Adaptation genomics: the next generation

Understanding the genetics of how organisms adapt to changing environments is a fundamental topic in modern evolutionary ecology. The field is currently progressing rapidly because of advances in genomics technologies, especially DNA sequencing. The aim of this review is to first briefly summarise how next generation sequencing (NGS) has transformed our ability to identify the genes underpinning adaptation. We then demonstrate how the application of these genomic tools to ecological model species means that we can start addressing some of the questions that have puzzled ecological geneticists for decades such as: How many genes are involved in adaptation? What types of genetic variation are responsible for adaptation? Does adaptation utilise pre-existing genetic variation or does it require new mutations to arise following an environmental change?     

Understanding genetic variation and function- the applications of next generation sequencing

Next generation sequencing (NGS) technology has had a transformatory effect upon population-level studies linking genetic variation to gene function. In this review, I briefly describe recent studies that have used top-down genome scanning and population genetic approaches to identify loci under recent selection, as well as some examples of how large NGS datasets can be deployed to detect the total level of deleterious, neutral and advantageous variation present in standing genetic variation. I then explore studies that have used some of these approaches to study gene function along with advances in sequencing populations under selection, QTL mapping techniques and emerging methodologies utilising targeted capture and NGS.     

The status of AFLP in the genomics era and a pipeline for converting AFLPs into single-locus markers

Even though next-generation sequencing (NGS) has now become the predominant state-of-the-art technique for genotyping populations, amplified fragment length polymorphism (AFLP) DNA fingerprinting is still a relevant method, thanks to its versatility, cost-effectiveness, independence of prior sequence information and broad applicability. Even though the number of AFLP studies reached its peak in 2012, it is still applied extensively for phylogenetic analysis, genotyping or identifying non-model species, which often feature complex and large genomes. For these purposes, tools continue to be developed for designing AFLP studies, scoring AFLPs or handling AFLP data. Moreover, AFLP studies embrace the NGS technology; for example, the whole-genome sequence of model species is used to design more efficient AFLP studies for non-model species. Conversely, in complexity reduction of polymorphic sequences and restriction site-associated DNA sequencing studies, polymorphisms are often found to be present in many restriction sites, which can still be studied as AFLPs. We discuss the latest advances in AFLP-based studies in the era of NGS and anticipate that AFLP will remain a relevant method in the near future, even for species with a known genome, owing to its many promising new features such as methylation-sensitive-AFLP. Here, we also present an optimized pipeline for converting AFLP markers into single-locus markers, which can be applied in both traditional AFLP and NGS studies.     

Next-generation sequencing and its potential impact on food microbial genomics

Recent efforts of researchers to elucidate the molecular mechanisms of biological systems have been revolutionized greatly with the use of high throughput and cost-effective techniques such as next generation sequencing (NGS). Application of NGS to microbial genomics is not just limited to predict the prevalence of microorganisms in food samples but also to elucidate the molecular basis of how microorganisms respond to different food-associated conditions, which in turn offers tremendous opportunities to predict and control the growth and survival of desirable or undesirable microorganisms in food. Concurrently, NGS has facilitated the development of new genome-assisted approaches for correlating genotype and phenotype. The aim of this review is to provide a snapshot of the various possibilities that these new technologies are opening up in area of food microbiology, focusing the discussion mainly on lactic acid bacteria and yeasts associated with fermented food. The contribution of NGS to a system level understanding of food microorganisms is also discussed.     

Application of Next-generation Sequencing Technology in Forensic Science

Next-generation sequencing （NGS） technology, with its high-throughput capacity and low cost, has developed rapidly in recent years and become an important analytical tool for many genomics researchers. New opportunities in the research domain of the forensic studies emerge by harnessing the power of NGS technology, which can be applied to simultaneously analyzing multi- ple loci of forensic interest in different genetic contexts, such as autosomes, mitochondrial and sex chromosomes. Furthermore, NGS technology can also have potential applications in many other aspects of research. These include DNA database construction, ancestry and phenotypic inference, monozygotic twin studies, body fluid and species identification, and forensic animal, plant and microbiological analyses. Here we review the application of NGS technology in the field of forensic science with the aim of providing a reference for future forensics studies and practice.     

tropiTree: An NGS-Based EST-SSR Resource for 24 Tropical Tree Species

The development of genetic tools for non-model organisms has been hampered by cost, but advances in next-generation sequencing (NGS) have created new opportunities. In ecological research, this raises the prospect for developing molecular markers to simultaneously study important genetic processes such as gene flow in multiple non-model plant species within complex natural and anthropogenic landscapes. Here, we report the use of bar-coded multiplexed paired-end Illumina NGS for the de novo development of expressed sequence tag-derived simple sequence repeat (EST-SSR) markers at low cost for a range of 24 tree species. Each chosen tree species is important in complex tropical agroforestry systems where little is currently known about many genetic processes. An average of more than 5,000 EST-SSRs was identified for each of the 24 sequenced species, whereas prior to analysis 20 of the species had fewer than 100 nucleotide sequence citations. To make results available to potential users in a suitable format, we have developed an open-access, interactive online database, tropiTree (http://bioinf.hutton.ac.uk/tropiTree), which has a range of visualisation and search facilities, and which is a model for the efficient presentation and application of NGS data.     

Applications of next-generation sequencing to phylogeography and phylogenetics

This is a time of unprecedented transition in DNA sequencing technologies. Next-generation sequencing (NGS) clearly holds promise for fast and cost-effective generation of multilocus sequence data for phylogeography and phylogenetics. However, the focus on non-model organisms, in addition to uncertainty about which sample preparation methods and analyses are appropriate for different research questions and evolutionary timescales, have contributed to a lag in the application of NGS to these fields. Here, we outline some of the major obstacles specific to the application of NGS to phylogeography and phylogenetics, including the focus on non-model organisms, the necessity of obtaining orthologous loci in a cost-effective manner, and the predominate use of gene trees in these fields. We describe the most promising methods of sample preparation that address these challenges. Methods that reduce the genome by restriction digest and manual size selection are most appropriate for studies at the intraspecific level, whereas methods that target specific genomic regions (i.e., target enrichment or sequence capture) have wider applicability from the population level to deep-level phylogenomics. Additionally, we give an overview of how to analyze NGS data to arrive at data sets applicable to the standard toolkit of phylogeography and phylogenetics, including initial data processing to alignment and genotype calling (both SNPs and loci involving many SNPs). Even though whole-genome sequencing is likely to become affordable rather soon, because phylogeography and phylogenetics rely on analysis of hundreds of individuals in many cases, methods that reduce the genome to a subset of loci should remain more cost-effective for some time to come.     

Next-generation hybridization and introgression

Hybridization has a major role in evolution-from the introgression of important phenotypic traits between species, to the creation of new species through hybrid speciation. Molecular studies of hybridization aim to understand the class of hybrids and the frequency of introgression, detect the signature of ancient hybridization, and understand the behaviour of introgressed loci in their new genomic background. This often involves a large investment in the design and application of molecular markers, leading to a compromise between the depth and breadth of genomic data. New techniques designed to assay a large sub-section of the genome, in association with next-generation sequencing (NGS) technologies, will allow genome-wide hybridization and introgression studies in organisms with no prior sequence data. These detailed genotypic data will unite the breadth of sampling of loci characteristic of population genetics with the depth of sequence information associated with molecular phylogenetics. In this review, we assess the theoretical and methodological constraints that limit our understanding of natural hybridization, and promote the use of NGS for detecting hybridization and introgression between non-model organisms. We also make recommendations for the ways in which emerging techniques, such as pooled barcoded amplicon sequencing and restriction site-associated DNA tags, should be used to overcome current limitations, and enhance our understanding of this evolutionary significant process.     

A pipeline for effectively developing highly polymorphic simple sequence repeats markers based on multi‐sample genomic data

Simple sequence repeats (SSRs) are widely used genetic markers in ecology, evolution, and conservation even in the genomics era, while a general limitation to their application is the difficulty of developing polymorphic SSR markers. Next‐generation sequencing (NGS) offers the opportunity for the rapid development of SSRs; however, previous studies developing SSRs using genomic data from only one individual need redundant experiments to test the polymorphisms of SSRs. In this study, we designed a pipeline for the rapid development of polymorphic SSR markers from multi‐sample genomic data. We used bioinformatic software to genotype multiple individuals using resequencing data, detected highly polymorphic SSRs prior to experimental validation, significantly improved the efficiency and reduced the experimental effort. The pipeline was successfully applied to a globally threatened species, the brown eared‐pheasant (Crossoptilon mantchuricum), which showed very low genomic diversity. The 20 newly developed SSR markers were highly polymorphic, the average number of alleles was much higher than the genomic average. We also evaluated the effect of the number of individuals and sequencing depth on the SSR mining results, and we found that 10 individuals and ~10X sequencing data were enough to obtain a sufficient number of polymorphic SSRs, even for species with low genetic diversity. Furthermore, the genome assembly of NGS data from the optimal number of individuals and sequencing depth can be used as an alternative reference genome if a high‐quality genome is not available. Our pipeline provided a paradigm for the application of NGS technology to mining and developing molecular markers for ecological and evolutionary studies.     

Epigenetics across the evolutionary tree: New paradigms from non-model animals

All animals have evolved solutions to manage their genomes, enabling the efficient organization of meters of DNA strands in the nucleus and allowing for nuanced regulation of gene expression while keeping transposable elements suppressed. Epigenetic modifications are central to accomplishing all these. Recent advances in sequencing technologies and the development of techniques that profile epigenetic marks and chromatin accessibility using reagents that can be used in any species has catapulted epigenomic studies in diverse animal species, shedding light on the multitude of epigenomic mechanisms utilized across the evolutionary tree. Now, comparative epigenomics is a rapidly growing field that is uncovering mechanistic aspects of epigenetic modifications and chromatin organization in non-model invertebrates, ranging from octopus to sponges. This review puts recent discoveries in the epigenetics of non-model invertebrates in historical context, and describes new insight into the patterning and functions of DNA methylation and other highly conserved epigenetic modifications.     

Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae)

Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.     

生态基因组学研究进展

生态基因组学是一个整合生态学、分子遗传学和进化基因组学的新兴交叉学科。生态基因组学将基因组学的研究手段和方法引入生态学领域,通过将群体基因组学、转录组学、蛋白质组学等手段与方法将个体、种群及群落、生态系统不同层次的生态学相互作用整合起来,确定在生态学响应及相互作用中具有重要意义的关键的基因和遗传途径,阐明这些基因及遗传途径变异的程度及其生态和进化后果的特征,从基因水平探索有机体响应天然环境(包括生物与非生物的环境因子)的遗传学机制。生态基因组学的研究对象可以分为模式生物与非模式生物两大类。拟南芥、酿酒酵母等模式生物在生态基因组学领域发挥了重要作用。随着越来越多基因组学技术的开发与完善,越来越多的非模式生物生态基因组学的研究将为生态学的发展提供重要的理论与实践依据。生态基因组学最核心的方法包括寻找序列变异、研究基因差异表达和分析基因功能等方法。生态基因组学已广泛渗透到生态学的相关领域中,将会在生物对环境的响应、物种间的相互作用、进化生态学、全球变化生态学、入侵生态学、群落生态学等研究领域发挥更大的作用。