Ernest F. Retzel

Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes.

BackgroundMeiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.ResultsA high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes.ConclusionThese findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.

Reaching the depth of the Chinese hamster ovary cell transcriptome

The high-throughput DNA sequencing Illumina Solexa GAII platform was employed to characterize the transcriptome of an antibody-producing Chinese hamster ovary (CHO) cell line. More than 55 million sequencing reads were generated and mapped to an existing set of CHO unigenes derived from expressed sequence tags (ESTs), as well as several public sequence databases. A very significant fraction of sequencing reads has not been previously seen. The frequency with which fragments of a unigene were sequenced was taken as an estimate of the abundance level of the corresponding transcripts. A wide dynamic range of transcript abundance levels was observed, spanning six orders of magnitude. However, the distribution of coverage across transcript lengths was found to vary, from relatively uniform to highly variable. This observation suggests that more challenges are yet to be resolved before direct sequencing can be used as a true quantitative measure of transcript level and for differential gene expression analysis. With the depth that high-throughput sequencing methods can reach, one can expect that the entire transcriptome of this industrially important organism will be decoded in the near future.

Male-specific region of the bovine Y chromosome is gene rich with a high transcriptomic activity in testis development

The male-specific region of the mammalian Y chromosome (MSY) contains clusters of genes essential for male reproduction. The highly repetitive and degenerative nature of the Y chromosome impedes genomic and transcriptomic characterization. Although the Y chromosome sequence is available for the human, chimpanzee, and macaque, little is known about the annotation and transcriptome of nonprimate MSY. Here, we investigated the transcriptome of the MSY in cattle by direct testis cDNA selection and RNA-seq approaches. The bovine MSY differs radically from the primate Y chromosomes with respect to its structure, gene content, and density. Among the 28 protein-coding genes/families identified on the bovine MSY (12 single- and 16 multicopy genes), 16 are bovid specific. The 1,274 genes identified in this study made the bovine MSY gene density the highest in the genome; in comparison, primate MSYs have only 31–78 genes. Our results, along with the highly transcriptional activities observed from these Y-chromosome genes and 375 additional noncoding RNAs, challenge the widely accepted hypothesis that the MSY is gene poor and transcriptionally inert. The bovine MSY genes are predominantly expressed and are differentially regulated during the testicular development. Synonymous substitution rate analyses of the multicopy MSY genes indicated that two major periods of expansion occurred during the Miocene and Pliocene, contributing to the adaptive radiation of bovids. The massive amplification and vigorous transcription suggest that the MSY serves as a genomic niche regulating male reproduction during bovid expansion.

ZNF280BY and ZNF280AY: autosome derived Y-chromosome gene families in Bovidae

BackgroundRecent progress in exploring the Y-chromosome gene content in humans, mice and cats have suggested that autosome-to-Y transposition of the male fertility genes is a recurrent theme during the mammalian Y-chromosome evolution. These transpositions are lineage-dependent. The purpose of this study is to investigate the lineage-specific Y-chromosome genes in bovid.ResultsWe took a direct testis cDNA selection strategy and discovered two novel gene families, ZNF280BY and ZNF280AY, on the bovine (Bos taurus) Y-chromosome (BTAY), which originated from the transposition of a gene block on the bovine chromosome 17 (BTA17) and subsequently amplified. Approximately 130 active ZNF280BY loci (and ~240 pseudogenes) and ~130 pseudogenized ZNF280AY copies are present over the majority of the male-specific region (MSY). Phylogenetic analysis indicated that both gene families fit with the birth-and-death model of evolution. The active ZNF280BY loci share high sequence similarity and comprise three major genomic structures, resulted from insertions/deletions (indels). Assembly of a 1.2 Mb BTAY sequence in the MSY ampliconic region demonstrated that ZNF280BY and ZNF280AY, together with HSFY and TSPY families, constitute the major elements within the repeat units. The ZNF280BY gene family was found to express in different developmental stages of testis with sense RNA detected in all cell types of the seminiferous tubules while the antisense RNA detected only in the spermatids. Deep sequencing of the selected cDNAs revealed that different loci of ZNF280BY were differentially expressed up to 60-fold. Interestingly, different copies of the ZNF280AY pseudogenes were also found to differentially express up to 10-fold. However, expression level of the ZNF280AY pseudogenes was almost 6-fold lower than that of the ZNF280BY genes. ZNF280BY and ZNF280AY gene families are present in bovid, but absent in other mammalian lineages.ConclusionsZNF280BY and ZNF280AY are lineage-specific, multi-copy Y-gene families specific to Bovidae, and are derived from the transposition of an autosomal gene block. The temporal and spatial expression patterns of ZNF280BY s in testis suggest a role in spermatogenesis. This study offers insights into the genomic organization of the bovine MSY and gene regulation in spermatogenesis, and provides a model for studying evolution of multi-copy gene families in mammals.

The expansion of the PRAME gene family in Eutheria.

The PRAME gene family belongs to the group of cancer/testis genes whose expression is restricted primarily to the testis and a variety of cancers. The expansion of this gene family as a result of gene duplication has been observed in primates and rodents. We analyzed the PRAME gene family in Eutheria and discovered a novel Y-linked PRAME gene family in bovine, PRAMEY, which underwent amplification after a lineage-specific, autosome-to-Y transposition. Phylogenetic analyses revealed two major evolutionary clades. Clade I containing the amplified PRAMEYs and the unamplified autosomal homologs in cattle and other eutherians is under stronger functional constraints; whereas, Clade II containing the amplified autosomal PRAMEs is under positive selection. Deep-sequencing analysis indicated that eight of the identified 16 PRAMEY loci are active transcriptionally. Compared to the bovine autosomal PRAME that is expressed predominantly in testis, the PRAMEY gene family is expressed exclusively in testis and is up-regulated during testicular maturation. Furthermore, the sense RNA of PRAMEY is expressed specifically whereas the antisense RNA is expressed predominantly in spermatids. This study revealed that the expansion of the PRAME family occurred in both autosomes and sex chromosomes in a lineage-dependent manner. Differential selection forces have shaped the evolution and function of the PRAME family. The positive selection observed on the autosomal PRAMEs (Clade II) may result in their functional diversification in immunity and reproduction. Conversely, selective constraints have operated on the expanded PRAMEYs to preserve their essential function in spermatogenesis.

The transcriptome landscape of early maize meiosis

BackgroundA major step in the higher plant life cycle is the decision to leave the mitotic cell cycle and begin the progression through the meiotic cell cycle that leads to the formation of gametes. The molecular mechanisms that regulate this transition and early meiosis remain largely unknown. To gain insight into gene expression features during the initiation of meiotic recombination, we profiled early prophase I meiocytes from maize (Zea mays) using capillary collection to isolate meiocytes, followed by RNA-seq.ResultsWe detected ~2,000 genes as preferentially expressed during early meiotic prophase, most of them uncharacterized. Functional analysis uncovered the importance of several cellular processes in early meiosis. Processes significantly enriched in isolated meiocytes included proteolysis, protein targeting, chromatin modification and the regulation of redox homeostasis. The most significantly up-regulated processes in meiocytes were processes involved in carbohydrate metabolism. Consistent with this, many mitochondrial genes were up-regulated in meiocytes, including nuclear- and mitochondrial-encoded genes. The data were validated with real-time PCR and in situ hybridization and also used to generate a candidate maize homologue list of known meiotic genes from Arabidopsis.ConclusionsTaken together, we present a high-resolution analysis of the transcriptome landscape in early meiosis of an important crop plant, providing support for choosing genes for detailed characterization of recombination initiation and regulation of early meiosis. Our data also reveal an important connection between meiotic processes and altered/increased energy production.

Comparative Transcriptomics of Early Meiosis in Arabidopsis and Maize

Though sexually reproductive plants share the same principle and most processes in meiosis, there are distinct features detectable. To address the similarities and differences of early meiosis transcriptomes from the dicot model system Arabidopsis and monocot model system maize, we performed comparative analyses of RNA-seq data of isolated meiocytes, anthers and seedlings from both species separately and via orthologous genes. Overall gene expression showed similarities, such as an increased number of reads mapping to unannotated features, and differences, such as the amount of differentially expressed genes. We detected major similarities and differences in functional annotations of genes up-regulated in meiocytes, which point to conserved features as well as unique features. Transcriptional regulation seems to be quite similar in Arabidopsis and maize, and we could reveal known and novel transcription factors and cis-regulatory elements acting in early meiosis. Taken together, meiosis between Arabidopsis and maize is conserved in many ways, but displays key distinctions that lie in the patterns of gene expression.

Characterization of a set of novel meiotically-active promoters in Arabidopsis

BackgroundHomologous recombination, together with selection, laid the foundation for traditional plant breeding. The recombination process that takes place during meiotic cell division is crucial for the creation of novel variations of highly desired traits by breeders. Gaining control over this process is important for molecular breeding to achieve more precise, large-scale and quicker plant improvement. As conventional ubiquitous promoters are neither tissue-specific nor efficient in driving gene expression in meiocytes, promoters with high meiotic activities are potential candidates for manipulating the recombination process. So far, only a few meiotically-active promoters have been reported. Recently developed techniques to profile the transcriptome landscape of isolated meiocytes provided the means to discover promoters from genes that are actively expressed in meiosis.ResultsIn a screen for meiotically-active promoters, we examined ten promoter sequences that are associated with novel meiotic candidate genes. Each promoter was tested by expressing a GFP reporter gene in Arabidopsis. Characterization of regulatory regions revealed that these meiotically-active promoters possessed conserved motifs and motif arrangement. Some of the promoters unite optimal properties which are invaluable for meiosis-directed studies such as delivering specific gene expression in early meiosis I and/or meiosis II. Furthermore, the examination of homologs of the corresponding genes within green plants points to a great potential of applying the information from Arabidopsis to other species, especially crop plants.ConclusionsWe identified ten novel meiotically-active promoters; which, along with their homologs, are prime candidates to specifically drive gene expression during meiosis in plants and can thus provide important tools for meiosis study and crop breeding.

The plant-specific protein FEHLSTART controls male meiotic entry, initializing meiotic synchronization in Arabidopsis.

Meiosis marks the transition from the sporophyte to the gametophyte generation in the life cycle of flowering plants, and creates genetic variations through homologous recombination. In most flowering plants, meiosis is highly synchronized within each anther, which is significant for efficient fertilization. To date, little is known about the molecular mechanisms of entry into meiosis and exit from it, and only a few genes in Arabidopsis have been characterized with a role in regulating meiotic progression. In this study, we report the functional characterization of a plant-specific basic helix-loop-helix (bHLH) protein, FEHLSTART (FST), a defect in which leads to premature meiotic entry and asynchronous meiosis, and results in decreased seed yield. Investigation of the time course of meiosis showed that the onset of leptotene, the first stage of prophase I, frequently occurred earlier in fst-1 than in the wild type. Asynchronous meiosis followed, which could manifest in the disruption of regular spindle structures and symmetric cell divisions in fst-1 mutants during the meiosis I/II transition. In accordance with frequently accelerated meiotic entry, whole-transcriptome analysis of fst-1 anthers undergoing meiosis revealed that 19 circadian rhythm genes were affected and 47 pollen-related genes were prematurely expressed at a higher level. Taken together, we propose that FST is required for normal meiotic entry and the establishment of meiotic synchrony.

A high-resolution comparative map of porcine chromosome 4 (SSC4)

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.