Next Article in Journal
The Human Tyrosyl-DNA Phosphodiesterase 1 (hTdp1) Inhibitor NSC120686 as an Exploratory Tool to Investigate Plant Tdp1 Genes
Previous Article in Journal
Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment
Previous Article in Special Issue
Genome Size Diversity and Its Impact on the Evolution of Land Plants
Open AccessArticle

Assembly of the Boechera retrofracta Genome and Evolutionary Analysis of Apomixis-Associated Genes

1
Dobzhansky Center for Genome Bioinformatics, St. Petersburg State Universit, Sredniy Prospekt, 41, Vasilievsky Island, 199004 St. Petersburg, Russia
2
All-Russia Research Institute for Agricultural Microbiology, Podbelskogo sh. 3, Pushkin, 196608 St. Petersburg, Russia
3
Department of Biology, Colorado State University, Fort Collins, CO 80523; USA
4
University and Jepson Herbaria, University of California, Berkeley, NC 94720; USA
5
Department of Biotechnology, Indian Institute of Technology. Sardar Patel road, 600036 Chennai, India
6
Department of Plant and Microbial Biology Zurich-Basel Plant Science Center, University of Zurich, Zollikerstrasse 107, 8008 Zurich; Switzerland
7
Department of Energy Joint Genome Institute, Walnut Creek, CA 94598; USA
8
HudsonAlpha Institute of Biotechnology, Huntsville, AL 35806; USA
9
Department of Biology, Duke University, Durham, NC 27708-0338; USA
10
Department of Plant Embryology and Reproductive Biology, Komarov Botanical Institute RAS, 197376 St. Petersburg, Russia
*
Author to whom correspondence should be addressed.
Genes 2018, 9(4), 185; https://doi.org/10.3390/genes9040185
Received: 14 February 2018 / Revised: 21 March 2018 / Accepted: 22 March 2018 / Published: 28 March 2018
(This article belongs to the Special Issue Evolution and Biodiversity of the Plant Genome Architecture)

Abstract

Closely related to the model plant Arabidopsis thaliana, the genus Boechera is known to contain both sexual and apomictic species or accessions. Boechera retrofracta is a diploid sexually reproducing species and is thought to be an ancestral parent species of apomictic species. Here we report the de novo assembly of the B. retrofracta genome using short Illumina and Roche reads from 1 paired-end and 3 mate pair libraries. The distribution of 23-mers from the paired end library has indicated a low level of heterozygosity and the presence of detectable duplications and triplications. The genome size was estimated to be equal 227 Mb. N50 of the assembled scaffolds was 2.3 Mb. Using a hybrid approach that combines homology-based and de novo methods 27,048 protein-coding genes were predicted. Also repeats, transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were annotated. Finally, genes of B. retrofracta and 6 other Brassicaceae species were used for phylogenetic tree reconstruction. In addition, we explored the histidine exonuclease APOLLO locus, related to apomixis in Boechera, and proposed model of its evolution through the series of duplications. An assembled genome of B. retrofracta will help in the challenging assembly of the highly heterozygous genomes of hybrid apomictic species.
Keywords: Boechera; Brassicaceae; genome; assembly; annotation; apomixis Boechera; Brassicaceae; genome; assembly; annotation; apomixis

1. Introduction

Among over the 370 genera belonging to the family Brassicaceae (Cruciferae), only the genus Boechera shows asexual reproduction by seeds [1,2,3,4]. Apomixis is defined as asexual reproduction through seeds that results in progeny identical to the maternal plant. The harnessing of apomixis is widely considered as a key enabling technology for crop improvement because it allows the fixation of any heterozygous genotype, leading to simpler and faster breeding schemes [5,6,7]. The Boechera genus includes 110 sexual and apomictic species, widely distributed in North America. Plants from the Boechera genus are represented by biannual and perennial herbs with a chromosome base number of n = 7 [8,9].
Apomixis in the Boechera genus is of special interest because it can occur at the diploid level, which is very rare [1,2,3,4,5,6,7,8]. Furthermore, the phylogenetic proximity of Boechera to the model plant Arabidopsis thaliana is attractive for potential functional studies. Although the genus Boechera includes both sexual and apomictic species and accessions that are of variable ploidy and geographical origin, search for homologous sequences are feasible across the genus [10]. The sexual accessions of Boechera are self-compatible and largely self-pollinating [11], unlike the sexual ancestors of most other apomicts, which are typically self-incompatible and cross-pollinating [12]. This inbreeding causes low heterozygosity in sexual Boechera species. Apomictic Boechera accessions have likely arisen through independent hybridization events [13]. Their hybridogenic origin is supported by the aberrant structure of their chromosomes, as they are often observed as a consequence of hybridization, leading to alloploidy, aneuploidy, the replacement of homeologous chromosomes, and aberrant chromosomes [13,14].
Certain apomictic Boechera accessions are hypothesized to have arisen through hybridization between sexual Boechera stricta and Boechera retrofracta (Figure 1). Boechera retrofracta was previously included within Boechera holboellii (sensu lato) [15]. Up to now only the genome sequence of B. stricta was available [16], while the genome of B. retrofracta has not been assembled yet.
In this paper, we present the assembly and annotation of the B. retrofracta genome. The availability of the B. retrofracta genome sequence together with the previously assembled B. stricta genome will greatly help in the assembly and annotation of related apomictic hybrid species and provide the basis to investigate the peculiarities of hybridization events, chromosomal organization, the stability of apomictic genomes, and the genetic factors underlying apomixis. The performed assembly and annotation allowed us to analyze of the APOLLO (APOmixis-Linked LOcus) genes, that are associated with apomixis in Boechera.

2. Materials and Methods

2.1. Sample Information

The reference B. retrofracta genotype was collected in Panther Creek, Lemhi County, Idaho, at 45°18′11.9″ N 114°22′35.9″ W, 1610 m elevation (Figure 1). Plant growth, DNA extraction, and library construction were the same as with B. stricta [16]. Briefly, seedlings were germinated in aseptic culture in half-strength Murashige and Skoog (MS) liquid medium. Cell nuclei were used for isolation of clean high-molecular-size nuclear DNA in Tris-EDTA (TE) buffer.

2.2. Sequencing Strategy

The B. retrofracta genome was sequenced within the JGI Community Sequencing Project to produce sequence data for the Boechera genus [17]. Six libraries were constructed and sequenced using three platforms including Illumina, Roche, and Sanger: one paired-end (PE) library, four mate pairs (MP) libraries and one Sanger bacterial artificial chromosome (BAC) end library. Read length and actual insert sizes for each library are given in Appendix A (Table A1). This sequencing scheme was specially developed for the initial contig assembly by the DISCOVAR assembler [18], followed by scaffolding. Construction of genomic libraries and sequencing were performed following Lee et al [16].

2.3. Raw Data Filtration and Pre-Processing

Filtration of the PE library LIB400 was performed in two stages. First, reads containing long adapter fragments were removed using Cookiecutter [19]. Then Trimmomatic [20] was used to filter out reads with short adapter fragments. However, according to the DISCOVAR requirements no trimming or quality filtration was performed at those two stages. Only whole reads contaminated by adapters were discarded.
To process Illumina MP libraries LIB5000 and LIB7000 the NextClip [21] tool was modified to handle Cre-Lox libraries. It is important to note that original NextClip uses a very simple algorithm to align linker sequences to reads. It takes into account only the number of matching bases. As the CreLox linker is significantly longer than the Nextera linker, the number of false hits may significantly increase. To mitigate this effect, a requirement for the presence of a continuous 9-bp core alignment was added. The modified tool was named CreClip and can be found in [22].
Reads from Roche MP Libraries LIB4000R and LIB24000R were split into “forward” and “reverse” segments separated by linker. Then, low quality ends were trimmed from “reverse” segments by Trimmomatic. Finally, reverse segments were reverse complemented to mimic to Illumina MP libraries.

2.4. Genome Size Estimation

Estimation of the genome size based on the 23-mer distribution (as well as other k-mer based statistics) was performed using the KrATER software [23] on the LIB400 library and further compared with the previous estimations of Boechera genus [24].

2.5. Genome Assembly and Quality Evaluation

At the assembly stage initial contigs were constructed from the filtered LIB400 reads by DISCOVAR. Then, the obtained contigs were extended using a BAC end sequencing (BES) library and the SSPACE scaffolder [25].
Before scaffolding the assessment of the actual (mean) insert size was performed. Filtered reads from all libraries were aligned to initial contigs by Burrows–Wheeler Aligner (BWA) [26]. For each library, only alignments to contigs with 3× length of the target insert size were used in the estimation (Table 1) to minimize alignment artifacts. Next, the extended contigs were scaffolded by SSPACE in two stages: at the first stage, all four MP libraries (LIB4000R, LIB5000, LIB7000, LIB24000R) were used to produce raw scaffolds, at the second stage, raw scaffolds were linked to the intermediate scaffolds using the BES library only. Scaffolding was carried out in several stages because different options were required to utilize the BES data. Gap closing in the intermediate scaffolds was performed using GapCloser (a module for SOAPdenovo2) [27] on the LIB400 library only. Finally, all scaffolds with a length of less than 250 bp (i.e., less than read length of LIB400, the library used for initial contig construction) were filtered out, as the corresponding short fragments most likely are the assembly artifacts. Integrity of the assembly was verified by Core Eukaryotic Genes Mapping Approach (CEGMA) [28] and Benchmarking Universal Single-Copy Orthologs (BUSCO) [29]. A schematic diagram of the assembly pipeline is shown in Figure A1 in Appendix A.

2.6. Repeats Analysis

A de novo repeat identification in the B. retrofracta genome was performed using RepeatModeler [30] with default parameters. The obtained repeat library was combined with Arabidopsis thaliana repeats from RepBase [31], and the merged library was used to annotate repeats by RepeatMasker [32]. Then repeats in the B. retrofracta genome were softmasked by Bedtools [33] for the prediction of protein coding genes. Also, masking of tandem and interspersed repeats by tandem repeats finder (TRF) [34] and WindowMasker [35], respectively, were performed.

2.7. Variants Calling and Genotyping

For variant calling and genotyping filtered reads were aligned to the assembled genome using BWA mem with default options. Next, the Genome Analysis Toolkit (GATK) pipeline [36] for variant calling was applied in the following way: duplicates were marked using Picard MarkDuplicates (Broad Institute, Cambridge, MA, USA), realigned reads at indels, and, finally, HaplotypeCaller (Broad Institute) was used to call variants. Only single nucleotide polymorphisms (SNPs) and indels were kept passing the following filtering criteria: QualByDepth (QD) > 2.0, FisherStrand (FS) < 20.0, RMSMappingQuality (MQ) > 40.0, MappingQualityRankSumTest (MQRankSum) > −12.5, ReadPosRankSumTest (ReadPosRankSum) > −8.0 for SNPs, and QualByDepth (QD) > 2.0, FisherStrand (FS) < 20.0, ReadPosRankSumTest (ReadPosRankSum) > −20.0 for indels, respectively. Finally, the variants falling into the repeats masked by RepeatMasker were excluded.

2.8. Prediction of Protein-Coding Genes and Non-Coding RNA

The prediction of protein-coding genes was performed using a combined approach that synthesizes both homology-based and de novo predictions, where de novo predictions are used only to fill gaps and to extend the homology-based predictions. Pure de novo predictions were filtered out.
As homology-based evidence for gene presence, we have used proteins and transcripts of five closely-related species. Proteins of the four reference species—Arabidopsis thaliana (assembly TAIR10), Brassica rapa (Brapa_1.0), Capsella rubella (Caprub1_0), and Eutrema salsugineum (Eutsalg1_0)—were aligned to the B. retrofracta assembly by Exonerate [37], using the Protein2Genome model with a maximum of three hits per protein. The obtained alignments were classified into the top (primary) and secondary hits; the coding sequence (CDS) fragments were cut from each side by 3 bp for the top hits and by 9 bp for the secondary hits. Transcripts of B. stricta (assembly v1.2, [16]) with marked CDS regions were also aligned to the B. retrofracta genome by Exonerate using the cDNA2Genome model leaving the other options unchanged. Alignments of CDS segments were not cut for top hits, but cut by 3 bp for secondary hits.
These truncated fragments were clustered and supplied as hints to the AUGUSTUS software package [38], and the CDS segments of genes were predicted in the soft-masked B. retrofracta assembly using A. thaliana gene models. Proteins were translated from the predicted genes and aligned by HMMER 3.1 [39] and BLAST [40] to the Pfam [41] and Swiss-Prot [42] databases, respectively. Only genes supported by the both hints and hits to one of the protein databases were retained; the rest were discarded. Transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were predicted by tRNAscan-SE v1.3.1 [43] and Barrnap v0.6 [44], respectively.

2.9. Phylogenetic Analysis

The longest proteins corresponding to each predicted gene of B. retrofracta and six other Brassicaceae species—B. stricta (assembly v1.2), A. thaliana (TAIR10), Arabidopsis lyrata (v.1.0), Capsella rubella (Caprub1_0), Cardamine hirsuta (v1.0), and Eutrema salsugineum (Eutsalg1_0)—were aligned to profile Hidden Markov Models (HMM) of the braNOG subset from the eggNOG database [45] using HMMER. The top hits from the alignments were extracted and used for assignment of the corresponding proteins to orthologous groups, followed by extraction of single-copy orthologs.
To verify topology concordance and get a basis for future studies of positive selection, a species tree reconstruction was performed. Single-copy orthologous proteins of the seven species included in the analysis were aligned by multiple alignment using fast Fourier transform (MAFFT) [46]. Based on the obtained protein alignments, a maximum likelihood tree was reconstructed by RAxML v8.2 [47] with the PROTGAMMAAUTO option, and the JTT fitting model was tested with 1000 bootstrap replications. The tree was rooted with E. salsugineum as an outgroup. The resulting tree was drawn with FigTree software [48].

2.10. APOLLO Evolution Analysis

The evolutionary history of APOLLO gene was inferred by using the Maximum Likelihood method. Initial alignment of corresponding CDS was performed using prank v.140110 [49] in codon-aware mode. The alignment result was further used for building phylogenetic tree basing on the Tamura-Nei model [50,51]. The tree with the highest log likelihood (−12,153.79) was selected (see Section 3.7, Figure 4). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated. There were a total of 1158 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [52].

2.11. Whole-Genome Comparison

As a preliminary step for the future whole-genome comparison of different Boechera species a whole genome alignment was performed via Cactus multiple genome aligner [53] and further visualized with web-tool ClicO FS [54] based on Circos [55].
For further information about the initial data and results, see Appendix B.

3. Results

3.1. k-mer Based Statistics

k-mer spectrum built by KrATER [23] is shown in Figure 2. The 23-mer distribution has a peak of erroneous 23-mers at 1× coverage corresponding to sequencing errors and one major peak at 371× coverage corresponding to diploid 23-mers (shared between homologous chromosomes), but no significant peak related to heterozygous genome positions was detected (Figure 2). However, we detected several small additional peaks at double (737×) and triple (1120×) depth, which are probably related to duplications and triplications, respectively.
The genome size of B. retrofracta was estimated to be close to 227 Mbp, which is close to the previous estimations of a minimal genome size of 200 Mbp in the Boechera genus [24].

3.2. Genome Assembly and Evaluation

We have achieved N50 of 2,297,899 bp, L50 of 25, and a total assembly length of 222.25 Mbp for the final scaffolds, which is very close to our 23-mer based estimation. Detailed statistics including N50 and total assembly values for every stage of the assembly pipeline are listed in Table 1 and Table 2. It is important to note that the final assembly (Table 1, column final scaffolds) has smaller size than previous intermediate assemblies due to the last filtration step. All scaffolds shorter than 250 bp (a read length of LIB400) were treated as artifacts of assembly and were removed. However, size of final assembly (222.25 Mbp) is closer to estimated genome size (226.87 Mbp) than the size of intermediate assemblies.
Evaluation of the assembly completeness was performed using CEGMA [28] and BUSCO [29]. In the assembled genome 242 (97.58%) complete core eukaryotic genes (CEGs) were identified. Out of 1440 BUSCO genes from the Embryophyta, set only 12 (0.8%) genes were not found, 6 were fragmented, 36 (2.5%) were duplicated and 1422 (98.8%) were complete. This high fraction of complete BUSCO genes suggests high completeness of the assembly and its integrity at least in gene-coding regions.

3.3. Repeats Annotation

In total approximately 85 Mbp (38.13%) of the assembly were masked. The detailed description of the annotated repeat types is listed in Table 3. It is important to note that a large number (10.96% of the assembly size) of interspersed repeats was not classified. The results are shown in Table 4.

3.4. Variant Calling and Genotyping

In the genome 3341 SNPs and 1317 indels were detected. Among these, 103 (3.08%) SNPs and 97 (7.37%) indels were homozygous and, therefore, most likely artifacts of alignment or assembly or SNP calling. Mean heterozygous SNP and indel densities in non-masked regions (138 Mbp in total) are 0.0235 SNP and 0.0089 indel per Kbp, respectively, suggesting a very low heterozygosity of the B. retrofracta genome.

3.5. Prediction of Protein-Coding Genes and Non-Coding RNAs

In total 27,048 genes with 28,269 transcripts were predicted. tRNA and rRNA genes predicted by tRNAscan-SE and Barrnap are given in Table 5 and Table 6 respectively.

3.6. Species Tree Reconstruction

In course of the assignment of proteins to orthologous groups 8959 single-copy orthologs were identified among the seven species (B. retrofracta, B. stricta, A. thaliana, A. lyrata, C. rubella, C. hirsuta, and E. salsugineum).
The corresponding phylogenetic tree was rooted with E. salsugineum as an outgroup (Figure 3). All nodes have a high support and no topology discordance was found with the tree reconstructed previously by Huang et al [52].

3.7. Analysis of Evolution of the APOLLO Locus

Results from Corral et al. [56] suggest that APOLLO (aspartate glutamate aspartate aspartate histidine exonuclease) is one of the important apomixis-related genes in Boechera. It was shown that the APOLLO locus has several alleles with apomixis-associated polymorphisms. All studied apomictic plants carry at least one of the “apoalleles”, while both copies in sexual genotypes were “sexalleles”.
In this study we decided to take a closer look to this locus in our assembly and other Brassicaceae species in this study. Along with an exact copy of the APOLLO locus, we also found two other, more distant copies, which may indicate past duplication events. We searched for these orthologs in other species, and reconstructed phylogenetic tree (Figure 4). All Brassicaceae genomes in the study also carried these three copies, related to the clusters of orthologous genes ENOG410BURN (APOLLO locus), ENOG410BUTR, and ENOG410C333 in the EggNOG database.
We observed that branches in the tree were grouped by genes rather than by species, suggesting that the triplication event took place before the separation of the Brassicaceae species in this study. It is worth noting that in Populus trichocarpa genome there is only one copy of these locus, which gives an upper-bound time estimate of the series of duplication events.
We also examined APOLLO alleles (both apo- and sex-alleles) described in Boechera ssp. by Corral et al. [56]. We can see that these alleles arise after the separation of the Boechera genus, and compose two separate clades. Given the fact that B.retrofracta and B.stricta are the sexual species, it was not surprising that in both cases all corresponding polymorphic sites were in the “sexallele”-state, and clustered with sex-alleles.
We calculated the Ka/Ks ratio for the internal branches in this tree and found that branch leading to apo-alleles is under positive selection (Ka/Ks 1.4646, the branch is shown in red in Figure 4), which is typical for paralogues that are required to serve a novel function.
The APOLLO gene was initially described in A. thaliana as an exonuclease, protein NEN3, Q9CA74 in Uniprot database [42], probably involved in enucleation of sieve elements, whereas two other copies were described as NEN1 (Q9FLR0) and NEN2 (Q0V842). Given that, we may suggest an evolutionary scenario where, after the series of duplications, one of the NEN protein copies in the common ancestor of Boechera spp. might have acquired alter regulation, and might induce development of the apomictic reproduction from the ancestral “sexual” state, following by separation of the apomictic lineages.
That could explain the phenomena of the diploid apomictic Boechera, emerged as a result of duplication events rather than polyploidy.

3.8. Whole-Genome Comparison

As an example of whole-genome comparison a Circos plot was built for B. retrofracta and B. stricta (Appendix C). Since both assemblies are performed on a scaffold level, it is difficult to highlight any large genome rearrangements. However, this plot is a visual way to represent the scatteredness of both assemblies.

4. Discussion

In this study we present a de novo assembly and annotation of the genome of Boechera retrofracta, a perennial flowering plant belonging to Brassicaceae family that is native to North America. The genome of B. retrofracta demonstrated a very low level of heterozygosity compare to the genomes of apomictic accessions [2,8,9,10,11,12,13,14,15,16]. Notably, repeats in the genome of B. retrofracta occupied almost 40% of the genome space. Nearly half of them were long terminal repeats (LTRs) (18.27%). The genome size was found to be 227 Mb, nearly two-fold larger than the Arabidopsis thaliana genome (Table 7). At the same time the amount of protein-coding genes in the genome of B. retrofracta is slightly less then in the B. stricta and A. thaliana genomes and much less than that in the A. lyrata genome (Table 1). Despite the largest genome size, the number of predicted transcripts in B. retrofracta is the smallest among the four Brassicaceae species compared (Table 1). The presence of a slightly greater number of genes in B. stricta compared with B. retrofracta, despite a smaller genome size, may be associated with aneuploidy of the chromosomal fragments, or genome rearrangements occurred as a result of interhybridization, which is characteristic of many Boechera species and accessions.
As an example of how the genome of the sexual species B. retrofracta could be used to study evolution and origin of apomixis, we performed an evolutionary analysis of the three alleles of the APOLLO (APOmixis-Linked LOcus) gene (apo- and sex-alleles) described by Corral et al [56]. We examined this gene in more detail in our assembly and in other Brassicaseae species. Along with the described copy of APOLLO, we also found two other, more distant copies, which evidently arose by two sequential gene duplications (triplication). The APOLLO phylogenetic tree may indicate that triplication event occurred before the separation of Brassicaceae species under study (Figure 4). We also analyzed the APOLLO alleles described in Boechera ssp. It was clear that these alleles arose after separation of the Boechera genus. In sexual B. retrofracta and B. stricta polymorphic sites corresponded to the “sexallele”-state and clustered with sex-alleles of the other species.
These results are compatible with an evolutionary scenario where, after the series of duplications, one of the NEN exonuclease protein (ancestor of APOLLO) copies in the common ancestor of Boechera spp. experiencing relaxed selection might be deregulated, promoting development of the apomictic reproduction from the ancestral “sexual” state, following by separation of the apomictic lineages. This model of evolution of APOLLO alleles might explain the phenomenon of apomictic development in Boechera in the diploid condition, emerged as a result of duplication events rather than polyploidy.
In conclusion, increasing number of sequenced genomes from the economically important Brassicaceae family will facilitate future genetic, genomic, evolutionary, and domestication studies in this family. B. retrofracta is thought to be an ancestor of certain hybrids including apomictic species, for example Boechera divaricarpa. Consequently, the genome assembly presented in this report may help with the challenging genome assembly of highly heterozygous hybrid Boechera species that are apomictic. Thus, the B. retrofracta genome reported here will provide a basis to decipher the hybridogenesis events that led to the formation of apomictic Boechera accessions.

Acknowledgments

This work was supported by grants 16-54-21014 and 15-54-45001 from the Russian Foundation for Basic Research and by grant 1.52.1647.2016 from St. Petersburg State University (to V.B.), grant IZLRZ3_163885 from the Swiss National Science Foundation (to U.G.) through the Scientific & Technological Cooperation Programme Switzerland-Russia (STCPSR). US National Science Foundation Doctoral Dissertation Improvement Grant 1311269 (to C.R.) and grant R01 GM086496 from the National Institutes of Health (USA) (to T.M.O.). Work conducted by the US Department of Energy Joint Genome Institute was supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. The research was carried out within the framework of the state assignment No. AAAA-A18-118030790063-6 to BIN RAS.

Author Contributions

T.M.O., V.B., D.S.R. and U.G. designed the study. T.M.O., C.R., J.S., D.S. and K.P. gathered samples, extracted and sequenced genomic DNA. S.K., A.K., D.Z., E.B. and M.R. performed genome assembly, annotation and phylogenetic analysis. S.K., V.B., M.R., E.B. and R.B. wrote manuscript with revision by all other authors. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Table A1. Sequencing scheme of Boechera retrofracta genome.
Table A1. Sequencing scheme of Boechera retrofracta genome.
IDLibrary TypePlatformRead LengthMean Insert Size (bp)Number of Reads Pairs
LIB400paired endsIllumina250402189788627
LIB4000Rmate pairsRoche-40143259085
LIB5000mate pairsIllumina150487719083787
LIB7000mate pairsIllumina150688234066282
LIB24000Rmate pairsRoche-24,332672098
BESBAC end sequencingSanger-147,70817775
Abbreviations: BAC, bacterial artificial chromosome; BES, BAC end sequencing.
Figure A1. Pipeline used to assembly genome of Boechera retrofracta.
Figure A1. Pipeline used to assembly genome of Boechera retrofracta.
Genes 09 00185 g0a1

Appendix B

The original data could be found at: http://public.dobzhanskycenter.ru/ad89dedc8b4674276c9b0760f29b07af/ or at NCBI, BioProject ID: PRJNA418376.

Appendix C

Figure C1. Comparison of Boechera stricta and Boechera retrofracta genomes on a scaffold level.
Figure C1. Comparison of Boechera stricta and Boechera retrofracta genomes on a scaffold level.
Genes 09 00185 g0c1

References

  1. Sharbel, T.F.; Mitchell-Olds, T. Recurrent polyploid origins and chloroplast phylogeography in the Arabis holboellii complex (Brassicaceae). Heredity 2001, 87, 59–68. [Google Scholar] [CrossRef] [PubMed]
  2. Schranz, M.E.; Dobes, C.; Koch, M.A.; Mitchell-Olds, T. Sexual reproduction, hybridization, apomixis and polyploidization in the genus Boechera (Brassicaceae). Am. J. Bot. 2005, 92, 1797–1810. [Google Scholar] [CrossRef] [PubMed]
  3. Naumova, T.N. Apomixis and amphimixis in flowering plants. Cytol. Genet. 2008, 3, 51–63. [Google Scholar] [CrossRef]
  4. Aliyu, O.M.; Schranz, M.E.; Sharbel, T.F. Quantitative variation for apomictic reproduction in the genus Boechera (Brassicaceae). Am. J. Bot. 2010, 97, 1719–1731. [Google Scholar] [CrossRef] [PubMed]
  5. Koltunow, A.M.; Grossniklaus, U. Apomixis: A developmental perspective. Annu. Rev. Plant Biol. 2003, 54, 547–574. [Google Scholar] [CrossRef] [PubMed]
  6. Rodríguez-Leal, D.; Vielle-Calzada, J.P. Regulation of apomixis: Learning from sexual experience. Curr. Opin. Plant Biol. 2012, 15, 549–555. [Google Scholar] [CrossRef] [PubMed]
  7. Brukhin, V. Molecular and genetic regulation of apomixis. Russ. J. Genet. 2017, 53, 943–964. [Google Scholar] [CrossRef]
  8. Windham, M.D.; Al-Shehbaz, I.A. New and noteworthy species of Boechera (Brassicaceae) II: Apomictic hybrids. Harv. Pap. Bot. 2007, 11, 257–274. [Google Scholar] [CrossRef]
  9. Windham, M.D.; Al-Shehbaz, I.A. New and noteworthy species of Boechera (Brassicaceae) III: Additional sexual diploids and apomictic hybrids. Harv. Pap. Bot. 2007, 12, 235–257. [Google Scholar] [CrossRef]
  10. Lovell, J.T.; Williamson, R.J.; Wright, S.I.; McKay, J.K.; Sharbel, T.F. Mutation Accumulation in an Asexual Relative of Arabidopsis. PLoS Genet. 2017, 13, e1006550. [Google Scholar] [CrossRef] [PubMed]
  11. Rushworth, C.A.; Song, B.H.; Lee, C.R.; Mitchell-Olds, T. Boechera, a model system for ecological genomics. Mol. Ecol. 2011, 20, 4843–4857. [Google Scholar] [CrossRef] [PubMed]
  12. Beck, J.B.; Alexander, P.J.; Allphin, L.; Al-Shehbaz, I.A.; Rushworth, C.; Bailey, C.D.; Windham, M.D. Does hybridization drive the transition to asexuality in diploid Boechera? Evolution 2011, 66, 985–995. [Google Scholar] [CrossRef] [PubMed]
  13. Kantama, L.; Sharbel, T.F.; Schranz, M.E.; Mitchell-Olds, T.; de Vries, S.; de Jong, H. Diploid apomicts of the Boechera holboellii complex display large-scale chromosome substitutions and aberrant chromosomes. Proc. Natl. Acad. Sci. USA 2007, 104, 14026–14031. [Google Scholar] [CrossRef] [PubMed]
  14. Mandáková, T.; Schranz, M.E.; Sharbel, T.F.; de Jong, H.; Lysak, M.A. Karyotype evolution in apomictic Boechera and the origin of the aberrant chromosomes. Plant J. 2015, 82, 785–793. [Google Scholar] [CrossRef] [PubMed]
  15. Windham, M.D.; Al-Shehbaz, I.A. New and noteworthy species of Boechera I: Sexual diploids. Harv. Pap. Bot. 2006, 11, 61–88. [Google Scholar] [CrossRef]
  16. Lee, C.R.; Wang, B.; Mojica, J.P.; Mandáková, T.; Prasad, K.V.S.K.; Goicoechea, J.L.; Perera, N.; Hellsten, U.; Hundley, H.N.; Johnson, J.; Grimwood, J.; et al. Young inversion with multiple linked QTLs under selection in a hybrid zone. Nat. Ecol. Evol. 2017, 1, 0119. [Google Scholar] [CrossRef] [PubMed]
  17. Why Sequence Boechera holboellii? Available online: http://jgi.doe.gov/why-sequence-boechera-holboellii/ (accessed on 1 November 2017).
  18. Weisenfeld, N.I.; Yin, S.; Sharpe, T.; Lau, B.; Hegarty, R.; Holmes, L.; Sogoloff, B.; Tabbaa, D.; Williams, L.; Russ, C.; Nusbaum, C.; et al. Comprehensive variation discovery in single human genomes. Nat. Genet. 2014, 46, 1350–1355. [Google Scholar] [CrossRef] [PubMed]
  19. Starostina, E.; Tamazian, G.; Dobrynin, P.; O’Brien, S.; Komissarov, A. Cookiecutter: A tool for kmer-based read filtering and extraction. bioRxiv 2015. [Google Scholar] [CrossRef]
  20. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [PubMed]
  21. Leggett, R.M.; Clavijo, B.J.; Clissold, L.; Clark, M.D.; Caccamo, M. NextClip: An analysis and read preparation tool for Nextera Long Mate Pair libraries. Bioinformatics 2013, 30, 566–568. [Google Scholar] [CrossRef] [PubMed]
  22. Kliver, S. CreClip. Available online: https://github.com/mahajrod/CreClip (accessed on 1 July 2016).
  23. Kliver, S.; Tamazian, G.; O’Brien, S.J.; Brukhin, V.; Komissarov, A. KrATER (K-mer Analysis Tool Easy to Run). Available online: https://github.com/mahajrod/KrATER (accessed on 1 November 2017).
  24. Anderson, J.T.; Willis, J.H.; Mitchell-Olds, T. Evolutionary genetics of plant adaptation. Trends Genet. 2011, 27, 258–266. [Google Scholar] [CrossRef] [PubMed]
  25. Boetzer, M.; Henkel, C.V.; Jansen, H.J.; Butler, D.; Pirovano, W. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 2010, 27, 578–579. [Google Scholar] [CrossRef] [PubMed]
  26. Li, H.; Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef] [PubMed]
  27. Luo, R.; Liu, B.; Xie, Y.; Li, Z.; Huang, W.; Yuan, J.; Tang, J. SOAPdenovo2: An empirically improved memory-efficient short-read de novo assembler. GigaScience 2012, 1, 18. [Google Scholar] [CrossRef] [PubMed]
  28. Parra, G.; Bradnam, K.; Korf, I. CEGMA: A pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 2007, 23, 1061–1067. [Google Scholar] [CrossRef] [PubMed]
  29. Simão, F.A.; Waterhouse, R.M.; Ioannidis, P.; Kriventseva, E.V.; Zdobnov, E.M. BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 2015, 31, 3210–3212. [Google Scholar] [CrossRef] [PubMed]
  30. Smit, A.F.A.; Hubley, R. RepeatModeler Open, version 1.0; Institute for Systems Biology: Seattle, WA, USA, 2008. [Google Scholar]
  31. Bao, W.; Kojima, K.K.; Kohany, O. Repbase Update a database of repetitive elements in eukaryotic genomes. Mob. DNA 2015, 6, 11. [Google Scholar] [CrossRef] [PubMed]
  32. Smit, A.F.A.; Hubley, R.; Green, P. RepeatMasker Open, version 4.0; Institute for Systems Biology: Seattle, WA, USA, 2013. [Google Scholar]
  33. Quinlan, A.R.; Hall, I.M. BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics 2010, 26, 841–842. [Google Scholar] [CrossRef] [PubMed]
  34. Benson, G. Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res. 1999, 27, 573–580. [Google Scholar] [CrossRef] [PubMed]
  35. Morgulis, A.; Gertz, E.M.; Schäffer, A.A.; Agarwala, R. WindowMasker: Window-based masker for sequenced genomes. Bioinformatics 2005, 22, 134–141. [Google Scholar] [CrossRef] [PubMed]
  36. Van der Auwera, G.A.; Carneiro, M.O.; Hartl, C.; Poplin, R.; del Angel, G.; Levy-Moonshine, A.; Jordan, T.; Shakir, K.; Roazen, D.; Thibault, J.; et al. From FastQ data to high-confidence variant calls: The genome analysis toolkit best practices pipeline. Curr. Protoc. Bioinform. 2013, 43. [Google Scholar] [CrossRef]
  37. Slater, G.; Birney, E. Automated generation of heuristics for biological sequence comparison. BMC Bioinform. 2005, 6. [Google Scholar] [CrossRef] [PubMed][Green Version]
  38. Stanke, M.; Keller, O.; Gunduz, I.; Hayes, A.; Waack, S.; Morgenstern, B. AUGUSTUS: Ab initio prediction of alternative transcripts. Nucleic Acids Res. 2006, 34. [Google Scholar] [CrossRef] [PubMed]
  39. Johnson, L.S.; Eddy, S.R.; Portugaly, E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinform. 2010, 11, 431. [Google Scholar] [CrossRef] [PubMed]
  40. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
  41. Bateman, A.; Coin, L.; Durbin, R.; Finn, R.D.; Hollich, V.; Griffiths-Jones, S.; Khanna, A.; Marshall, M.; Moxon, S.; Sonnhammer, E.L.; et al. The Pfam protein families database. Nucleic Acids Res. 2004, 32 (Suppl. 1), D138–D141. [Google Scholar] [CrossRef] [PubMed]
  42. UniProt Consortium. UniProt: The universal protein knowledgebase. Nucleic Acids Res. 2017, 45, D158–D169. [Google Scholar] [CrossRef]
  43. Lowe, T.M.; Eddy, S.R. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997, 25, 955–964. [Google Scholar] [CrossRef] [PubMed]
  44. Victorian Bioinformatics Consortium. Barrnap. Available online: http://www.vicbioinformatics.com/software.barrnap.shtml (accessed on 1 November 2017).
  45. Huerta-Cepas, J.; Szklarczyk, D.; Forslund, K.; Cook, H.; Heller, D.; Walter, M.C.; Rattei, T.; Mende, D.R.; Sunagawa, S.; Kuhn, M.; et al. eggNOG 4.5: A hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. 2015, 44, D286–D293. [Google Scholar] [CrossRef] [PubMed]
  46. Katoh, K.; Standley, D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013, 30, 772–780. [Google Scholar] [CrossRef] [PubMed]
  47. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef] [PubMed]
  48. FigTree. Available online: http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 1 November 2017).
  49. Löytynoja, A. Phylogeny-aware alignment with PRANK. Methods Mol. Biol. 2014, 1079, 155–170. [Google Scholar] [CrossRef] [PubMed]
  50. Talavera, G.; Castresana, J. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 2007, 56, 564–577. [Google Scholar] [CrossRef] [PubMed]
  51. Tamura, K.; Nei, M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 1993, 10, 512–526. [Google Scholar] [CrossRef] [PubMed]
  52. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed]
  53. Paten, B.; Earl, D.; Nguyen, N.; Diekhans, M.; Zerbino, D.; Haussler, D. Cactus: Algorithms for genome multiple sequence alignment. Genome Res. 2011, 21, 1512–1528. [Google Scholar] [CrossRef] [PubMed]
  54. Cheong, W.H.; Tan, Y.C.; Yap, S.J.; Ng, K.P. ClicO FS: An interactive web-based service of Circos. Bioinformatics 2015, 31, 3685–3687. [Google Scholar] [CrossRef] [PubMed]
  55. Krzywinski, M.; Schein, J.E.; Birol, I.; Connors, J.; Gascoyne, R.; Horsman, D.; Jones, S.J.; Marra, M.A. Circos: An Information Aesthetic for Comparative Genomics. Genome Res. 2009, 19, 1639–1645. [Google Scholar] [CrossRef] [PubMed]
  56. Corral, J.M.; Vogel, H.; Aliyu, O.M.; Hensel, G.; Thiel, T.; Kumlehn, J.; Sharbel, T.F. A conserved apomixis-specific polymorphism is correlated with exclusive exonuclease expression in premeiotic ovules of apomictic Boechera species. Plant Physiol. 2013, 163, 1660–1672. [Google Scholar] [CrossRef] [PubMed]
  57. Phytozome v12.1 Database. Available online: https://phytozome.jgi.doe.gov/pz/portal.html (accessed on 30 January 2018).
Figure 1. Plant (a) and flower (b) of Boechera retrofracta.
Figure 1. Plant (a) and flower (b) of Boechera retrofracta.
Genes 09 00185 g001
Figure 2. Distribution of 23-mers for PE LIB400 library. Only one major peak at 371× coverage is present, however there are detectable duplications and triplications at 737× and 1120× coverage (upper plot, Y axis is on a logarithmic scale).
Figure 2. Distribution of 23-mers for PE LIB400 library. Only one major peak at 371× coverage is present, however there are detectable duplications and triplications at 737× and 1120× coverage (upper plot, Y axis is on a logarithmic scale).
Genes 09 00185 g002
Figure 3. Phylogenetic tree of seven Brassicaceae species used for analysis. Maximum likelihood tree was reconstructed by RAxML using 8959 single copy orthologs and was tested with 1000 bootstrap replicates. Numbers near nodes represent corresponding bootstrap support.
Figure 3. Phylogenetic tree of seven Brassicaceae species used for analysis. Maximum likelihood tree was reconstructed by RAxML using 8959 single copy orthologs and was tested with 1000 bootstrap replicates. Numbers near nodes represent corresponding bootstrap support.
Genes 09 00185 g003
Figure 4. Phylogenetic tree of the isoforms of APOLLO locus (exonuclease NEN) in seven species of interest and alleles of APOLLO locus of apomictic Boechera species from Corral et al (2013) [56]. Sequences of Populus trichocarpa, Vitus vinifera and Glycine max were used as outgroup. The clade related to the APOLLO locus is shown in green, with apo-alleles shown in red. Numbers near nodes represent corresponding bootstrap support.
Figure 4. Phylogenetic tree of the isoforms of APOLLO locus (exonuclease NEN) in seven species of interest and alleles of APOLLO locus of apomictic Boechera species from Corral et al (2013) [56]. Sequences of Populus trichocarpa, Vitus vinifera and Glycine max were used as outgroup. The clade related to the APOLLO locus is shown in green, with apo-alleles shown in red. Numbers near nodes represent corresponding bootstrap support.
Genes 09 00185 g004
Table 1. General statistics for all stages of the assembly pipeline.
Table 1. General statistics for all stages of the assembly pipeline.
ParameterContigsExtended ContigsRaw ScaffoldsIntermediate ScaffolfsGap Closed ScaffoldsFinal Scaffolds
Longest contig791,985792,3408,101,2569,045,7069,049,0809,049,080
Ns28,10028,10011,890,51916,366,99412,409,18912,409,189
Total length225,649 216226,402,628236,469,041240,945,496241,014,839222,253,471
Table 2. N50 values for all stages of the assembly pipeline and several different cutoffs for minimal scaffold length.
Table 2. N50 values for all stages of the assembly pipeline and several different cutoffs for minimal scaffold length.
Scaffold Length CutoffContigsExtended ContigsRaw ScaffoldsIntermediate ScaffolfsGap Closed ScaffoldsFinal Scaffolds
all85,28684,6481,256,5341,898,0061,898,9852,297,899
≥10085,28684,6481,256,5341,898,0061,898,9852,297,899
≥250101,388100,3931,442,4212,296,4842,297,8992,297,899
≥500115,732115,4861,538,7952,678,8572,680,9412,680,941
≥1000122,300121,6781,704,0642,678,8572,680,9412,680,941
Table 3. Repeats found by RepeatMasker.
Table 3. Repeats found by RepeatMasker.
ClassNumber of ElementsTotal Length (bp)Fraction of Assembly (%)
SINEs577125,2980.06
LINEs70754,351,2411.96
LTR elements51,04040,608,19518.27
DNA elements31,63812,868,6845.79
Unclassified82,69324,363,13510.96
Total interspersed repeats-82,316,55337.04
Small RNA54611,599,3540.72
Satellites1541573,0260.26
Simple repeats2044363,6420.16
Low complexity5674560
Table 4. Results of repeat masking performed by three different tools: RepeatMasker [32], TRF [34], WindowMasker [35].
Table 4. Results of repeat masking performed by three different tools: RepeatMasker [32], TRF [34], WindowMasker [35].
ToolNumber of RepeatsTotal Length (Mbp)
RepeatMasker173,02382.31
TRF100,59317.41
Windowmasker1,104,65064.20
Table 5. Annotated transfer RNAs (tRNAs).
Table 5. Annotated transfer RNAs (tRNAs).
tRNA TypeNumber
tRNAs decoding standard 20 AA1126
Selenocysteine tRNAs (TCA)0
Possible suppressor tRNAs (CTA,TTA)3
tRNAs with undetermined isotypes5
Resolution of Brassicaceae Phylogeny Using Nuclear Genes
Uncovers Nested Radiations and Supports Convergent
Morphological Evolution Predicted pseudogenes
32
Total tRNAs1166
Table 6. Annotated ribosomal RNAs (rRNAs).
Table 6. Annotated ribosomal RNAs (rRNAs).
rRNAComplete (≥80% of Expected Length)Partial (<80% of Expected Length)
5.8S17853
5S601104
28S01782
18S11458
12S0173
16S0607
Table 7. Comparison of genome characteristics of Boechera retrofracta with previously sequenced Boechera stricta and Arabidopsis thaliana genomes. Source for B.retrofracta—this paper, B.stricta, Arabidopsis lyrata and A.thaliana—Phytozome v12.1 database [57].
Table 7. Comparison of genome characteristics of Boechera retrofracta with previously sequenced Boechera stricta and Arabidopsis thaliana genomes. Source for B.retrofracta—this paper, B.stricta, Arabidopsis lyrata and A.thaliana—Phytozome v12.1 database [57].
Boechera retrofractaBoechera stricta v.1.2Arabidopsis lyrata v2.1Arabidopsis thaliana TAIR10
Total length227 M184 M207 Mb135 Mb
Chromosomesn = 7n = 7n = 8n = 5
Protein-coding loci 27,04827,41631,07327,416
Transcripts28,26929,81233,13235,386
Back to TopTop