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September 22, 2019  |  

Genus-wide sequencing supports a two-locus model for sex-determination in Phoenix.

The date palm tree is a commercially important member of the genus Phoenix whose 14 species are dioecious with separate male and female individuals. To identify sex determining genes we sequenced the genomes of 15 female and 13 male Phoenix trees representing all 14 species. We identified male-specific sequences and extended them using phased single-molecule sequencing or BAC clones. We observed that only four genes contained sequences conserved in all analyzed Phoenix males. Most of these sequences showed similarity to a single genomic locus in the closely related monoecious oil palm. CYP703 and GPAT3, two single copy genes present in males and critical for male flower development in other monocots, were absent in females. A LOG-like gene appears translocated into the Y-linked region and is suggested to play a role in suppressing female flowers. Our data are consistent with a two-mutation model for the evolution of dioecy in Phoenix.

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September 22, 2019  |  

A PECTIN METHYLESTERASE gene at the maize Ga1 locus confers male function in unilateral cross-incompatibility.

Unilateral cross-incompatibility (UCI) is a unidirectional inter/intra-population reproductive barrier when both parents are self-compatible. Maize Gametophyte factor1 (Ga1) is an intraspecific UCI system and has been utilized in breeding. However, the mechanism underlying maize UCI specificity has remained mysterious for decades. Here, we report the cloning of ZmGa1P, a pollen-expressed PECTIN METHYLESTERASE (PME) gene at the Ga1 locus that can confer the male function in the maize UCI system. Homozygous transgenic plants expressing ZmGa1P in a ga1 background can fertilize Ga1-S plants and can be fertilized by pollen of ga1 plants. ZmGa1P protein is predominantly localized to the apex of growing pollen tubes and may interact with another pollen-specific PME protein, ZmPME10-1, to maintain the state of pectin methylesterification required for pollen tube growth in Ga1-S silks. Our study discloses a PME-mediated UCI mechanism and provides a tool to manipulate hybrid breeding.

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September 22, 2019  |  

Ma orthologous genes in Prunus spp. shed light on a noteworthy NBS-LRR cluster conferring differential resistance to root-knot nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

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September 22, 2019  |  

Cloning of the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase family.

Yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating fungal disease threatening much of global wheat production. Race-specific resistance (R)-genes are used to control rust diseases, but the rapid emergence of virulent Pst races has prompted the search for a more durable resistance. Here, we report the cloning of Yr15, a broad-spectrum R-gene derived from wild emmer wheat, which encodes a putative kinase-pseudokinase protein, designated as wheat tandem kinase 1, comprising a unique R-gene structure in wheat. The existence of a similar gene architecture in 92 putative proteins across the plant kingdom, including the barley RPG1 and a candidate for Ug8, suggests that they are members of a distinct family of plant proteins, termed here tandem kinase-pseudokinases (TKPs). The presence of kinase-pseudokinase structure in both plant TKPs and the animal Janus kinases sheds light on the molecular evolution of immune responses across these two kingdoms.

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September 22, 2019  |  

The sequence of a male-specific genome region containing the sex determination switch in Aedes aegypti.

Aedes aegypti is the principal vector of several important arboviruses. Among the methods of vector control to limit transmission of disease are genetic strategies that involve the release of sterile or genetically modified non-biting males, which has generated interest in manipulating mosquito sex ratios. Sex determination in Ae. aegypti is controlled by a non-recombining Y chromosome-like region called the M locus, yet characterisation of this locus has been thwarted by the repetitive nature of the genome. In 2015, an M locus gene named Nix was identified that displays the qualities of a sex determination switch.With the use of a whole-genome bacterial artificial chromosome (BAC) library, we amplified and sequenced a ~200 kb region containing the male-determining gene Nix. In this study, we show that Nix is comprised of two exons separated by a 99 kb intron primarily composed of repetitive DNA, especially transposable elements.Nix, an unusually large and highly repetitive gene, exhibits features in common with Y chromosome genes in other organisms. We speculate that the lack of recombination at the M locus has allowed the expansion of repeats in a manner characteristic of a sex-limited chromosome, in accordance with proposed models of sex chromosome evolution in insects.

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September 22, 2019  |  

Deletions linked to PROG1 gene participate in plant architecture domestication in Asian and African rice.

Improving the yield by modifying plant architecture was a key step during crop domestication. Here, we show that a 110-kb deletion on the short arm of chromosome 7 in Asian cultivated rice (Oryza sativa), which is closely linked to the previously identified PROSTRATE GROWTH 1 (PROG1) gene, harbors a tandem repeat of seven zinc-finger genes. Three of these genes regulate the plant architecture, suggesting that the deletion also promoted the critical transition from the prostrate growth and low yield of wild rice (O. rufipogon) to the erect growth and high yield of Asian cultivated rice. We refer to this locus as RICE PLANT ARCHITECTURE DOMESTICATION (RPAD). Further, a similar but independent 113-kb deletion is detected at the RPAD locus in African cultivated rice. These results indicate that the deletions, eliminating a tandem repeat of zinc-finger genes, may have been involved in the parallel domestication of plant architecture in Asian and African rice.

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September 22, 2019  |  

The genomic basis of color pattern polymorphism in the Harlequin ladybird.

Many animal species comprise discrete phenotypic forms. A common example in natural populations of insects is the occurrence of different color patterns, which has motivated a rich body of ecological and genetic research [1-6]. The occurrence of dark, i.e., melanic, forms displaying discrete color patterns is found across multiple taxa, but the underlying genomic basis remains poorly characterized. In numerous ladybird species (Coccinellidae), the spatial arrangement of black and red patches on adult elytra varies wildly within species, forming strikingly different complex color patterns [7, 8]. In the harlequin ladybird, Harmonia axyridis, more than 200 distinct color forms have been described, which classic genetic studies suggest result from allelic variation at a single, unknown, locus [9, 10]. Here, we combined whole-genome sequencing, population-based genome-wide association studies, gene expression, and functional analyses to establish that the transcription factor Pannier controls melanic pattern polymorphism in H. axyridis. We show that pannier is necessary for the formation of melanic elements on the elytra. Allelic variation in pannier leads to protein expression in distinct domains on the elytra and thus determines the distinct color patterns in H. axyridis. Recombination between pannier alleles may be reduced by a highly divergent sequence of ~170 kb in the cis-regulatory regions of pannier, with a 50 kb inversion between color forms. This most likely helps maintain the distinct alleles found in natural populations. Thus, we propose that highly variable discrete color forms can arise in natural populations through cis-regulatory allelic variation of a single gene. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

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September 22, 2019  |  

The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat sequences. The new sequences span the entire repeat regions with good coverage revealing their length, variation in repeat sequences and their internal organization. The sequences obtained were used to compare with available sequences from whole genome sequencing projects indicating variation in number of repeats and their internal organization between individuals. The lack of these sequences has limited the association of genetic alterations with disease. The full sequences of these mucins will now allow such studies, which could be of importance for inflammatory bowel diseases for MUC2 and gastric ulcer diseases for MUC6 where deficient mucus protection is assumed to play an important role.

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September 22, 2019  |  

Evolutionary conservation of Y Chromosome ampliconic gene families despite extensive structural variation.

Despite claims that the mammalian Y Chromosome is on a path to extinction, comparative sequence analysis of primate Y Chromosomes has shown the decay of the ancestral single-copy genes has all but ceased in this eutherian lineage. The suite of single-copy Y-linked genes is highly conserved among the majority of eutherian Y Chromosomes due to strong purifying selection to retain dosage-sensitive genes. In contrast, the ampliconic regions of the Y Chromosome, which contain testis-specific genes that encode the majority of the transcripts on eutherian Y Chromosomes, are rapidly evolving and are thought to undergo species-specific turnover. However, ampliconic genes are known from only a handful of species, limiting insights into their long-term evolutionary dynamics. We used a clone-based sequencing approach employing both long- and short-read sequencing technologies to assemble ~2.4 Mb of representative ampliconic sequence dispersed across the domestic cat Y Chromosome, and identified the major ampliconic gene families and repeat units. We analyzed fluorescence in situ hybridization, qPCR, and whole-genome sequence data from 20 cat species and revealed that ampliconic gene families are conserved across the cat family Felidae but show high transcript diversity, copy number variation, and structural rearrangement. Our analysis of ampliconic gene evolution unveils a complex pattern of long-term gene content stability despite extensive structural variation on a nonrecombining background.© 2018 Brashear et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019  |  

Glyphosate resistance and EPSPS gene duplication: Convergent evolution in multiple plant species.

One of the increasingly widespread mechanisms of resistance to the herbicide glyphosate is copy number variation (CNV) of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. EPSPS gene duplication has been reported in eight weed species, ranging from 3-5 extra copies to more than 150 extra copies. In the case of Palmer amaranth (Amaranthus palmeri), a section of >300 kb containing EPSPS and many other genes has been replicated and inserted at new loci throughout the genome, resulting in significant increase in total genome size. The replicated sequence contains several classes of mobile genetic elements including helitrons, raising the intriguing possibility of extra-chromosomal replication of the EPSPS-containing sequence. In kochia (Kochia scoparia), from three to more than 10 extra EPSPS copies are arranged as a tandem gene duplication at one locus. In the remaining six weed species that exhibit EPSPS gene duplication, little is known about the underlying mechanisms of gene duplication or their entire sequence. There is mounting evidence that adaptive gene amplification is an important mode of evolution in the face of intense human-mediated selection pressure. The convergent evolution of CNVs for glyphosate resistance in weeds, through at least two different mechanisms, may be indicative of a more general importance for this mechanism of adaptation in plants. CNVs warrant further investigation across plant functional genomics for adaptation to biotic and abiotic stresses, particularly for adaptive evolution on rapid time scales.© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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September 21, 2019  |  

Potato late blight field resistance from QTL dPI09c is conferred by the NB-LRR gene R8.

Following the often short-lived protection that major nucleotide binding, leucine-rich-repeat (NB-LRR) resistance genes offer against the potato pathogen Phytophthora infestans, field resistance was thought to provide a more durable alternative to prevent late blight disease. We previously identified the QTL dPI09c on potato chromosome 9 as a more durable field resistance source against late blight. Here, the resistance QTL was fine-mapped to a 186 kb region. The interval corresponds to a larger, 389 kb, genomic region in the potato reference genome of Solanum tuberosum Group Phureja doubled monoploid clone DM1-3 (DM) and from which functional NB-LRRs R8, R9a, Rpi-moc1, and Rpi_vnt1 have arisen independently in wild species. dRenSeq analysis of parental clones alongside resistant and susceptible bulks of the segregating population B3C1HP showed full sequence representation of R8. This was independently validated using long-range PCR and screening of a bespoke bacterial artificial chromosome library. The latter enabled a comparative analysis of the sequence variation in this locus in diverse Solanaceae. We reveal for the first time that broad spectrum and durable field resistance against P. infestans is conferred by the NB-LRR gene R8, which is thought to provide narrow spectrum race-specific resistance.

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September 21, 2019  |  

Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

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July 7, 2019  |  

Do echinoderm genomes measure up?

Echinoderm genome sequences are a corpus of useful information about a clade of animals that serve as research models in fields ranging from marine ecology to cell and developmental biology. Genomic information from echinoids has contributed to insights into the gene interactions that drive the developmental process at the molecular level. Such insights often rely heavily on genomic information and the kinds of questions that can be asked thus depend on the quality of the sequence information. Here we describe the history of echinoderm genomic sequence assembly and present details about the quality of the data obtained. All of the sequence information discussed here is posted on the echinoderm information web system, Echinobase.org. Copyright © 2015 Elsevier B.V. All rights reserved.

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July 7, 2019  |  

Comparative Analysis of the Shared Sex-Determination Region (SDR) among Salmonid Fishes.

Salmonids present an excellent model for studying evolution of young sex-chromosomes. Within the genus, Oncorhynchus, at least six independent sex-chromosome pairs have evolved, many unique to individual species. This variation results from the movement of the sex-determining gene, sdY, throughout the salmonid genome. While sdY is known to define sexual differentiation in salmonids, the mechanism of its movement throughout the genome has remained elusive due to high frequencies of repetitive elements, rDNA sequences, and transposons surrounding the sex-determining regions (SDR). Despite these difficulties, bacterial artificial chromosome (BAC) library clones from both rainbow trout and Atlantic salmon containing the sdY region have been reported. Here, we report the sequences for these BACs as well as the extended sequence for the known SDR in Chinook gained through genome walking methods. Comparative analysis allowed us to study the overlapping SDRs from three unique salmonid Y chromosomes to define the specific content, size, and variation present between the species. We found approximately 4.1 kb of orthologous sequence common to all three species, which contains the genetic content necessary for masculinization. The regions contain transposable elements that may be responsible for the translocations of the SDR throughout salmonid genomes and we examine potential mechanistic roles of each one. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019  |  

The assembly and characterisation of two structurally distinct cattle MHC class I haplotypes point to the mechanisms driving diversity.

In cattle, there are six classical MHC class I genes that are variably present between different haplotypes. Almost all known haplotypes contain between one and three genes, with an allele of Gene 2 present on the vast majority. However, very little is known about the sequence and therefore structure and evolutionary history of this genomic region. To address this, we have refined the MHC class I region in the Hereford cattle genome assembly and sequenced a complete A14 haplotype from a homozygous Holstein. Comparison of the two haplotypes revealed extensive variation within the MHC class Ia region, but not within the flanking regions, with each gene contained within a conserved 63- to 68-kb sequence block. This variable region appears to have undergone block gene duplication and likely deletion at regular breakpoints, suggestive of a site-specific mechanism. Phylogenetic analysis using complete gene sequences provided evidence of allelic diversification via gene conversion, with breakpoints between each of the extracellular domains that were associated with high guanine-cytosine (GC) content. Advancing our knowledge of cattle MHC class I evolution will help inform investigations of cattle genetic diversity and disease resistance.

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