Primitive Genepools of Asian Pears and Their Complex Hybrid Origins Inferred from Fluorescent Sequence-Specific Amplification Polymorphism (SSAP) Markers Based on LTR Retrotransposons

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Abstract

Recent evidence indicated that interspecific hybridization was the major mode of evolution in Pyrus. The genetic relationships and origins of the Asian pear are still unclear because of frequent hybrid events, fast radial evolution, and lack of informative data. Here, we developed fluorescent sequence-specific amplification polymorphism (SSAP) markers with lots of informative sites and high polymorphism to analyze the population structure among 93 pear accessions, including nearly all species native to Asia. Results of a population structure analysis indicated that nearly all Asian pear species experienced hybridization, and originated from five primitive genepools. Four genepools corresponded to four primary Asian species: P. betulaefolia, P. pashia, P. pyrifolia, and P. ussuriensis. However, cultivars of P. ussuriensis were not monophyletic and introgression occurred from P. pyrifolia. The specific genepool detected in putative hybrids between occidental and oriental pears might be from occidental pears. The remaining species, including P. calleryana, P. xerophila, P. sinkiangensis, P. phaeocarpa, P. hondoensis, and P. hopeiensis in Asia, were inferred to be of hybrid origins and their possible genepools were identified. This study will be of great help for understanding the origin and evolution of Asian pears.

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The genome of the pear ( Pyrus bretschneideri Rehd.)

Jun Jun Wu, Zhiwen Wang, Zebin Shi … (2013)

The draft genome of the pear ( Pyrus bretschneideri ) using a combination of BAC-by-BAC and next-generation sequencing is reported. A 512.0-Mb sequence corresponding to 97.1% of the estimated genome size of this highly heterozygous species is assembled with 194× coverage. High-density genetic maps comprising 2005 SNP markers anchored 75.5% of the sequence to all 17 chromosomes. The pear genome encodes 42,812 protein-coding genes, and of these, ∼28.5% encode multiple isoforms. Repetitive sequences of 271.9 Mb in length, accounting for 53.1% of the pear genome, are identified. Simulation of eudicots to the ancestor of Rosaceae has reconstructed nine ancestral chromosomes. Pear and apple diverged from each other ∼5.4–21.5 million years ago, and a recent whole-genome duplication (WGD) event must have occurred 30–45 MYA prior to their divergence, but following divergence from strawberry. When compared with the apple genome sequence, size differences between the apple and pear genomes are confirmed mainly due to the presence of repetitive sequences predominantly contributed by transposable elements (TEs), while genic regions are similar in both species. Genes critical for self-incompatibility, lignified stone cells (a unique feature of pear fruit), sorbitol metabolism, and volatile compounds of fruit have also been identified. Multiple candidate SFB genes appear as tandem repeats in the S -locus region of pear; while lignin synthesis-related gene family expansion and highly expressed gene families of HCT , C3′H , and CCOMT contribute to high accumulation of both G-lignin and S-lignin. Moreover, alpha-linolenic acid metabolism is a key pathway for aroma in pear fruit.

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Nested retrotransposons in the intergenic regions of the maize genome.

N Motchoulskaia, Jodi Edwards, John M. Lee … (1996)

The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

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The diversity of LTR retrotransposons

Ericka Havecker, Xiang Gao, Daniel F. Voytas (2004)

Transposons are mobile genetic elements that can multiply in the genome using a variety of mechanisms. Retrotransposons replicate through reverse transcription of their RNA and integration of the resulting cDNA into another locus. This mechanism of replication is shared with retroviruses, with the difference that retrotransposons do not form infectious particles that leave the cell to infect other cells. The long terminal repeat (LTR) retrotransposons, one of the main groups of retroelements (which include both LTR and non-LTR retrotransposons as well as retroviruses), are among the most abundant constituents of eukaryotic genomes. The LTRs are the direct sequence repeats that flank the internal coding region, which - in all autonomous (functional) LTR retrotransposons - includes genes encoding both structural and enzymatic proteins. The gag gene encodes structural proteins that form the virus-like particle (VLP), inside which reverse transcription takes place. The pol gene encodes several enzymatic functions, including a protease that cleaves the Pol polyprotein, a reverse transcriptase (RT) that copies the retrotransposon's RNA into cDNA, and an integrase that integrates the cDNA into the genome. Much of what we know about the mechanisms of LTR retrotransposition (Figure 1) comes from work on yeast retrotransposons [1,2], but it is generally assumed that the mechanism is very similar among LTR retrotransposons from divergent hosts. First, a retrotransposon's RNA is transcribed by the cellularly encoded RNA polymerase II from a promoter located within the 5' LTR. The RNA is then translated in the cytoplasm to give the proteins that form the VLP and carry out the reverse transcription and integration steps. Typically, two RNA molecules are packaged into one virus-like particle, and the RNA is subsequently made into a full-length DNA copy through a reverse transcription reaction that is first primed from a tRNA that pairs to a sequence near the 5' LTR (the primer-binding site). The resulting partial cDNA (called 'strong stop' DNA) is transferred from the 5' LTR to the 3' LTR, where reverse transcription proceeds. A second priming event initiates at a polypurine tract near the 3' LTR. The cDNA primed from the polypurine tract undergoes an additional strand transfer, ultimately giving rise to a double-stranded cDNA molecule. Finally, the cDNA is integrated back into the host DNA, adding another copy of the retrotransposon to the genome. LTR retrotransposon diversity As genome-sequence data has accumulated for a large number of eukaryotes, it has become clear that the genomes of most organisms contain LTR retrotransposons from multiple distinct lineages. Although all are flanked by LTRs and encode gag and pol genes, the lineages diverge considerably in their DNA sequences and genomic organization. The International Committee on Taxonomy of Viruses has attempted to provide a taxonomic framework for understanding the relationships among the vast numbers of retrotransposons that have come to light through genome-sequence analysis [3,4] (Figure 2); this framework is based on relationships among the amino-acid sequences of the RT protein, the most highly conserved of the retrotransposon proteins. Two retrotransposon families - the Pseudoviridae and the Metaviridae - have been described in detail; both are found in most eukaryotes. The two families are also distinguished by the order of the coding regions within their pol genes (see Figure 3). Discovery of the Gmr1 retrotransposon from Atlantic cod and related elements has shown that some members of the Pseudoviridae (on the basis of RT sequence) have a gene order characteristic of Metaviridae [5]. As with any taxonomic framework, the LTR retrotransposon classification system undergoes frequent revision as diverse elements are identified. This is particularly true for the genera that make up the two main families. Three genera have been proposed for the Pseudoviridae (Figure 2): pseudoviruses, hemiviruses and sireviruses (whose names do not necessarily indicate that they are viruses; Figure 2). The sireviruses derive from plant hosts and make up a distinct lineage according to their RT amino-acid sequences; the pseudoviruses and hemiviruses are distinguished by the primer used for reverse transcription (a full tRNA or a half tRNA, respectively). Note that this classification does not correspond directly with the phylogenetic relationships of the retrotransposons, so that the pseudoviruses make up three distinct lineages (Figure 2). The Metaviridae also comprises three genera - the metaviruses, the errantiviruses and the semotiviruses - which can be discriminated by phylogenetic analysis of RT amino-acid sequences. A distinct lineage of elements, the DIRS group (named after the founding member from Dictyostelium discoideum), has yet to be placed within the taxonomic framework. In addition to having characteristic RT sequences, the DIRS elements have some unusual features: they lack a protease and have a tyrosine recombinase instead of an integrase [6,7]. Organization of the gag and the pol genes Whereas RT amino-acid sequences and the order of domains within pol are sufficiently conserved to be used to classify the LTR retrotransposons, the ways in which gag and pol are organized and expressed vary considerably. As multiple proteins are encoded on one mRNA, the gag and pol genes in some LTR retrotransposons are separated by a frameshift or a stop codon, and occasionally these breaks in the reading frame are ignored by the translational machinery. Much more Gag than Pol is needed for productive VLP formation and consequently for replication of the retrotransposon; the use of either a stop codon that is occasionally ignored or ribosomal frameshifting (strategies called recoding) are used to regulate the ratio of the two proteins. We [8] have analyzed the genome sequences of Caenorhabditis elegans, Schizosaccharomyces pombe, Drosophila melanogaster, Candida albicans and Arabidopsis thaliana to predict the strategies used to express their gag and the pol genes. By analyzing the genomic structure and the nucleotide sequences surrounding the gag-pol junction, the type of recoding used for translation of the Pol protein could be inferred [8]. The results indicated that the mechanism used to express Pol is related to the host from which the retrotransposon originates. For example, about 50% of the retrotransposons identified in the study had a single open reading frame (ORF) fusing Gag and Pol, and this organization was the one found most often in plant elements. A single Gag-Pol ORF does not undergo recoding per se but is subjected to other mechanisms, such as differential protein degradation, to ensure a high ratio of Gag to Pol. Retrotransposons in the Metaviridae from the animal kingdom preferentially used -1 frameshifting to regulate Pol protein production. In contrast, a +1 frameshift was more rarely observed but was distributed equally among kingdoms and among Pseudoviridae and Metaviridae. Finally, stop-codon suppression was found in a total of only two possible cases. Additional open reading frames in LTR retrotransposons Although retrotransposon gag and pol genes are believed to be necessary and sufficient for transposition, a number of retrotransposon families with aberrant genomic organizations have now been identified (Figure 3). One frequent structural change is the addition of coding information. Retrotransposons with 'env-like' genes One of the main differences between retrotransposons (with a wholly intracellular life-cycle) and their infectious retrovirus cousins is the presence of an envelope (env) gene in the latter, which allows a virus particle to infect another cell. A number of retroelements have an extra ORF in the same position as the env gene found in retrovirus genomes (Figure 3). The best characterized examples of env-containing retroelements are the Drosophila errantiviruses, including gypsy and ZAM [9,10]. The life-cycle of these elements has been examined in detail, and gypsy has been shown to be infectious [11,12]. The presence of an env gene within a retroelement is not limited to the errantiviruses; genomic studies have revealed that env-like ORFs are widespread among retrotransposons in both the Pseudoviridae (sireviruses) and Metaviridae (errantiviruses, metaviruses and semotiviruses) [13,14]. Elements contaning an env-like ORF in each of these lineages also originate from diverse host species. The retroelement most recently shown to have an env-like ORF, Boudicca, is a metavirus from a human blood fluke [15]. Other examples of metaviruses include the Athila elements, which represent a large proportion of the retroelements in Arabidopsis [16]. In a related element in barley, Bagy-2, the env-like transcript is spliced, similarly to the env transcripts of retroviruses [17]. Members of the sirevirus group make up half of the approximately 400 Pseudoviridae sequences present in GenBank, and of these, about one third have an env-like ORF (X.G. and D.V., unpublished observation). Semotiviruses (also called BEL retrotransposons) with env-like ORFs have also been described in nematode genomes as well as in pufferfish and Drosophila [18,19]. Do Env-like proteins enable these diverse retroelements to become infectious? In a few cases, the env-like genes have been shown to be significantly similar in sequence to genes of different viruses, suggesting that they were acquired by retrotransposons through transduction of a cellular gene [13]. Except for some errantiviruses, where the Env-like protein has been implicated in infection, the function of the Env-like proteins remains unclear. The amino-acid sequences of these proteins are highly divergent, making it difficult to assess whether or not they have a common function. That said, many Env-like proteins have predicted transmembrane domains (like retroviral Env proteins), although this is not a universal feature. It is possible that retroviral activity has evolved several times in the history of retrotransposons, or that these genes may confer novel function(s), such as movement between tissues of an organism (as suggested for the gypsy elements) or movement within cells (such as between the cytoplasm and the nucleus). Alternatively, the Env-like proteins could serve as chaperone proteins to facilitate replication. Functional studies are required to discern the biological roles of these interesting genes. Other additional ORFs Other novel coding regions have also been identified within various retrotransposons, but it is unclear how broadly these coding sequences are conserved. For example, RIRE2 of rice - a metavirus - has a small ORF of unknown function upstream of its gag gene [20]. Some plant retrotransposons carry ORF(s) that are antisense to the genomic RNA transcript (Figure 3), including the metaviruses RIRE2 of rice and Grande1 of maize [21,22]. The functions of the antisense ORFs are also unknown. In a few cases, retrotransposons have acquired sequences that probably do not have any role in the life cycle of the elements. The Bs1 retrotransposon of maize, for example, has transduced a cellular gene sequence - in this case a part of a gene encoding an ATPase [23,24]. LTR retrotransposons lacking ORFs An intriguing story is emerging about the presence of non-autonomous LTR retrotransposons in many eukaryotic genomes. Non-autonomous elements do not encode the proteins necessary for transposition; instead, they are mobilized in trans by proteins provided from functional (autonomous) elements. This mechanism is well documented for DNA transposons [25], and recent genome-mining studies have revealed many types of non-autonomous retrotransposons, suggesting that the process also occurs among retrotransposons. Typically, these elements lack all coding capacity but have retained LTRs, a primer-binding site and a polypurine tract (Figure 3). These are the minimal features required for replication, because the LTRs contain the promoter needed to produce a template RNA, and the primer-binding site and the polypurine tract are needed to prime reverse transcription. The success of some non-autonomous elements is staggering; for example, the non-autonomous Dasheng and Zeon-1 elements are each represented by around 1,000 copies in the maize genome [26,27]. For most non-autonomous retrotransposons, it is unclear which autonomous element is involved in mobilization. Striking similarities between the non-autonomous Dasheng element and the autonomous RIRE2 element, however, make it very probable that RIRE2 provides the proteins needed to move Dasheng [28]. The evidence for this, mostly provided by the emerging rice genome sequence, includes a high degree of sequence similarity within and adjacent to the LTRs (suggesting that the promoters and/or sequences necessary for reverse transcription are the same), a similar distribution of RIRE2 and Dasheng along the rice chromosomes (suggesting that they may be integrated by the same enzyme), the presence of chimeric Dasheng/RIRE2 elements (suggesting that RNAs from both elements are packaged within a single virus-like particle), and the presence of young Dasheng and RIRE2 elements (suggesting that these elements could be co-expressed). The non-autonomous Dasheng elements are large, ranging in size from 5.5 kilobases (kb) to 8.5 kb [28]. Large non-autonomous elements like Dasheng have now been named 'large retrotransposon derivatives' (LARDS) [29]. The LARDs identified in barley and other members of the Triticeae have LTRs of 4.5 kb and an internal domain of 3.5 kb. The internal domain of the LARDs contains conserved non-coding DNA that may provide important secondary structure to the mRNA, although it is not known how these non-coding sequence features function in the life cycle of the LARDs. On the basis of sequence identity, it seems that barley LARDs may be mobilized by a retrotransposon related to the metaviruses Erika-1 of the wheat Triticum monococcum and RIRE3 of rice. Finally, a second class of non-autonomous LTR retrotransposons has been identified in plants, called 'terminal-repeat retrotransposons in miniature' (TRIMs; Figure 3). They were originally identified in a potato urease gene intron and subsequently found in the Arabidopsis genome, where the founding element was named Katydid [30]. TRIMs also lack an internal coding domain but, in contrast to the LARD type of non-autonomous retrotransposon, TRIMs are very small - less than 540 bp overall. There are TRIMs in both monocotyledonous and dicotyledonous plants, but no autonomous partner has been found or proposed. The location of TRIMs within promoters and introns indicates that these elements have been important in restructuring plant genomes. Non-coding information in LTR retrotransposons Variation in retrotransposon genomic organization is not limited to the presence or absence of coding information. Some retrotransposons contain a large amount of conserved non-coding sequence. The barley LARD element with 3.5 kb of non-coding DNA (mentioned above) is one example; another is a group of plant metaviruses that carry several kilobases of non-coding DNA between pol and the 3' LTR. Among these are the maize Cinful [31] and Grande1 [22] elements, RIRE2 from rice [21] and Tat1 from Arabidopsis [32]. For Grande1 and RIRE2, antisense ORFs have been described, but they do not account for the entire segment of non-coding DNA [21,22]. In addition, many retrotransposons, including the Grande1 and Cinful elements, have a series of short tandem repeats very close to the 3' end of the pol gene, or at a putative pol-env junction. This may suggest a potential function for the tandem repeats: they may facilitate recombination and acquisition of new coding information through gene transduction [31]. In support of this hypothesis, repeated non-coding information seems to be found between the env-like ORF and the 3' LTR in both the SIRE1 [33] and Athila retrotransposons [16]. In the retrotransposons with env-like ORFs, the repeats show similarity to polypurine tracts, suggesting that they might instead have a role in reverse transcription. The sequenced eukaryotic genomes have provided a new appreciation of the diversity among LTR retrotransposons. As sequence data accumulate, additional novel elements are likely to be revealed. The challenge in the future will be to understand how diversity in retrotransposon genome organization and coding sequences reflects differences in retrotransposition mechanisms and strategies employed by these elements to colonize their host genomes.

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Takaya Moriguchi: Role: Editor

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Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 12 February 2016

Publication date Collection: 2016

Volume: 11

Issue: 2

Electronic Location Identifier: e0149192

Affiliations

[1 ]Department of Horticulture, The State Agricultural Ministry Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Hangzhou, Zhejiang 310058, China

[2 ]Forest & Fruit Tree Institute, Shanghai Academy of Agricultural Sciences, Shanghai, 201403, China

[3 ]Institute of Horticulture and Landscape, College of Ecology, Lishui University, Lishui, Zhejiang 323000, China

[4 ]Department of Biotechnology, Mirpur University of Science and Technology (MUST), Main Allama Iqbal Road, Mirpur, Azad Kashmir-10250, Pakistan

[5 ]Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province, China

NARO Institute of Fruit Tree Science, JAPAN

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: SJ YT DC. Performed the experiments: SJ PY MA. Analyzed the data: SJ XZ XY. Wrote the paper: SJ YT DC.

* E-mail: ywteng@ 123456zju.edu.cn (YT); dycai@ 123456zju.edu.cn (DC)

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Publisher ID: PONE-D-15-53195

DOI: 10.1371/journal.pone.0149192

PMC ID: 4752223

PubMed ID: 26871452

SO-VID: 56d7f94c-ccfc-4a2b-a15c-9e1626f52584

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This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 8 December 2015

Date accepted : 28 January 2016

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Figures: 4, Tables: 3, Pages: 17

Funding

This research was funded by a Grant from the National Natural Science Foundation of China (No. 31201592, http://www.nsfc.gov.cn/) awarded to DC, and a Specialized Research Fund for the Doctoral Program of Higher Education (20110101110091, http://www.cutech.edu.cn/cn/index.htm) and a Grant for Innovative Research Team of Zhejiang Province of China (2013TD05, http://www.zjnsf.gov.cn/) awarded to YT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Primitive Genepools of Asian Pears and Their Complex Hybrid Origins Inferred from Fluorescent Sequence-Specific Amplification Polymorphism (SSAP) Markers Based on LTR Retrotransposons

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Most cited references 15

The genome of the pear ( Pyrus bretschneideri Rehd.)

Nested retrotransposons in the intergenic regions of the maize genome.

The diversity of LTR retrotransposons

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