisons between samples. The blue shaded region represents miRNAs that are regulated by hsf-1 during HS. The q-value is the FDR-adjusted p-value of the test statistic as determined by the Benjamini-Hochberg correction for multiple testing (B) Relative abundance of the miRNAs normally upregulated by HSF-1 upon HS. The log2 fold change from the miRNA-seq data, of the miRNAs determined via Venn diagram analysis to be regulated by HSF-1 upon HS as compared to the hsf-1(+);-HS control, shows the miRNAs determined to be significantly different compared to each treatment condition. Significance was determined with the Benjamini-Hochberg correction where ‘*’;q-value<0.05. (C) miRNAs determined to normally be upregulated by HSF-1 during HS. The miRNAs that had a significant difference between the hsf-1(+);+HS and hsf-1(-);+HS treatment conditions in (B), as determined by the Benjamini-Hochberg correction for multiple testing, are listed.((TIF)Click here for additional data file.S5 FigA) Venn diagram of the miRNAs significantly downregulated compared to the control. The Venn diagram shows the overlap among miRNAs that were found to be significantly downregulated  as compared to the hsf-1(+);-HS control for each of the indicated comparisons between samples. The blue shaded region represents miRNAs that are normally downregulated by hsf-1 during HS. The q-value is the FDR-adjusted p-value of the test statistic as determined by the Benjamini-Hochberg correction for multiple testing (B) Relative abundance of the miRNAs normally downregulated by HSF-1 upon HS. The log2 fold change from the miRNA-seq data, of the miRNAs determined via Venn diagram analysis to be regulated by HSF-1 upon HS as compared to the hsf-1(+);-HS control, shows the miRNAs determined to be significantly different compared to each treatment condition. Significance was determined with the Benjamini-Hochberg correction where ‘*’;q-value<0.05. (C) miRNAs determined to normally be downregulated by HSF-1 during HS. The miRNAs that had a significant difference between the hsf-1(+);+HS and hsf-1(-);+HS treatment conditions in (B) as determined by the Benjamini-Hochberg correction for multiple testing are listed.((TIF)Click here for additional data file.S6 FigA) Venn diagram analysis of the miRNAs significantly upregulated as compared to the control. The Venn diagram shows the overlap among miRNAs that were found to be significantly downregulated  as compared to the hsf-1(+);-HS control for each of the indicated comparisons between samples. A miRNA that is downregulated in response to hsf-1 RNAi is considered to normally be upregulated by HSF-1. The yellow shaded region thus represents miRNAs that are normally upregulated by hsf-1 independently of HS. The q-value is the FDR-adjusted p-value of the test statistic as determined by the Benjamini-Hochberg correction for multiple testing (B) Relative abundance of the miRNAs normally upregulated by HSF-1 independently of HS. The log2 fold change from the miRNA-seq data, of the miRNAs determined via Venn diagram analysis to be regulated by HSF-1 independently of HS as compared to the hsf-1(+);-HS control, shows the miRNAs determined to be significantly different compared to each treatment condition. Significance was determined with the Benjamini-Hochberg correction where ‘*’;q-value<0.05. (C) miRNAs determined to normally be upregulated by HSF-1 independently of HS. The miRNAs that did not have a significant difference between the hsf-1(-);+HS and hsf-1(-);-HS treatment conditions in (B), as determined by the Benjamini-Hochberg correction for multiple testing, are listed. (D) Venn diagram analysis of the miRNAs significantly downregulated as compared to the control. The Venn diagram analysis shows the overlap among miRNAs that were found to be significantly upregulated  as compared to the hsf-1(+);-HS control for each of the indicated comparisons between samples. A miRNA that is upregulated in response to hsf-1 RNAi is considered to normally be downregulated by HSF-1. The yellow shaded region thus represents miRNAs that are normally downregulated by hsf-1 independently of HS. The q-value is the FDR-adjusted p-value of the test statistic as determined by the Benjamini-Hochberg correction for multiple testing (E) Relative abundance of the miRNAs normally downregulated by HSF-1 independently of HS. The log2 fold change from the miRNA-seq data, of the miRNAs determined via Venn diagram analysis to be regulated by HSF-1 independently of HS as compared to the hsf-1(+);-HS control, shows the miRNAs determined to be significantly different compared to each treatment condition. Significance was determined with the Benjamini-Hochberg correction where ‘*’;q-value<0.05. (F) miRNAs determined to normally be downregulated by HSF-1 independently of HS. The miRNAs that did not have a significant difference between the hsf-1(+);+HS and hsf-1(-);+HS treatment conditions in (E), as determined by the Benjamini-Hochberg correction for multiple testing, are listed.((TIF)Click here for additional data file.S7 FigA) Processes normally downregulated by HSF-1-regulated miRNAs during HS. The genes predicted to be suppressed by HSF-1-regulated miRNAs during HS were classified by Gene Ontology terms using DAVID. Processes with an enrichment score>~2.5 are listed. (B) Processes normally upregulated by HSF-1-regulated miRNAs during HS. The genes predicted to be induced by HSF-1-regulated miRNAs during HS were classified by Gene Ontology terms using DAVID. The top 4 processes are listed.((TIF)Click here for additional data file.S8 FigA) Processes normally downregulated by HSF-1-regulated miRNAs independently of HS. The genes predicted to be suppressed by HSF-1-regulated miRNAs independently of HS were classified by Gene Ontology terms using DAVID. Processes with an enrichment score>~2.48 are listed. (B) Processes normally upregulated by HSF-1-regulated miRNAs independently of HS. The genes predicted to be induced by HSF-1-regulated miRNAs during HS were classified by Gene Ontology terms using DAVID. The top 3 processes are listed.((TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Choerodon rubescens, towards the cooler (southern) end of its distribution. Here, we use a genome wide set of 14 559 single-nucleotide polymorphisms (SNPs) to identify the likely source population for this recruitment event. Most loci (76%) showed low genetic divergence across the species’ range, indicating high levels of gene flow and confirming previous findings using neutral microsatellite markers. However, a small proportion of loci showed strong patterns of differentiation and exhibited patterns of population structure consistent with local adaptation. Clustering analyses based on these outlier loci indicated that recruits at the southern end of C. rubescens’ range originated 400 km to the north, at the centre of the species’ range, where average temperatures are up to 3 °C warmer. Survival of these recruits may be low because they carry alleles adapted to an environment different to the one they now reside in, but their survival is key to establishing locally adapted populations at and beyond the range edge as water temperatures increase with climate change.Understanding source-sink dynamics is important for conservation management, particularly when climatic events alter species’ distributions. Following a 2011 ‘marine heatwave’ in Western Australia, we observed high recruitment of the endemic fisheries target species  The need to identify source-sink dynamics of fish populations for fisheries and conservation management3 has gained particular importance in light of recent species distribution shifts in response to climate change6. Distributional shifts can act as a buffer against potential extinctions from rising temperatures7 by allowing access to cooler areas and opening novel habitats for expansion9. But such shifts require that populations can disperse and successfully adapt to the novel local environmental conditions12. Understanding larval connectivity patterns and the local adaptive capacity of populations is therefore crucial for assessing species potential for distributional shifts in response to climate change.The majority of marine species exist as discrete adult populations that are connected by a dispersive larval stage13. Oceanography in WA is largely governed by the poleward flowing Leeuwin Current (LC), which during this heatwave was stronger than in the two previous centuries, increasing maximum sea-surface temperatures by up to 3 °C along the entire WA coast14. This anomalous oceanographic event resulted in mortality to fishes, crustaceans and corals16, and numerous ecological changes17. High temperatures and strong LC flow were largely maintained during 2012 and 2013 when an unusually high recruitment event of the baldchin groper Choerodon rubescens, a highly targeted fish species endemic to WA, was documented towards its cooler (southern) range edge18. This recruitment event was viewed as a possible indication of a poleward distributional shift in response to warming oceans and stronger LC flow, but such high recruitment events are often episodic and may fail to establish the new populations necessary for such a shift to occur19. Identifying the origin of these recruits and assessing whether there is evidence for local adaptive capacity in this species can assist our understanding of source-sink dynamics and the potential response of this commercially important species to rapid environmental change. This information has important implications for the design of conservation management measures in this species and the large number of species undergoing distributional shifts at a global scale20.A recent ‘marine heatwave’ along the coast of Western Australia (WA) drastically changed local oceanographic conditions, simulating future climate change scenariosC. rubescens are characterized by high levels of gene flow21 maintained primarily by a highly dispersive larval phase and counter-flowing currents22. High levels of gene flow and associated lack of population structure make it difficult to assign recruits to their natal populations23, particularly given the challenges in obtaining complete sampling of the putative parent populations that is necessary for parentage studies24. However, population genomic studies involving high numbers of single-nucleotide polymorphisms (SNPs) have revealed that despite considerable gene flow and an apparent lack of population structure, many fish species show significant levels of differentiation at highly structured ‘outlier’ loci that are putatively under directional selection28. This is particularly the case when samples are collected across strong environmental gradients29. Genome-wide scans for outlier loci putatively under selection can therefore be useful for assigning recruits in high gene flow species where neutral genetic markers fail to show population structure30.A previous study using neutral microsatellite markers indicated that populations of C. rubescens, a high-gene flow endemic species distributed across a temperate-tropical transition zone that could be used to assign recruits to their most likely natal population. This study presents two sets of information important for developing climate adaptive management measures for this endemic fisheries target species: (1) a scan for genomic signatures of local adaptation, and (2) identification of the geographical origin of recruits towards the cooler range-edge of this species, during an anomalous marine heatwave event. Since shifts in distributions of marine species occur mostly along ecosystems influenced by poleward boundary currents20, the applicability of the results from this study can be extended to global conservation management and bring insight into the exchange of larvae between populations that differ in their locally adaptive genetic signatures.In this study, we specifically test for the presence of outlier loci in adult populations of C. rubescens samples collected from four different locations spanning the species’ geographical range  of 0.131. Removal of these outliers and those under balancing selection , resulted in a neutral dataset consisting of 11 055 loci. None of the loci in the neutral data set deviated significantly (p < 0.05) from Hardy Weinberg equilibrium consistently in any of the sampling locations. Levels of linkage disequilibrium (LD) were low overall with less than 1% of neutral loci showing significant LD (p < 0.05), after correcting for multiple comparisons via the Benjamini & Hochberg method32. For these reasons, all 11 055 loci in the neutral data set were retained for further analyses.Single-nucleotide polymorphism (SNP) loci were called across 65 nge Fig. ; Table 133 and BAYESCENV34 identified a lower number of outlier loci than LOSITAN, but these loci were always a subset of those identified via LOSITAN, demonstrating high consistency between methods. BAYESCAN33 identified 0.06% of all SNP loci (n = 9) as being under directional selection. Outlier analyses with BAYESCENV34 identified 0.15% of all SNP loci (n = 22), and further revealed a significant association with average sea surface temperatures (SST) along the latitudinal gradient sampled during both summer  and winter .Genome-wide scans for selection with both BAYESCANvs outlier loci datasets .As expected, we found substantial differences in population genetic structure patterns inferred from analyses based on the neutral ets Fig. . Discrim38. DAPC plots with a priori information of K = 4 (the number of populations sampled), revealed distinct populations for Cape Naturaliste and Kalbarri, which separated from a group of overlapping populations formed by Abrolhos Islands adults and Garden Island recruits  to proteins involved in important cellular processes such as growth and metabolism, membrane transport and signal transmission Table . Other oC. rubescens across 12 degrees of latitude in Western Australia. By contrast, genome-wide scans for selection identified 282 outlier loci putatively under directional selection that showed significant population structure between adult sampling regions and thus could be used to assign juveniles from an anomalous recruitment event at the southern edge of the species distribution to a population over 400 km to the north at the Abrolhos Islands, where average sea surface temperatures are up to 3 °C warmer. It is important to note, however, that while our results indicate that the Abrolhos Islands are part of the same genetic source population as the Garden Island recruits, sampling of the adult populations was too coarse to definitely pinpoint the geographic origin of these recruits. Increased strength of poleward current flow, together with warmer temperatures associated with a recent marine heatwave, may have assisted larvae originating from warm water sites to disperse to, settle and persist at the cooler end of the species distribution. Recruit settlement was traced to autumn and winter 2012  end of the species range may have originated from a highly mixed larval pool derived from populations spread throughout the species range and that intense post-settlement selection in response to warmer than long-term average water temperatures at the site where recruits were collected may have driven the observed genetic similarity in outlier loci between the recruits at Garden Island and adult fish from warmer water sites to the north.Population genetic analyses using a dataset comprised of 11 055 neutral single nucleotide polymorphisms identified an absence of genetic structure in populations of the highly targeted fish species 41. Lack of structure in neutral loci indicates that C. rubescens populations are connected via high levels of gene flow across the thermal and habitat gradients sampled, and between offshore islands and the mainland, consistent with a previous microsatellite study of this species21. Larval connectivity is promoted by a pelagic larval phase and counter-flowing current systems along south-western WA which promote larval mixing (poleward flowing Leeuwin Current (LC) vs the northward flowing inshore currents of Ningaloo and the Capes22). Despite these high levels of gene flow, outlier loci revealed patterns of population structure that support post-settlement selection and suggest that strong selective forces are acting on a mixed pool of recruits resulting in the selective mortality of juveniles originating from cooler environments. Although strong selection appears to be acting at a local scale, connectivity is present between cooler and warmer water populations promoted by counter-flowing currents in WA, making it difficult to distinguish between the processes of dispersal and selection. While the selective forces causing local adaptation are unknown, an environmental gradient in sea surface temperature is a likely factor43. The association of outlier loci with genes that play a direct role in growth and metabolism47, processes that are highly regulated by temperature in marine teleosts49, provides some support for this hypothesis. It is further supported by the significant association between outlier loci and sea surface temperature found in this study . Several outlier loci also had best blast hits with genes associated with temperature. For example, genes of the ubiquitin family and neural epidermal growth factor-like like (NELL) have been shown to upregulate in response to heat stress52. However, we cannot rule out the influence of other factors correlated with temperature such as latitude, depth and habitat54.We found contrasting patterns of genetic variation in neutral and outlier loci suggesting the presence of possible local adaptation amidst high levels of gene flow in an important endemic fishery species in WA56, once temperatures return to normal conditions following the heatwave. Recruit ageing demonstrated that juveniles have survived one winter of warmer waters  could actually increase the adaptive capacity of populations at higher latitudes58. As C. rubescens has relatively long generation times , evolutionary responses to increasing temperatures are likely to be slow and may require frequent introductions of adaptive variation in order to maintain local fitness optima61. Expansion of our sampling to include the full range of C. rubescens with a focus on specifically determining local adaptive capacity in response to temperature is recommended.Using outlier loci to assign recruits to source populations provides a novel and powerful approach to understanding population connectivity and the evolution of geographic ranges in marine species with a dispersive larval stage. The genetic mismatch at outlier loci between recruits at Garden Island and adults from a neighbouring population at Cape Naturaliste with a similar temperature profile, suggests the possibility that recruits may be maladapted to their new environment at the southern range edge62 along the Capes region, which are becoming increasingly warmer with climate change63. Poleward range-shift facilitation via successful colonization and adaptation may therefore be likely for this and other fish species in the region. However, because the LC is expected to weaken in the next five decades64, the Capes counter-current will likely flow at greater strength promoting higher self-recruitment and/or northward larval dispersal65, and could ultimately restrict the extension of species to their cooler range margins in this region (i.e. the Capes region). Coupled with increasing temperatures at the warmer (northern) range edge, geographical distributions could ultimately be reduced rather than extended. It is therefore crucial to better understand dispersal pathways across the full range of C. rubescens, with emphasis on the Abrolhos Islands, where it is currently most abundant and which appears to be an important larval source. This study applied powerful genomic techniques to better understand processes of dispersal and recruitment in a high gene flow species and highlights the importance of such studies in monitoring changes in source-sink dynamics associated with a rapidly changing climate.We found evidence of adaptation to local environmental gradients amidst high levels of gene flow and poleward larval connectivity in a highly targeted endemic species in WA. These characteristics suggest an adaptive capacity to changing environments via successful colonization of novel habitats at cooler locationsChoerodon rubescens (Labridae) is a large bodied wrasse and an important fisheries target41, distributed along a latitudinal gradient spanning ~1 400 km of the WA coast in habitats that range from tropical coral to temperate rocky-kelp reefs66 66 and 25 years41. It is protogynous, with females at the centre of its range typically attaining sexual maturity at ~3 years60, and later changing to males when they are 12 years59. Reproduction peaks during the austral spring to mid-summer (October to January) and larvae typically settle onto the reef in the austral summer and early autumn (December to March)59. Juveniles and adults are essentially sedentary68, but connectivity between populations is maintained via dispersive planktonic larvae with a pelagic larval duration (PLD) of ~23 days  were undertaken along a 2 km sampling area at seven reefs (2 to 35 m water depth) in three locations: Kalbarri, Abrolhos Islands and Cape Naturaliste  range edge and high fishing pressure. Sampling recruits and adults from the same location is ideal and highly desired, however this may be challenging for many species due to the above reasons and also because recruits often occupy different habitats to adults. Tissue samples were obtained from fin clips and/or muscle tissue of each individual, preserved in 100% ethanol and stored at room temperature. Total genomic DNA was isolated for each sample using DNeasy blood and tissue kits  following the manufacturer’s protocols.We sampled 70. The GBS method is a procedure based on high-throughput next-generation sequencing of genomic subsets targeted by restriction enzymes, and is ideal for non-model organisms that lack genomic resources70. Libraries were sequenced with paired-end 100-bp reads on an Illumina HiSeq. 2000 sequencer . SNP calling was then performed using the TASSEL UNEAK pipeline with default settings72. We then selected only loci with a genotyping success rate greater than 85% and retained those for further analyses.Single-nucleotide polymorphism (SNP) loci were genotyped at the ACRF Biomolecular Resource Facility, John Curtin School of Medical Research (JCSMR), Australian National University (ANU) via the genotyping by sequencing (GBS) methodFDIST approach of Beaumont and Nichols (1996)73 as implemented in LOSITAN31. This method uses expected heterozygosities and unbiased FST values for each locus74 to generate a global neutral distribution for FST under Wright’s Island model75. The probability of each locus FST belonging to this neutral distribution is then used to classify loci into one of three selection categories: neutral (0.1–0.9), under balancing selection (<0.1) and under positive selection (>0.995)25. If a large dataset of neutral loci is used to create empirical p-values, this method has high performance and considerably reduces the amount of false-positives77. Outlier analyses were based on 1 000 000 simulations assuming an infinite alleles mutation model and using Neutral mean FST, 0.95 confidence intervals and an FDR of 0.1. Ten independent runs were performed to further reduce the amount of false-positives. Outlier analyses were performed on adult fish only . Two separate datasets for all samples (adults and recruits) were then generated: one dataset for neutral loci and another for outlier loci under positive selection.To identify loci under directional selection, we used the 33. Furthermore, we used BAYESCENV34, a genome-scan method that extends the BAYESCAN approach by including environmental data and using it to identify outliers, based on specific hypothesis about the drivers of local adaptation. We used satellite derived monthly average sea surface temperature data at the sampling unit level (reef) for 2002–2013  to carry out a genome scan and identify outliers significantly correlated with the temperature gradient sampled. We used default settings in BAYESCENV, tested for model convergence and set the FDR to 0.1. Temperature has been identified as the most important variable determining fish distributions along the coast of WA, where physiological gradients are consistent and the seascape is climatically buffered, relatively stable in geological time-scales and highly oligotrophic53. For this reason and given the relatively limited scope of our sampling and low sample sizes along the WA coast, we chose to limit our linkage of genotypic differences at outlier loci to temperature, rather than undertaking a more detailed landscape approach54.We also identified outlier loci in adult fish by applying a more rigorous Bayesian simulation-based approach implemented in BAYESCAN 2.1 with default settings and an FDR of 0.1. This approach identifies outlier loci under possible natural selection by using differences in allele frequencies between populations and directly estimating the probability that each locus is subject to selection78 in R79 and 100 simulations following Monte-Carlo permutation procedures. If loci deviated significantly (p ≤ 0.05) from HWE in all regions, they were removed from the SNP data set. We also estimated the extent of linkage disequilibrium (LD) between pairs of loci by calculating significance values for each pairwise comparison and assessing the correlation coefficient for each comparison, using the ‘genetics’ package80 in R79. In order to correct p-values for multiple comparisons and control false discovery rates, we used the p.adjust function selecting the Benjamini & Hochberg correction method32.We tested for significant deviations from Hardy-Weinberg equilibrium (HWE) for each neutral locus using the ‘adegenet’ package81. The DAPC method identifies and describes clusters of genetically related individuals from large datasets and allows the optimal visualization of between-population differentiation in multivariate space82. Furthermore, by using Bayesian Information Criterion (BIC) to assess the best supported model for identifying groups of individuals, the method provides a measure of the optimal number of genetic clusters (K) across a range of K values82. We used both the outlier and neutral loci datasets to test for population structure, and selected the model with the lowest BIC to identify the optimal number of K. The optimal number of principle components and discriminant functions to use in DAPC plots was determined by maximization of the α-score, which measures the bias between observed and random discrimination and provides a measure of discrimination ability and stability of the DAPC35. For all analyses we retained five principal components (PCA) and five linear discriminants (DA).To investigate population structure, individual-based discriminant analysis of principal components (DAPC) was conducted for neutral and outlier SNP data as identified via LOSITAN in R, using the package ‘adegenet’39 with a burn-in period of 10 000 and 100 000 MCMC iterations, and under the assumption that populations were admixed and allele frequencies correlated between populations. Analyses were run for K = 1 to 5, each replicated 10 independent times and without assuming prior genetic structure. The most likely number of genetic clusters (K) was chosen based on results from STRUCTURE HARVESTER37 by comparing the likelihood of the data for different values of K and using the ΔK method83. Results were then averaged using CLUMPP to minimize variance across iterations84, before graphics were generated.In addition to the DAPC analyses, Bayesian inference of genetic partitioning was implemented in the program STRUCTUREK) identified via BIC in both DAPC analyses82 and STRUCTURE37, as described in the previous section. Outlier loci identified via LOSITAN were used for this assignment. For DAPC analyses, ordination plots were produced with and without a-priori information of different values of K in order to better visualize recruit assignment.Recruits were assigned to parent populations by assessing membership probabilities of individual juveniles  to different groups and the proximity of individuals to the different clusters  and Uniprot’s Swiss-Prot databases to query BLASTn top hits. The number of significant matches did not change greatly when a less conservative E-value was used .To determine the possible function of outlier loci, we examined loci identified via any of the three outlier detection methods used  for alignment with publicly available genomes using the NCBI program BLASTn. We followed Gaither Supplementary Information


The Norwegian Lundehund is a highly endangered native dog breed. Low fertility and high frequency predisposition to intestinal disorder imply inbreeding depression. We assessed the genetic diversity of the Lundehund population from pedigree data and evaluated the potential of optimal contribution selection and cross-breeding in the long-term management of the Lundehund population. The current Norwegian Lundehund population is highly inbred and has lost 38.8% of the genetic diversity in the base population. Effective population size estimates varied between 13 and 82 depending on the method used. Optimal contribution selection alone facilitates no improvement in the current situation in the Lundehund due to the extremely high relatedness of the whole population. Addition of (replacement with) 10 breeding candidates of foreign breed to 30 Lundehund breeders reduced the parental additive genetic relationship by 40–42% (48–53%). Immediate actions are needed to increase the genetic diversity in the current Lundehund population. The only option to secure the conservation of this rare breed is to introduce individuals from foreign breeds as breeding candidates. Fratercula arctica, lunde in Norwegian) from their nests in screes and burrows on steep mountainsides along the Northern Norwegian coast [th century [The Norwegian Lundehund is a highly endangered dog breed, native to Norway , listed an coast ,2. There century . Lundehu century . In the  century . Since tIt is expected that the bottlenecks have caused a great loss of genetic diversity in the Lundehund population. Visual evidence for this loss of diversity is the reduced variation of coat colours. Before the Second World War black and white Lundehund existed, whereas at present these variants are extinct . ReducedIn the 1960s breeders discovered that many dogs were suffering from intestinal problems, later diagnosed as intestinal lymphangiectasia (IL) . Due to et al. [IS (0.87) indicating close relatedness between the individuals. In comparison, the heterozygosity in five Danish dog breeds was estimated to be between 0.27 and 0.36 [Molecular genetic studies have verified extremely low genetic diversity in the Lundehund. Melis et al.  reportedet al.  found an. [IS 0.8 indicatiThe Norwegian Lundehund club, the organization responsible for breeding and conservation of the Lundehund, has initiated a cross-breeding project to improve health and welfare of the population. In particular, it is critical to reduce the risk of developing IL and improve fertility without compromising the preservation of the distinctive characteristics of the breed. The Norwegian Kennel Club, the head organization for breed organizations, has granted permission to cross three phenotypically similar breeds into the Lundehund: Norwegian Buhund, Icelandic sheepdog and Norrbottenspets. To date, only a few attempts of crossbreeding have been made, and only two of those matings have resulted in litters.The sustainable management of small populations is dependent on the long term control of increases in additive genetic relationships. Optimal contribution selection (OCS) is a method that allows maximisation of genetic merit of a cohort of animals, while setting constraints on the average relationships between the individuals. OCS has proven to be a robust method also in complex breeding scenarios, imposed by various restrictions, realising most of the long-term genetic gain obtained by OCS without restrictions . Many smOur overall goal was to evaluate the conservation possibilities of the current Lundehund population. Consequently, we assessed the genetic diversity of the Lundehund population from pedigree data in terms of levels of inbreeding and relationships, probability of gene origin, and effective population size. We furthermore evaluated the potential of OCS and cross-breeding in the long-term management of the Lundehund population.Pedigree data from 1930 to 2015 comprising records of 5433 individuals was obtained from the Norwegian Lundehund Club. The data included identification of the individual (number and name), identification number of sire and dam, country of origin, country of residence, sex, birth date, information whether the dog is alive or not, and number of offspring. A total of 1220 individuals were registered as alive, of which 50.4% were living in Norway. The largest populations of Lundehund outside Norway are situated in Sweden (N = 156), Finland (N = 106) and Denmark (N = 78). The distribution of the current global Lundehund population is presented in The current pedigree dates back to the 1930s and these founder dogs were considered as unrelated. If the dogs used to construct the current population were related, the level of relatedness and average inbreeding in the current population will be underestimated.e. The effective population size based on different definitions of ΔF was computed as Population analyses relative to inbreeding, additive genetic relationships and pedigree completeness were carried out using EVA 2.1 ,9. The eF from the full data set (b) was used as the increase in inbreeding between two generations. Consequently, Ne was estimated as b) of the individual inbreeding coefficients over the equivalent generations was used to approximate et al. [b) and the average generation interval (l): l × b. From this, Ft is the average inbreeding of the population in question. Additionally, values of log(1 − Fi) were regressed on birth years (method IV a) or complete generation equivalents (method IV b) (Pérez-Enciso 1995) and First, three simple regression based methods were used for estimating Δdata set . Individs Ne=12b . The regz et al. , the inbe via individual increases in inbreeding (ΔFi) was estimated according to Gutiérrez et al.[Fi is the individual inbreeding coefficient, t the generation coefficient of individual i, and N is number of individuals in the reference population [e (method VI). Averaging out the increase in co-ancestry in all pairs of individuals in the reference population provides an estimate of et al. [et al. [In method V, Nez et al.. “Realizpulation ,14. The z et al. ,14. See, [et al.  for a cofe) is defined as the number of equally contributing founders that would be expected to result in the same genetic diversity as in the population under study, thus accounting for the loss in genetic variability due to unequal founder contributions [pi) of the founders fa) is defined as the minimum number of ancestors (not necessarily founders) explaining the complete genetic diversity of the population. Parameter fa is calculated as fe but replacing pi with marginal ancestral contributions (mi), and thus unlike fe, accounts for potential bottlenecks in the pedigree [fge [fge which may be obtained from the average co-ancestry of a predefined reference population t as fe, fge and fne were used to derive measures of genetic diversity. The degree of genetic diversity relative to the base accounting for loss of diversity due to unequal founder contribution was calculated as Parameters derived from the probabilities of gene origin were used as complementary to increase in inbreeding and effective population size to describe genetic diversity in the Lundehund population. The effective number of founders or the founder equivalent  and 12% (2014). Two incidences of drops in the average level of inbreeding coincide with these declines in PCI10. The first case is a result of “unknown” parentage of an imported individual causing an underestimation of the level of inbreeding in that cohort, as all Lundehund originate from Norway and thus are related to each other. The second case, in 2014, is a real fall in inbreeding as a consequence of using a Norwegian Buhund dam in breeding. High PCI of the Lundehund pedigree data ensures reliable estimation of the average levels of inbreeding in the current population.f) in the data set. If an animal with one unknown parent was defined as a half founder, the actual base population consisted of 43.5 individuals. Out of these, only 11 contributed to the last cohort of 2015 (percentage of genetic contribution in parentheses). Eight of the founders (7.0%) are parents born in the 1930s, each having one contributing offspring. One male founder (4.7%) has three offspring born in the 1960s, of which two have further contributed to the last cohort. The founder male contributing most to the last cohort has 10 offspring all born in the 1960s, which all have further contributed to the population. This individual is the second most contributing ancestor in the pedigree, with 35% contribution to the last cohort.There were 49 founder animals  throughout the pedigree is 1.0% per generation if linear regression of F over time was used for estimation. The average co-ancestry and inbreeding coefficient in the last fully registered cohort (2014/2) was 35.6% and 33.9%, respectively. The development of the level of inbreeding (F) between 1964/1 and 2015/1 together with PCI10 is shown in g was 4% .In total, 33 individuals registered in the pedigree had an inbreeding coefficient equal to or larger than 0.5. One generation of self-fertilisation or three generations of full-sib mating result in inbreeding coefficients of 0.5. Of the total number of matings, 0.98% (N = 53), 2.04% (N = 111) and 0.81% (N = 44) were full-sib, half-sib or parent-offspring matings, respectively.The global Lundehund population is highly inbred. To our knowledge, this is the second highest level of inbreeding reported for dogs in the scientific literature. Since 1996 the average within cohort inbreeding has been relatively stable between 36–38%, with the exception of two cohorts , where importation and the use of a foreign breed lowered the average inbreeding to 34% , panel ASeveral studies have reported average inbreeding coefficients for various dog breeds either assessed by genealogical or molecular data. Only one pedigree-based estimate of inbreeding for Lundehund has been reported prior to our study; Pfahler and Distl  have rece with different methods. Regression based methods estimated up to over 6-fold Ne (34–82) compared to estimates based on individual increase in inbreeding or co-ancestry in the Lundehund (13) [e estimation, whereas the regression on birth date was very sensitive to the choice of the time period . Based on the number of breeding animals, the effective population size for the Lundehund has earlier been reported to be approximately 80 [e from a molecular data of 28 individuals of Lundehund. Their estimate for the last 7–200 generations was 10–13, in good agreement with our pedigree based estimate, supporting a genetically small and closed population [Nbreeding . No earlery low 1. Based oe have been reported for a large group of dog breeds [et al. [e of 46–2136 in 61 dog breeds in France [et al. [e varied between 17 (Greyhound) and 114 (Labrador retriever) in10 dog breeds registered in the UK Kennel Club since 1970. In a more recent paper, Lewis et al. [et al. [e estimates based on both individual increase in inbreeding (3.2–829.1) and individual increase in co-ancestry (4.5–334.9) in total of 23 Belgian dog breeds. The variability in the reported estimates of Ne arises from the breed history, the amount and quality of the pedigree information, and in particular, from the method used to obtain the estimates. In regression based methods, inbreeding coefficients are regressed on either generations or year of birth. Consequently, the choice of the time period used for calculation of Ne becomes decisive: flat or negative trends of inbreeding will result in illogical estimates of Ne [e calculation [Regression based and highly variable estimates of Ng breeds ,23–26,29 [et al.  reportedn France . Accordi [et al.  reportedfe, fa, fge and fne. Only a small proportion of the base population contributed to the current Lundehund population, reflected by low ratio of fe to f (0.12). Comparison of fe and fa in Lundehund indicates substantial loss of genetic variation due to bottlenecks. The parameter fe is not very useful for assessing genetic diversity. First, it only reflects the inequalities in the proportional contributions of founders and does not account for genetic drift [fge is diminished by both unequal proportional contribution of founders and by genetic drift, that is, discounts fe to account for the irreversible losses of founder alleles [fge represents the cumulative loss of genetic diversity since the base population and directly relates to Ne [Parameters of probability of gene origin were estimated to be 6, 3, 1.3 and 1.6 for ic drift , and secic drift  as the g alleles . The vales to Ne ,19. Raties to Ne ,24,30, bfge has been constant, as has the total loss in genetic diversity.Genetic diversity measures GD, GD* and GD*-GD were used to quantify the impacts of unequal founder contribution, bottlenecks and random genetic drift. It was estimated that 8.3% of the variation in the base population was lost due to unequal founder contribution (GD* = 0.917) and additional 30.5% due to genetic drift accumulated over non-founder populations (GD*-GD). Consequently, 38.8% of the genetic diversity in the base population was lost from the current living Lundehund population. Proportionally, 78.5% of the total loss was due to random genetic drift and 21.5% due to unequal founder contributions. In fact, the total loss in genetic diversity is simply the average co-ancestry of the chosen reference population , which hMolecular genetic studies have showed extremely low genetic diversity in the Lundehund ,6,31 andVery similar results of OCS were obtained from the data sets global, Nordic and Norway, and only the latter is presented here . RelaxinWhen the individuals of cross-bred origin  were excluded as breeding candidates, OCS had no effect. The different scenarios of restricted male use resulted in identical levels of average relatedness and inbreeding in the next generation as observed in the last cohort, 76% and 38%, respectively.The results of the population analysis of Lundehund quantify the narrow genetic base well documented in the breed’s history . AlthougDue to the extremely high relatedness, OCS alone facilitates no improvement in the current situation in the Lundehund. This was clearly demonstrated by invariant OCS solutions over regions . If cross-bred individuals were excluded as breeding candidates, no favourable changes in relatedness were achieved independent on the number of matings allowed per male.In situations where genetic variation is depleted in a population, migration is the only method to introduce new variation into the population and enable sustainable management and selection . Based oa, of 0.77), introduction of additional 10 parents of foreign breed reduced the average parental a by approximately 40%, down to 0.42–0.48  were introduced. If instead of adding new candidates, ten purebred individuals were replaced by parents from other breeds, a reduction of 48–53% in parental a was observed. When breeders of foreign breeds were added to the default Lundehund parental group, the number of breeds and different levels of a and F of the foreign populations seemed to have a minor effect on the parental a, whereas the number of individuals introduced was decisive Click here for additional data file.