Fund: Croatian Science Foundation
Project type: Research project
Project Code: IP-2018-01-8708
Project title: Application of NGS methods in the assessment of genomic variability in ruminants - ANAGRAMS.
Project duration: 1.12.2018. to 30.05.2023.
Institution in which the project is carried out:
University of Zagreb Faculty of Agriculture
Prof. Ivica Kisić, PhD
Svetošimunska cesta 25, 10000 Zagreb, Croatia
Phone: +385 1 239 3779
Fax: +385 1 231 5300
E-mail: dekanat@agr.hr
Project Manager:
University of Zagreb Faculty of Agriculture
Department of Animal Science
Prof. Vlatka Čubrić Čurik, PhD
Phone: +385 1 239 4008
E-mail: vcubric@agr.hr
Associates from the Faculty of Agriculture:
Prof. Ino Čurik, PhD.H
Assist. Prof. Vladimir Brajković, PhD
Ivana Držaić, PhD
Assoc. Prof. Maja Ferenčaković, PhD
Mario Shihabi, MSc
External associate:
Dinko Radić, PhD, Culture Center Vela Luka, Vela Luka, Croatia
assist. prof. Strahil Ristov, PhD, Ruđer Bošković Institute, Zagreb, Hrvatska
assist. prof. Boris Lukić, Faculty of Agrobiotechnical Sciences, Osijek
prof. Stašo Forenbaher, PhD, Institute for Anthropological Research, Zagreb
Consultants:
prof. Peter Dovč, PhD, University of Ljubljana, Biotechnical Faculty, Domžale, Slovenia
Gregor Gorjanc, PhD, The University of Edinburgh, College of Medicine and Veterinary Medicine, Edinburgh, United Kingdom
prof. Meng-Hua Li, PhD, Chinese Academy of Sciences, Institute of Zoology, Beijing, China
prof. Preston T Miracle, PhD, University of Cambridge, St John’s College, Cambridge, United Kingdom
Siniša Radović, PhD, Croatian Academy of Science and Arts, The Institute for the Paleontology and Geology of the Quaternary Period, Zagreb, Croatia
assist. prof. Mojca Simčič, PhD, University of Ljubljana, Biotechnical Faculty, Domžale, Slovenia
prof. Johann Sölkner, PhD, University of Natural Resources and Life Sciences, Division of Livestock Sciences, Vienna, Austria
Summary
The extremely rapid development of next-generation sequencing (NGS) and computational capabilities has opened a range of new opportunities in various scientific fields (human and veterinary medicine, agriculture, forestry, ecology, informatics and even archeology). Unfortunately, the successful application of NGS and its use in various disciplines depends heavily on expertise in different fields. Here, we have brought together experts in molecular genetics, population genomics, animal husbandry, informatics, and archeology to answer several questions related to conservation genomics (effective population size, inbreeding, and deleterious genome load) and genomic biodiversity (mixing and population structure) of ruminants. We will also analyze ancient bones (over 3000 years old) of cattle, goats and sheep to improve our understanding of genomic changes in the domestication process (phylogeny of mitogenomes). Our project proposal has a strong component of theoretical outputs to estimate inbreeding, effective population size, and individual intervention, which will be analyzed by computer simulations (all sequences will be replicated). In addition, NGS analyzes (high throughput SNP beadchip and whole genome sequences) will be performed on several indigenous breeds (cattle, goats and sheep) with the following objectives: (i) ensuring the "genomic positioning" of Croatian breeds among other world breeds, (ii) assessing the conservation status of Croatian breeds (inbreeding level, effective population size, deleterious stress and genetic diversity) and (iii) identifying genomic uniquenesses (genomic regions) specific to Croatian native breeds. The obtained results will provide a basis for future management of breed conservation. The application of NGS has a strong potential economic impact on society in the future. Therefore, we will organize an open course "Genomics for the protection of animals", write several popularizing articles and train two PhD students.
Current situation in the field of research
The extremely rapid development in the field of molecular genetics (Kobold et al., 2013, Header and Chain, 2016), known as Next Generation Sequencing (NGS), and the increasing computational power and capacity (Eisnestein, 2015) have opened countless opportunities in scientific fields such as human and veterinary medicine (Manolio, 2013), agriculture (Van Tassell et al., 2005), ecology (Ekblom and Galindo, 2011), computer science (Tripathi et al., 2016), etc. Today, even archeology as a part of social sciences and humanities is drastically influenced by the development of NGS and new related computer programs (MacHugh et al., 2017; Marciniak and Perry, 2017). At the same time, this rapid change is opening up a range of economic opportunities that were not previously available or foreseeable. Unfortunately, effective use of all these technological advances relies heavily on knowledge from multiple disciplines (molecular biology, informatics, and population genetics) that is rarely offered in regular university programs. One explanation is that the development of NGS is very rapid, whereas the development and modification of academic curricula takes more time. Another explanation is that the required knowledge is interdisciplinary, from molecular genetics to informatics (bioinformatics, management of large databases) to whole disciplines related to its application (e.g. medicine, agriculture, archeology, etc.), which is difficult to harmonize in one program. Consequently, in Croatia and similarly developed countries, there is not a large number of scientific groups or commercial entities that can use the possibilities of NGS. With regard to knowledge transfer and its application in industry, a more active approach should be promoted.
In this project, we have assembled a MIT disciplinary (Stock and Burton, 2011) team of Croatian scientists with expertise in molecular genetics (Vlatka Čubrić Čurik, Vladimir Brajković), population genetics (Dragica Šalamon, Ino Čurik, Maja Ferenčaković), applied animal breeding (Maja Ferenčaković, Ino Čurik), computer science (Strahil Ristov), and archeology (Dinko Radić). While most of the team has collaborative experience from previous and some other projects, two new PhD students and one postdoc will be trained in the application of NGS. With funding from this project, an already enrolled PhD student Ivana Držaić, a young researcher at the Department Animal Sciences (Faculty of Agriculture, University of Zagreb), will be able to write her PhD thesis based on NGS analyzes. Another PhD student and a postdoc will be trained in the use of NGS analysis as part of the regular recruitment process for this project.
The core of the project is NGS analysis of native ruminant populations as an effective management technique for conservation. Recently, SNP chips for cattle (Van Tassell et al., 2005; Matukumalli et al., 2009), sheep (Kijas et al., 2014; Xu et al., 2017) and goats (Nicoloso et al., 2015; Tosser-Klopp et al., 2015) have been developed and effectively used for: (i) assessing the conservation status of populations, i.e. to evaluate the degree of inbreeding (Ferenčaković et al., 2013 a, b, Čurik et al., 2014), different effective population sizes (Flury et al., 2010; Barbato et al., 2015; Mészáros et al., 2015; Rodríguez-Ramilo et al., 2015; Wang, 2016), genetic diversity (Gautier et al, 2010; Kijas et al, 2014), (ii) determining the specificities of population structure (Decker et al., 2014,) and intervention (Frkonja et al., 2012; Khayatzadeh et al., 2016) and (iii) determining the genomic regions responsible for adaptation (Sabeti et al., 2002; Qanbari et al., 2012; Utsunomiya et al., 2012). Assessment of all these genomic parameters is important in selected populations as it provides a reference that is a prerequisite for future biodiversity management. Simultaneous whole-genome sequencing of native breeds provides a genomic description of the breed over a long period of time (Bruford et al., 2015) and allows the assessment of pollution levels (Bosse et al., 2015; Zhang et al., 2015). In addition, a significant part of the project will be based on computer simulations and the development of new theoretical measurements related to: (i) estimating effective population size from genomic inbreeding, (ii) comparing correlation with effective population size from gamete imbalance, (iii) providing information on individual interventions, and (iv) on inbreeding assessment for sex chromosomes. The mouflon population from the experiment "Kalifront Mouflon" is analyzed as an example of a population living under natural conditions and hunted with a high degree of inbreeding.
We believe that the information provided will help to understand the impact of wildlife population reduction. Croatia is known for its rich zooarchaeological remains (mainly bones) of animals (Miracle and Brajković, 1992; Miracle et al., 2010; Lenardić et al., 2017) from different stages of the domestication process (pre-domestication, early and later stages of domestication.) ). The analysis within the archaeogenetics project will contribute to a better understanding of the domestication process, especially when Croatia is located on the main migration routes from Middle East to Europe (Zilihao 2001; Beja-Pereira et al., 2006). There are two opposing hypotheses, one supports the migration of domesticated animals including interbreeding of domestic and wild animals (Loftus et al., 1999; Cymbrion et al., 2005) and the other supports the complexity of the domestication process including inbreeding of domestic and wild animals (Beja-Pereira et al., 2006). Thus, given the domestication of cattle, we expect to find traces of the mitogenome of the population of the ancient aurochs that once inhabited the area below the sea level of the Adriatic Sea. In contrast, we expect to confirm that the mitochondrial haplotypes of the "ovidocaprids" (sheep and goats) are very similar to the haplotypes of the Fertile Crescent. Archaeogenetics is a very young scientific discipline that is developing rapidly, and several successful papers have been presented recently (Brajkovic et al., ISAG meeting poster).
Relation of the project to the current situation in the research field
The core of the research group involved in this project (hereafter referred to as the "AGB group") consists of seven researchers from Department Animal Sciences (Faculty of Agriculture, University of Zagreb) who are responsible for research and teaching in the field of animal breeding and genetics. In the last two decades, the members of the "AGB group" have published a number of articles in well-known journals on genetics and heredity (Nature Genetics, PLoS Genetics, Heredity, Genetic Selection Evolution, Animal Genetics, PLoS One, BMC Genomics, Conservation Genetics, Journal of Animal Breeding and Genetics, etc.) With the development of NGS, the research activities of "AGB group", in collaboration with many foreign scientists, focus on the use of information from high-throughput SNP chips on topics related to genomics of farm animal populations, and their research is internationally recognized. Here we would like to highlight the pioneering contribution to the assessment of genomic inbreeding in livestock populations (Ferenčaković et al., 2013a, b, Curik et al., 2014, Zavarez et al., 2015,) and inbreeding depression (Ferenčaković et al., 2017, Curik et al. al., 2017), where three scientific papers were recently published (Ferenčaković et al., 2013a, b, Curik et al., 2014a), cited 89 times (Web of Science Core Collection).
In addition, the research of Frkonja et al. (2012) has been cited 25 times (Web of Science Core Collection) and is one of the reference works on perturbation methodology among researchers in domestic animal genomics. In addition, members of the "AGB group" participated in the evaluation of effective population size, where a very interesting approach was presented (Curik et al., 2014b, Kukučkova et al., 2017, CHINA; PORTUGAL), but it was not fully evaluated at the theroetic level by computer simulations (proposed in this project). Unfortunately, apart from a pilot study (Curik et al., 2014b), this study was not performed on the domestic animal population, as two previous applications of the Croatian Science Fund did not recognize this. Although we have been successful in SNP chip-based research, we are strategically in transition to conduct high quality whole genome sequence (WGS) research as well as research related to archaeogenetics. In our current project "MitoTAUROMics" funded by Croatian Science Foundation, we have learned to type mitogens with NGS as well as analyze sequences, including extraction of mitogen data from publicly available WGSs (EAAP, ISAG), and as part of the project we have built the largest database of bovine mitogenome data (currently we have 850 sequences from more than 100 breeds).
The most attractive result of the "MitoTAUROMics" project was the demonstration of the presence of the maternal P Aurocha lineage in the modern cattle population (Cubric Curik et al., 2017 a, b). Based on the results and sequences obtained in "MitoTAUROMics", where a large number of European breeds were typed, we prepared background information for analyzes with ancient specimens (planned as part of this project). For the needs of "MitoTAUROMics", the "AGB group" established cooperation with a computer expert, Strahil Ristov, from the Ruđer Bošković Institute (Čačić et al., 2014, Ristov et al., 2016). The experience of a computer expert has been shown to be crucial for the efficient performance of computer-complex NGS analyzes. A very important part of the project proposal relates to the analysis of ancient DNA (from bone). "AGB-Group" is currently participating in an EU project entitled "Smart Integration of Genetics with the Sciences of the Past in Croatia: Minding and Mending the Gap" (MendTheGap). This was the top ranked project out of 546 projects in the H2020-TWINN-2015 competition, Twinning: Spreading excellence and Widening Participation. While the MendTheGap project promotes collaboration between genetics and past science in Croatia, the support from the Ministry of Science, Education and Sports in the call "Developing and strengthening synergies with horizontal activities of HORIZON 2020: twinning and ERA Chairs" (KK.01.1.1.06) was related to Structural Funds resources, MendTheGap does not allow direct research costs. Therefore, the HRZZ RFP is an opportunity for us to complement the effectiveness of the MendTheGap project with research in archaeogenetics. Archeological expertise in the project is provided by two experts Dinko Radić from the Vela Luka Cultural Center and two consultants, Siniša Radović from the Croatian Academy of Sciences and Arts and Preston Miracle from the University of Cambridge, scientists who are recognized archeologists and two zooarchaeology experts who have expertise in tree analysis of bones (Radović and Miracle) and work on archeological sites in Croatia (Miracle and Brajković, 1992; Miracle et al., 2010; Farbstein et al., 20112; Lenardić et al., 2017) can provide.
In general, we are aware that the project proposal is ambitious and requires additional training and/or consultation with experts in the field. Nevertheless, a large number of researchers are involved in this project, two PhD studies are planned, and we already have a number of nice publications in the field of domestic animal genomics. In addition, many of our collaborators and strategic partners are involved in this project as consultants to provide expertise when needed.
Methodology
The project will be implemented in 6 separate work packages (WPs), which are large units with one or more objectives.
Work package 1 (WP1), NGS analysis and database construction
In this random package, NGS genotyping (using high-density Illumina-based SNP bead chips) is performed for the following indigenous ruminant breeds in the Republic of Croatia: Istrian Cattle (25), Buša (25), Slavonian-Sirmian Podolian Cattle (25), Croatian Spotted Goat (25 ), Pag Sheep (25), Istrian Sheep (25), Ore (25) and Dalmatian Pramenka (25), as well as the population of mouflon (25) from the area "Kalifront polygon" on Rab (which is a small closed population). The planned number of individuals is 25 per breed / population at the price of 120 EUR per sample. This is still a small number of individuals to assess conservation status, but we hope that the price will drop and allow us to perform more analyzes. Whole genome sequencing for Istrian Cattle (5), Buša (5), Slavonian-Sirmian Podolian Cattle (3), Croatian Spotted Goat (5) and Califront Mouflon (5) will be possible while we will take part of the sequence from the repository. Sampling is not necessary for these analyzes as the AGB group has a good collection of Croatian native breeds from the MitoTAUROmics project. Sequencing will take place in the second year of the project, so the number may increase. After NGS genotyping, we will combine the data with other cattle breeds from Gautier et al., 2010; Kijas et al., 2012; Decker et al., 2014. We have experience in merging the bases of world breeds with local breeds (Kukučkova et al., 2017 and Curik at al., 2017 - 1.
Work package 2 (WP2), New methods and development of new conservation measures
The research will be based on computer simulations and ideas derived from our work over the last seven years. Some ideas have already been presented with good feedback (e.g. ROH inbreeding effective size was presented by Curik et al., (2016 - Conference on Conservation Genomics in Portugal) and Curik et al, (2017 - 1st World Conference on sheep in China). For good publications it is necessary to perform computer simulations.The idea is to estimate effective population size with gamete imbalance (Wang, 2016) based on linkage disequilibrium (Flury et al, 2010) with statistical prediction.We are aware that the evaluation of inbreeding effective size proposed by Ferenčaković et al. (2013b) proposed assessment of inbreeding effective size can be used to evaluate the admixture of an individual.The idea is based on the fact that long ROHs can come from parents that have a distant past, but this requires computer simulations and The base population is important in estimating the inbreeding coefficient by ROH (Curik et al, 2014).The segregation of the length of nearest ancestor ROHs is different for autosomes than for sex chromosomes. Therefore, it is not known how to interpret the ROH inbreeding coefficient estimated by sex chromosomes. An additional idea is to collect empirical data on the relationship between conservation measures, e.g. genetic variability, historical effective population size (Li and Durbin, 2011), inbreeding levels (Zhang et al., 2015a) and deleterious accumulations (Bosse et al., 2015; Zhang et al., 2015b and Nuijten et al, 2016) obtained from the whole genome sequence.
Work Package 3 (WP3), Genomic Characterization from Croatian Indigenous ruminant
The results of the research in WP3 will not have a large response factor as the usual methodology will be used, as the work influences the methodology of Kukučkova et al. (2017). All three studies will be important for conservation management of genomic variability and future selection programs.
Work package 4 (WP4), Archaeogenetic research of Croatian ruminant mitogenomes.
Extraction of aDNA will be performed using the classical phenol-chloroform method with prior physical purification of samples. Special attention will be paid to contamination. We will start with 50 samples, from which we hope to successfully isolate 20 to 30 aDNAs. Using the "capture enrichment method" (Maricic et al. 2010), an mtDNA library will be created for NGS sequencing. From 20-30 aDNAs, we expect at least 15 libraries to have 5-10 whole mitogenomes after NGS sequencing. The resulting sequences will be compared with those from genetic repositories using various software such as: Clustal Omega (Sievers et al., 2011), MEGA 7 (Kumar et al., 2016), Network version 5.0.0.3. (Bandelt et al., 1999), PopArt (Leigh and Bryant 2015), Galaxy platform (Afghan et al., 2016), Mitotoolpy (Peng et al., 2015), and SAS software version 9.3 (SAS Institute, Cary, North Carolina, USA). A large base of mitogenomes is assembled. BEAST 2 (Bouckaert et al., 2014), a Bayesian phylogenetic analysis program (Rieux and Khatchikian 2017), is used. The program evaluates rooted (Duchêne et al., 2011), (Rieux and Khatchikian 2017) time-measured phylogeny using "strict" or "relaxed" molecular clock models. BEAST2 uses the Markov chain Monte Carlo (MCMC) method so that each tree is proportionally determined according to its posterior probability.
Work Package 5 (WP5), Training and Dissemination and Work Package 6 (WP6), Project Management
As WP5 and WP6 refer to training, dissemination and project management, they are transparent and do not need to be explained.
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