Sorting duplicated loci disentangles complexities of polyploid genomes masked by genotyping by sequencing

Many plants and animals of polyploid origin are currently enjoying a genomics explosion enabled by modern sequencing and genotyping technologies. However, routine filtering of duplicated loci in most studies using genotyping by sequencing introduces an unacceptable, but often overlooked, bias when detecting selection. Retained duplicates from ancient whole‐genome duplications (WGDs) may be found throughout genomes, whereas retained duplicates from recent WGDs are concentrated at distal ends of some chromosome arms. Additionally, segmental duplicates can be found at distal ends or nearly anywhere in a genome. Evidence shows that these duplications facilitate adaptation through one of two pathways: neo‐functionalization or increased gene expression. Filtering duplicates removes distal ends of some chromosomes, and distal ends are especially known to harbour adaptively important genes. Thus, filtering of duplicated loci impoverishes the interpretation of genomic data as signals from contiguous duplicated genes are ignored. We review existing strategies to genotype and map duplicated loci; we focus in detail on an overlooked strategy of using gynogenetic haploids (1N) as a part of new genotyping by sequencing studies. We provide guidelines on how to use this haploid strategy for studies on polyploid‐origin vertebrates including how it can be used to screen duplicated loci in natural populations. We conclude by discussing areas of research that will benefit from better inclusion of polyploid loci; we particularly stress the sometimes overlooked fact that basing genomic studies on dense maps provides value added in the form of locating and annotating outlier loci or colocating outliers into islands of divergence.     

Screening of duplicated loci reveals hidden divergence patterns in a complex salmonid genome

A whole‐genome duplication (WGD) doubles the entire genomic content of a species and is thought to have catalysed adaptive radiation in some polyploid‐origin lineages. However, little is known about general consequences of a WGD because gene duplicates (i.e., paralogs) are commonly filtered in genomic studies; such filtering may remove substantial portions of the genome in data sets from polyploid‐origin species. We demonstrate a new method that enables genome‐wide scans for signatures of selection at both nonduplicated and duplicated loci by taking locus‐specific copy number into account. We apply this method to RAD sequence data from different ecotypes of a polyploid‐origin salmonid (Oncorhynchus nerka) and reveal signatures of divergent selection that would have been missed if duplicated loci were filtered. We also find conserved signatures of elevated divergence at pairs of homeologous chromosomes with residual tetrasomic inheritance, suggesting that joint evolution of some nondiverged gene duplicates may affect the adaptive potential of these genes. These findings illustrate that including duplicated loci in genomic analyses enables novel insights into the evolutionary consequences of WGDs and local segmental gene duplications.     

Evolution of the active sequences of the HpaI short interspersed elements

Ninety-nine members of the salmonid HpaI and AvaIII families of short interspersed repetitive elements (SINEs) were aligned and a general consensus sequence was deduced. The presence of 26 correlated changes in nucleotides (diagnostic nucleotides) from those in the consensus sequence allowed us to divide the members of the HpaI family into 12 subfamilies and those of the AvaIII family into two subfamilies. On the basis of the average sequence divergences and the phylogenetic distributions of the subfamilies, the relative antiquity of the subfamilies and the process of sequential changes in the respective source sequences were inferred. Despite the higher mutation rates of CG dinucleotides in individual dispersed members, no hypermutability of CG positions was observed in changes in the source sequences. This result suggests that sequences of SINEs located in a nonmethylated or hypomethylated genomic region could have been selected as source sequences for retroposition and/or that some CG sites are the parts of recognition sequences of retropositional machineries. Correspondence to: N. Okada     

Paralogs are revealed by proportion of heterozygotes and deviations in read ratios in genotyping‐by‐sequencing data from natural populations

Whole‐genome duplications have occurred in the recent ancestors of many plants, fish, and amphibians, resulting in a pervasiveness of paralogous loci and the potential for both disomic and tetrasomic inheritance in the same genome. Paralogs can be difficult to reliably genotype and are often excluded from genotyping‐by‐sequencing (GBS) analyses; however, removal requires paralogs to be identified which is difficult without a reference genome. We present a method for identifying paralogs in natural populations by combining two properties of duplicated loci: (i) the expected frequency of heterozygotes exceeds that for singleton loci, and (ii) within heterozygotes, observed read ratios for each allele in GBS data will deviate from the 1:1 expected for singleton (diploid) loci. These deviations are often not apparent within individuals, particularly when sequence coverage is low; but, we postulated that summing allele reads for each locus over all heterozygous individuals in a population would provide sufficient power to detect deviations at those loci. We identified paralogous loci in three species: Chinook salmon (Oncorhynchus tshawytscha) which retains regions with ongoing residual tetrasomy on eight chromosome arms following a recent whole‐genome duplication, mountain barberry (Berberis alpina) which has a large proportion of paralogs that arose through an unknown mechanism, and dusky parrotfish (Scarus niger) which has largely rediploidized following an ancient whole‐genome duplication. Importantly, this approach only requires the genotype and allele‐specific read counts for each individual, information which is readily obtained from most GBS analysis pipelines.     

GENE‐DOSAGE EFFECTS ON FITNESS IN RECENT ADAPTIVE DUPLICATIONS: ace‐1 IN THE MOSQUITO CULEX PIPIENS

Gene duplications have long been advocated to contribute to the evolution of new functions. The role of selection in their early spread is more controversial. Unless duplications are favored for a direct benefit of increased expression, they are likely detrimental. In this article, we investigated the case of duplications favored because they combine already functionally divergent alleles. Their gene‐dosage/fitness relations are poorly known because selection may operate on both overall expression and duplicates relative dosage. Using the well‐documented case of Culex pipiens resistance to insecticides, we compared strains with various ace‐1 allele combinations, including two duplicated alleles carrying both susceptible and resistant copies. The overall protein activity was nearly additive, but, surprisingly, fitness correlated better with the relative proportion of susceptible and resistant copies rather than any absolute measure of activity. Gene dosage is thus crucial, duplications stabilizing a “heterozygote” phenotype. It corroborates the view that these were favored because they fix a permanent heterosis, thereby solving the irreducible trade‐off between resistance and synaptic transmission. Moreover, we showed that the contrasted successes of the two duplicated alleles in natural populations depend on genetic changes unrelated to ace‐1, confirming the probable implication of recessive sublethal mutations linked to structural rearrangements in some duplications.     

Simultaneous quantification of total and conjugated ubiquitin levels in a single immunoblot

Ubiquitin (Ub) is a posttranslational modifier, and total Ub (Ub_T) is always in dynamic equilibrium among free Ub (Ub_F), activated Ub (Ub_A), and conjugated Ub (Ub_C) in the forms of mono-Ub, thioester-bond-linked Ub, and peptide-bond-linked Ub, respectively. In this study, we developed a simple method to simultaneously determine the levels of Ub_T, Ub_F + Ub_A, and Ub_C in a single immunoblot and demonstrated its reliability and reproducibility by determining [Ub_T], [Ub_F + Ub_A], and [Ub_C] in various mouse tissues and cultured cells.     

An analysis of retroposition in plants based on a family of SINEs from Brassica napus

The identification of a family of SINE retroposons dispersed in the genome of oilseed rape Brassica napus has provided the basis for an evolutionary analysis of retroposition in plants. The repetitive elements (called S1_Bn) are 170 by long and occupy roughly 500 loci by haploid genome. They present characteristic features of SINE retroposons such as a 3 terminal A-rich region, two conserved polymerase III motifs (box A and B), flanking direct repeats of variable sizes, and a primary and secondary sequence homology to several tRNA species. A consensus sequence was made from the alignment of 34 members of the family. The retroposon population was divided into five subfamilies based on several correlated sets of mutations from the consensus. These precise separations in subfamilies based on diagnostic mutations and the random distribution of mutations observed inside each subfamily are consistent with the master sequence model proposed for the dispersion of mammalian retroposons. An independent analysis of each subfamily provides strong evidence for the coexpression of at least three subfamily master sequences (SMS). In contrast to mammalian retroposition, diagnostic positions are not shared between SMS. We therefore propose that SMS were all derived from a general master sequence (GMS) and independently activated for retroposition after a variable period of random drift. Possible models for plant retroposition are discussed.Abbreviations SMS subfamily master sequence - GMS general master sequence Correspondence to: J.-M. Deragon     

Patterns of gene duplication in the plant SKP1 gene family in angiosperms: evidence for multiple mechanisms of rapid gene birth

Gene duplication plays important roles in organismal evolution, because duplicate genes provide raw materials for the evolution of mechanisms controlling physiological and/or morphological novelties. Gene duplication can occur via several mechanisms, including segmental duplication, tandem duplication and retroposition. Although segmental and tandem duplications have been found to be important for the expansion of a number of multigene families, the contribution of retroposition is not clear. Here we show that plant SKP1 genes have evolved by multiple duplication events from a single ancestral copy in the most recent common ancestor (MRCA) of eudicots and monocots, resulting in 19 ASK (Arabidopsis SKP1-like) and 28 OSK (Oryza SKP1-like) genes. The estimated birth rates are more than ten times the average rate of gene duplication, and are even higher than that of other rapidly duplicating plant genes, such as type I MADS box genes, R genes, and genes encoding receptor-like kinases. Further analyses suggest that a relatively large proportion of the duplication events may be explained by tandem duplication, but few, if any, are likely to be due to segmental duplication. In addition, by mapping the gain/loss of a specific intron on gene phylogenies, and by searching for the features that characterize retrogenes/retrosequences, we show that retroposition is an important mechanism for expansion of the plant SKP1 gene family. Specifically, we propose that two and three ancient retroposition events occurred in lineages leading to Arabidopsis and rice, respectively, followed by repeated tandem duplications and chromosome rearrangements. Our study represents a thorough investigation showing that retroposition can play an important role in the evolution of a plant gene family whose members do not encode mobile elements.     

Testing Mendelian inheritance from field-collected parasites: Revealing duplicated loci enables correct inference of reproductive mode and mating system

Cryptic aspects of parasite population biology, e.g., mating systems, are increasingly being inferred from polymorphic and co-dominant genetic markers such as microsatellite loci. Underlying the use of such co-dominant markers is the assumption of Mendelian inheritance. The failure to meet this assumption can lead to artifactual statistics and erroneous population inferences. Here, we illustrate the importance of testing the Mendelian segregation and assortment of genetic markers and demonstrate how field-collected samples can be utilised for this purpose. To examine the reproductive mode and mating system of hermaphroditic parasites, we developed microsatellites for the cestode, Oochoristica javaensis. Among loci, we found a bimodal distribution of F_IS (a fixation index that quantifies the deviation from Hardy–Weinberg equilibrium within subpopulations) values where loci were either highly negative (close to −1) or highly positive (∼0.8). By conducting tests of Mendelian segregation from natural crosses, we determined that loci with negative F_IS values were in fact duplicated loci that were amplified by a single primer pair. Genetic crosses also provided linkage data and indicated that the duplicated loci most likely arose via tandem duplications rather than whole genome/chromosome duplications. By correcting for the duplicated loci, we were able to correctly infer that O. javaensis has sexual reproduction, but the mating system is highly inbred. To assist others in testing Mendelian segregation and independent assortment from natural samples, we discuss the benefits and limitations, and provide guidelines for particular parasite systems amenable to the methods employed here.     

Evolutionary dynamics of leucine-rich repeat receptor-like kinases and related genes in plants：A phylogenomic approach

Leucine-rich repeat （LRR） receptor-like kinases （RLKs）, evolutionarily related LRR receptor-like proteins （RLPs） and receptor-like cytoplasmic kinases （RLCKs） have important roles in plant signaling, and their gene subfamilies are large with a complicated history of gene duplication and loss. In three pairs of closely related lineages, including Arabidopsis thaliana and A. lyrata （Arabidopsis）, Lotus japonicus, and Medicago truncatula （Legumes）, Oryza sativa ssp. japonica, and O. sativa ssp. indica （Rice）, we find that LRR RLKs comprise the largest group of these LRR-related subfamilies, while the related RLCKs represent the smal est group. In addition, comparison of orthologs indicates a high frequency of reciprocal gene loss of the LRR RLK/LRR RLP/RLCK subfamilies. Furthermore, pairwise comparisons show that reciprocal gene loss is often associated with lineage-specific duplication（s） in the alternative lineage. Last, analysis of genes in A. thaliana involved in development revealed that most are highly conserved orthologs without species-specific duplication in the two Arabidopsis species and originated from older Arabidopsis-specific or rosid-specific duplications. We discuss＆amp;nbsp;potential pitfal s related to functional prediction for genes that have undergone frequent turnover （duplications, losses, and domain architecture changes）, and conclude that prediction based on phylogenetic relationships wil likely outperform that based on sequence similarity alone.     

Protein Tyrosine Kinase cDNAs from Amphioxus, Hagfish, and Lamprey: Isoform Duplications Around the Divergence of Cyclostomes and Gnathostomes

Animals evolved a variety of gene families involved in cell–cell communication and developmental control by gene duplication and domain shuffling. Each family is made up of several subtypes or subfamilies with distinct structures and functions, which diverged by gene duplications and domain shufflings before the divergence of parazoans and eumetazoans. Since the separation from protostomes, vertebrates expanded the multiplicity of members (isoforms) in the same subfamily by further gene duplications in their early evolution before the fish–tetrapod split. To know the dates of isoform duplications more closely, we have conducted isolation and sequencing cDNAs encoding the fibroblast growth factor receptor, Eph, src, and platelet-derived growth factor receptor subtypes belonging to the protein tyrosine kinase family from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Lampetra reissneri, a lamprey. From a phylogenetic tree of each subfamily inferred from a maximum likelihood (ML) method, together with a bootstrap analysis based on the ML method, we have shown that the isoform duplications frequently occurred in the early evolution of vertebrates around or just before the divergence of cyclostomes and gnathostomes by gene duplications and possibly chromosomal duplications. Received: 28 April 1998 / Accepted: 30 June 1999     

Linked by Ancestral Bonds: Multiple Whole-Genome Duplications and Reticulate Evolution in a Brassicaceae Tribe

Pervasive hybridization and whole-genome duplications (WGDs) influenced genome evolution in several eukaryotic lineages. Although frequent and recurrent hybridizations may result in reticulate phylogenies, the evolutionary events underlying these reticulations, including detailed structure of the ancestral diploid and polyploid genomes, were only rarely reconstructed. Here, we elucidate the complex genomic history of a monophyletic clade from the mustard family (Brassicaceae), showing contentious relationships to the early-diverging clades of this model plant family. Genome evolution in the crucifer tribe Biscutelleae (∼60 species, 5 genera) was dominated by pervasive hybridizations and subsequent genome duplications. Diversification of an ancestral diploid genome into several divergent but crossable genomes was followed by hybridizations between these genomes. Whereas a single genus (Megadenia) remained diploid, the four remaining genera originated by allopolyploidy (Biscutella, Lunaria, Ricotia) or autopolyploidy (Heldreichia). The contentious relationships among the Biscutelleae genera, and between the tribe and other early diverged crucifer lineages, are best explained by close genomic relatedness among the recurrently hybridizing ancestral genomes. By using complementary cytogenomics and phylogenomics approaches, we demonstrate that the origin of a monophyletic plant clade can be more complex than a parsimonious assumption of a single WGD spurring postpolyploid cladogenesis. Instead, recurrent hybridization among the same and/or closely related parental genomes may phylogenetically interlink diploid and polyploid genomes despite the incidence of multiple independent WGDs. Our results provide new insights into evolution of early-diverging Brassicaceae lineages and elucidate challenges in resolving the contentious relationships within and between land plant lineages with pervasive hybridization and WGDs.     

Tangled up in two: a burst of genome duplications at the end of the Cretaceous and the consequences for plant evolution

Genome sequencing has demonstrated that besides frequent small-scale duplications, large-scale duplication events such as whole genome duplications (WGDs) are found on many branches of the evolutionary tree of life. Especially in the plant lineage, there is evidence for recurrent WGDs, and the ancestor of all angiosperms was in fact most likely a polyploid species. The number of WGDs found in sequenced plant genomes allows us to investigate questions about the roles of WGDs that were hitherto impossible to address. An intriguing observation is that many plant WGDs seem associated with periods of increased environmental stress and/or fluctuations, a trend that is evident for both present-day polyploids and palaeopolyploids formed around the Cretaceous–Palaeogene (K–Pg) extinction at 66 Ma. Here, we revisit the WGDs in plants that mark the K–Pg boundary, and discuss some specific examples of biological innovations and/or diversifications that may be linked to these WGDs. We review evidence for the processes that could have contributed to increased polyploid establishment at the K–Pg boundary, and discuss the implications on subsequent plant evolution in the Cenozoic.     

A rare case of plastid protein‐coding gene duplication in the chloroplast genome of Euglena archaeoplastidiata (Euglenophyta)

Gene duplication is an important evolutionary process that allows duplicate functions to diverge, or, in some cases, allows for new functional gains. However, in contrast to the nuclear genome, gene duplications within the chloroplast are extremely rare. Here, we present the chloroplast genome of the photosynthetic protist Euglena archaeoplastidiata. Upon annotation, it was found that the chloroplast genome contained a novel tandem direct duplication that encoded a portion of RuBisCO large subunit (rbcL) followed by a complete copy of ribosomal protein L32 (rpl32), as well as the associated intergenic sequences. Analyses of the duplicated rpl32 were inconclusive regarding selective pressures, although it was found that substitutions in the duplicated region, all non‐synonymous, likely had a neutral functional effect. The duplicated region did not exhibit patterns consistent with previously described mechanisms for tandem direct duplications, and demonstrated an unknown mechanism of duplication. In addition, a comparison of this chloroplast genome to other previously characterized chloroplast genomes from the same family revealed characteristics that indicated E. archaeoplastidiata was probably more closely related to taxa in the genera Monomorphina, Cryptoglena, and Euglenaria than it was to other Euglena taxa. Taken together, the chloroplast genome of E. archaeoplastidiata demonstrated multiple characteristics unique to the euglenoid world, and has justified the longstanding curiosity regarding this enigmatic taxon.     

Molecular Evolution of Prolactin Gene Family in Rodents

In this study, we identified two novel members of prolactin gene family in rat by blast searches against the published genomic database. A further analysis showed that gene duplications leading to PRL gene family in rodents occurred after rodents diverged from other mammals. Major reorganization of the gene loci in rodents was largely completed before the split of rat and mouse. But PL-I and PL-II genes are the exceptions, which have clustered in a species-specific manner in the phylogenetic tree. By combining results from gene conversion testing, relative chromosomal location comparison and estimated time for gene duplication, we believe that rodent PL-I and PL-II genes are species-specific and are the results of serial duplications which occurred after the divergence of mouse and rat. Our analysis also reveals that continual gene duplication and divergence occurred during the evolution of rodent PRL gene family.     

ATG8 lipidation and ATG8‐mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci

Autophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin‐fold proteins Autophagy‐related (ATG)‐8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8‐PE adduct, we also show that ATG8 lipidation requires the ATG12–ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12–ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12–ATG5 adduct is essential for ATG8‐mediated autophagy in plants by promoting ATG8 lipidation.     

Evolutionary Dynamics of Recently Duplicated Genes: Selective Constraints on Diverging Paralogs in the <Emphasis Type="Italic">Drosophila pseudoobscura</Emphasis> Genome

Duplicated genes produce genetic variation that can influence the evolution of genomes and phenotypes. In most cases, for a duplicated gene to contribute to evolutionary novelty it must survive the early stages of divergence from its paralog without becoming a pseudogene. I examined the evolutionary dynamics of recently duplicated genes in the Drosophila pseudoobscura genome to understand the factors affecting these early stages of evolution. Paralogs located in closer proximity have higher sequence identity. This suggests that gene conversion occurs more often between duplications in close proximity or that there is more genetic independence between distant paralogs. Partially duplicated genes have a higher likelihood of pseudogenization than completely duplicated genes, but no single factor significantly contributes to the selective constraints on a completely duplicated gene. However, DNA-based duplications and duplications within chromosome arms tend to produce longer duplication tracts than retroposed and inter-arm duplications, and longer duplication tracts are more likely to contain a completely duplicated gene. Therefore, the relative position of paralogs and the mechanism of duplication indirectly affect whether a duplicated gene is retained or pseudogenized. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.