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Knowledge of how individuals are related is important in many areas of research, and numerous methods for inferring pairwise relatedness from genetic data have been developed. However, the majority of these methods were not developed for situations where data are limited. Specifically, most methods rely on the availability of population allele frequencies, the relative genomic position of variants and accurate genotype data. But in studies of non‐model organisms or ancient samples, such data are not always available. Motivated by this, we present a new method for pairwise relatedness inference, which requires neither allele frequency information nor information on genomic position. Furthermore, it can be applied not only to accurate genotype data but also to low‐depth sequencing data from which genotypes cannot be accurately called. We evaluate it using data from a range of human populations and show that it can be used to infer close familial relationships with a similar accuracy as a widely used method that relies on population allele frequencies. Additionally, we show that our method is robust to SNP ascertainment and applicable to low‐depth sequencing data generated using different strategies, including resequencing and RADseq, which is important for application to a diverse range of populations and species.

Non-model plants i.e., the species which have one or all of the characters such as long life cycle, difficulty to grow in the laboratory or poor fecundity, have been schemed out of sequencing projects earlier, due to high running cost of Sanger sequencing. Consequently, the information about their genomics and key biological processes are inadequate. However, the advent of fast and cost effective next generation sequencing (NGS) platforms in the recent past has enabled the unearthing of certain characteristic gene structures unique to these species. It has also aided in gaining insight about mechanisms underlying processes of gene expression and secondary metabolism as well as facilitated development of genomic resources for diversity characterization, evolutionary analysis and marker assisted breeding even without prior availability of genomic sequence information. In this review we explore how different Next Gen Sequencing platforms, as well as recent advances in NGS based high throughput genotyping technologies are rewarding efforts on de-novo whole genome/transcriptome sequencing, development of genome wide sequence based markers resources for improvement of non-model crops that are less costly than phenotyping.

Understanding the genetic basis of adaptation is one of the central questions of evolutionary genetics. In the face of selective pressure, individuals with adaptive phenotypes survive and pass on their beneficial alleles that conferred this fitness advantage. Over sufficient time scales and under strong selection, differences accumulate between taxa in the form of physical and/or behavioral traits that have been shaped differently. By looking at the transcriptomes of related taxa we can identify sequence and regulatory differences that represent molecular signatures of these different selection regimes. When we study these differences between taxa that have evolved under opposing selective forces we can identify the genetic underpinnings of adaptation to those conditions. Species from the Heteromyidae form an exemplary system for studying the genetics of adaptation in a comparative framework. This new world family of rodents is comprised of three subfamilies (the Dipodomyinae, Perognathinae, and Heteromyinae) that range from the temperate/arid habitat of the southwestern United States down through Mexico and the mesic habitat of Central America into northern South America. Members of the Dipodomyinae and Perognathinae largely populate desert habitat and have developed numerous behavioral and physiological adaptations to combat the issue of water loss. This response to selection has manifested itself in extraordinarily efficient kidney function that forms a textbook example of adaptation. However, despite knowledge of their kidney physiology, the genetic basis of this ability to retain water during waste production is unknown. In contrast to the other Heteromyids, members of the Heteromyinae largely inhabit wet tropical and subtropical forest without the same need for efficient water use. This, coupled with a near simultaneous radiation into the three subfamilies provides a controlled framework to identify differences between species from dry and wet habitat as well as between temperate and tropical species. In this dissertation I utilize 2nd generation sequencing technologies to study these differences by characterizing the genes expressed in biologically interesting tissues of three Heteromyid species. In sequencing the kidney and spleen transcriptomes of adult Dipodomys spectabilis (banner-tailed kangaroo rat), Chaetodipus baileyi (Bailey's pocket mouse), and Heteromys desmarestianus (forest spiny pocket mouse), I have sampled a representative of the Dipodomyinae, Perognathinae, and Heteromyinae, respectively. In chapter 1, I took a first pass at characterizing the kidney transcriptome of D. spectabilis. I identified a priori candidate genes important to kidney function from genetic model rodent species in an effort to identify potential drivers of D. spectabilis' recorded ability to retain water during the production of highly concentrated waste. I hypothesized that those candidates identified in D. spectabilis coupled with genes upregulated in this adapted organ would contain signatures of selection and have drastically divergent patterns of expression as hallmarks of their role in adaptation to a desert lifestyle. In chapter 2, I sought to identify if these or other genes exhibited clear patterns of differential expression and differentiation from the mesic H. desmarestianus that were driven by the different selective regimes on desert and mesic species. I also compared both species to C. baileyi, hypothesizing that any consistent differences seen in both C. baileyi and D. spectabilis relative to H. desmarestianus would be attributable to adaptation to arid habitat. In this chapter I identified a large number of genes in each species (roughly 11,000 per species) and filtered this set down to a subset of genes that either due to expression patterns or positive selection have evidence to be possible drivers of efficient osmoregulation in desert Heteromyids. For chapter 3, I focused on the genes expressed in spleen as an important tissue for immune response. Again I utilized the comparative framework of the Heteromyid family history in comparing the tropical H. desmarestianus to the temperate D. spectabilis and C. baileyi. My hypothesis was that greater selective pressure on immune response from increased pathogen diversity and burden in the tropics would fashion sequence evolution and different levels of gene regulation for immunity genes in H. desmarestianus. As in chapter 2, I generated a large amount of sequence data while winnowing down to a small number of solid candidate genes displaying positive selection and differential gene expression between the tropical and temperate Heteromyids. Due to the consistent patterns seen in the comparisons of D. spectabilis and C. baileyi against H. desmarestianus I predict that a subset of the genes identified here from kidney and spleen RNA-seq will prove to play a significant adaptive role in other vertebrates. Namely, the genes identified in chapters 1 and 2 will play roles in adaptation to water scarcity and those identified in chapter 3 will be consistently affected by the immune challenge of increased pathogen exposure in tropical species.

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins. Electronic supplementary material The online version of this article (doi:10.1007/s00425-010-1121-1) contains supplementary material, which is available to authorized users.