Solitary bees – Potential vectors for gene flow from cultivated to wild strawberries

The genus Fragaria (Rosaceae) contains 24 plant species, including hybrid species such as the octoploid garden strawberry (F. × ananassa). Natural hybridization between Fragaria species has repeatedly been reported, and the potential future cultivation of genetically modified strawberries has made the study of hybridization potential between F. × ananassa and its wild relatives increasingly important. In Europe, F. × ananassa is the only octoploid species present, and the most likely candidate for hybridization is the common diploid woodland strawberry (F. vesca). To date, it is unknown whether pollinator spectra of the two Fragaria species overlap and thus might promote interspecific gene flow. We carried out a survey of flower visitors in northwestern Switzerland to identify major flower visitors of F. vesca and F. × ananassa. This survey indicated that wild bees are the most important shared flower visitors of F. × ananassa and F. vesca. Therefore, we studied flower choice behavior of the common wild bee Osmia bicornis in a greenhouse experiment. Osmia bicornis did not discriminate between F. × ananassa and F. vesca flowers. We conclude that wild bees are important shared flower visitors of both F. × ananassa and F. vesca and are potential vectors for gene flow between cultivated and wild strawberries.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

A ddRAD Based Linkage Map of the Cultivated Strawberry,Fragaria xananassa

The cultivated strawberry (Fragaria ×ananassa Duch.) is an allo-octoploid considered difficult to disentangle genetically due to its four relatively similar sub-genomic chromosome sets. This has been alleviated by the recent release of the strawberry IStraw90 whole genome genotyping array. However, array resolution relies on the genotypes used in the array construction and may be of limited general use. SNP detection based on reduced genomic sequencing approaches has the potential of providing better coverage in cases where the studied genotypes are only distantly related from the SNP array’s construction foundation. Here we have used double digest restriction-associated DNA sequencing (ddRAD) to identify SNPs in a 145 seedling F₁ hybrid population raised from the cross between the cultivars Sonata (♀) and Babette (♂). A linkage map containing 907 markers which spanned 1,581.5 cM across 31 linkage groups representing the 28 chromosomes of the species. Comparing the physical span of the SNP markers with the F. vesca genome sequence, the linkage groups resolved covered 79% of the estimated 830 Mb of the F. ×ananassa genome. Here, we have developed the first linkage map for F. ×ananassa using ddRAD and show that this technique and other related techniques are useful tools for linkage map development and downstream genetic studies in the octoploid strawberry.     

Searching for gene flow from cultivated to wild strawberries in Central Europe

Background and Aims

Experimental crosses between the diploid woodland strawberry (Fragaria vesca L.) and the octoploid garden strawberry (F. × ananassa Duch.) can lead to the formation of viable hybrids. However, the extent of such hybrid formation under natural conditions is unknown, but is of fundamental interest and importance in the light of the potential future cultivation of transgenic strawberries. A hybrid survey was therefore conducted in the surroundings of ten farms in Switzerland and southern Germany, where strawberries have been cultivated for at least 10 years and where wild strawberries occur in the close vicinity.

Methods

In 2007 and 2008, 370 wild F. vesca plants were sampled at natural populations around farms and analysed with microsatellite markers. In 2010, natural populations were revisited and morphological traits of 3050 F. vesca plants were inspected. DNA contents of cell nuclei of morphologically deviating plants were estimated by flow cytometry to identify hybrids. As controls, 50 hybrid plants from interspecific hand-crosses were analysed using microsatellite analysis and DNA contents of cell nuclei were estimated by flow cytometry.

Key Results

None of the wild samples collected in 2007 and 2008 contained F. × ananassa microsatellite markers, while all hybrids from hand-crosses clearly contained markers of both parent species. Morphological inspection of wild populations carried out in 2010 and subsequent flow cytometry of ten morphologically deviating plants revealed no hybrids.

Conclusions

Hybrid formation or hybrid establishment in natural populations in the survey area is at best a rare event.     

Development and preliminary evaluation of a 90?K Axiom? SNP array for the allo-octoploid cultivated strawberry Fragaria × ananassa

Background

A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca ‘Hawaii 4’ reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array.

Results

About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing “haploSNPs” (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative “codon-based” SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix’s “SNPolisher” R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family ‘Holiday’ × ‘Korona’ with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM.

Conclusions

The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array’s high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1310-1) contains supplementary material, which is available to authorized users.     

DNA Complexity of the Root-knot Nematode (Meloidogyne spp.) Genome

Cot curves derived from renaturation kinetics of sheared denatured DNA indicated that the genome of six populations representing the four most common root-knot nematode species (Meloidogyne incognita, M. arenaria, M. javanica, and M. hapla) is composed of 20% repetitive and 80% nonrepetitive sequences of DNA. Cot curves were almost identical, indicating that all populations had a haploid genome of approximately the same size. Calculations from an average Cot curve gave an estimate of 0.51 x 108 nucleotide base pairs for the haploid genome of the four Meloidogyne species. This genome is about 12-13 times larger than the genome of the E. coli strain used as a control.     

Population genomics of Sinorhizobium medicae based on low-coverage sequencing of sympatric isolates

We investigated the genomic diversity of a local population of the symbiotic bacterium Sinorhizobium medicae, isolated from the roots of wild Medicago lupulina plants, in order to assess genomic diversity, to identify genomic regions influenced by duplication, deletion or strong selection, and to explore the composition of the pan-genome. Partial genome sequences of 12 isolates were obtained by Roche 454 shotgun sequencing (average 5.3 Mb per isolate) and compared with the published sequence of S. medicae WSM 419. Homologous recombination appears to have less impact on the polymorphism patterns of the chromosome than on the chromid pSMED01 and megaplasmid pSMED02. Moreover, pSMED02 is a hot spot of insertions and deletions. The whole chromosome is characterized by low sequence polymorphism, consistent with the high density of housekeeping genes. Similarly, the level of polymorphism of symbiosis genes (low) and of genes involved in polysaccharide synthesis (high) may reflect different selection. Finally, some isolates carry genes that may confer adaptations that S. medicae WSM 419 lacks, including homologues of genes encoding rhizobitoxine synthesis, iron uptake, response to autoinducer-2, and synthesis of distinct polysaccharides. The presence or absence of these genes was confirmed by PCR in each of these 12 isolates and a further 27 isolates from the same population. All isolates had rhizobitoxine genes, while the other genes were co-distributed, suggesting that they may be on the same mobile element. These results are discussed in relation to the ecology of Medicago symbionts and in the perspective of population genomics studies.     

Survey of genome sequences in a wild sweet potato,Ipomoea trifida (H. B. K.) G. Don

Ipomoea trifida (H. B. K.) G. Don. is the most likely diploid ancestor of the hexaploid sweet potato, I. batatas (L.) Lam. To assist in analysis of the sweet potato genome, de novo whole-genome sequencing was performed with two lines of I. trifida, namely the selfed line Mx23Hm and the highly heterozygous line 0431-1, using the Illumina HiSeq platform. We classified the sequences thus obtained as either ‘core candidates’ (common to the two lines) or ‘line specific’. The total lengths of the assembled sequences of Mx23Hm (ITR_r1.0) was 513 Mb, while that of 0431-1 (ITRk_r1.0) was 712 Mb. Of the assembled sequences, 240 Mb (Mx23Hm) and 353 Mb (0431-1) were classified into core candidate sequences. A total of 62,407 (62.4 Mb) and 109,449 (87.2 Mb) putative genes were identified, respectively, in the genomes of Mx23Hm and 0431-1, of which 11,823 were derived from core sequences of Mx23Hm, while 28,831 were from the core candidate sequence of 0431-1. There were a total of 1,464,173 single-nucleotide polymorphisms and 16,682 copy number variations (CNVs) in the two assembled genomic sequences (under the condition of log₂ ratio of >1 and CNV size >1,000 bases). The results presented here are expected to contribute to the progress of genomic and genetic studies of I. trifida, as well as studies of the sweet potato and the genus Ipomoea in general.     

Comparisons of Mitochondrial DNA from the Sibling Species Heterodera glycines and H. schachtii

Restriction fragment patterns of mitochondrial DNA from sibling species of cyst nematodes Heterodera glycines and H. schachtii were examined. Fourteen restriction endonucleases recognizing four, five, and six base-pair sequences yielded a total of 90 scorable fragments of which 10% were shared by both species. Mitochondrial genome sizes for H. glycines and H. schachtii were estimated to be 22.5-23.5 kb and 23.0 kb, respectively. A single wild type mitochondrial genome was identified in all populations of H. glycines examined, although other mitochondrial genomes were present in some populations. The H. schachtii genome exhibited 57 scorable fragments, compared with 33 identified in the H. glycines wild type genome. The estimated nucleotide sequence divergence between the two species was p = 0.145. This estimate suggests these species diverged from a common ancestor 7.3-14.8 million years ago.     

Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species

Background

Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology.

Results

Draft genome sequences of Nocardia asteroides NBRC 15531^T, Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402^T, and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4–11, 7–13, and 1–6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text.

Conclusion

We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied, and 4) different N. brasiliensis strains have some different gene clusters of PKS-I/NRPS, although the rest of the clusters are common within the N. brasiliensis strains. Genome sequencing suggested that Nocardia strains are highly promising resources in the search of novel secondary metabolites.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-323) contains supplementary material, which is available to authorized users.     

Unraveling the Complex Hybrid Ancestry and Domestication History of Cultivated Strawberry

Cultivated strawberry (Fragaria × ananassa) is one of our youngest domesticates, originating in early eighteenth-century Europe from spontaneous hybrids between wild allo-octoploid species (Fragaria chiloensis and Fragaria virginiana). The improvement of horticultural traits by 300 years of breeding has enabled the global expansion of strawberry production. Here, we describe the genomic history of strawberry domestication from the earliest hybrids to modern cultivars. We observed a significant increase in heterozygosity among interspecific hybrids and a decrease in heterozygosity among domesticated descendants of those hybrids. Selective sweeps were found across the genome in early and modern phases of domestication—59–76% of the selectively swept genes originated in the three less dominant ancestral subgenomes. Contrary to the tenet that genetic diversity is limited in cultivated strawberry, we found that the octoploid species harbor massive allelic diversity and that F. × ananassa harbors as much allelic diversity as either wild founder. We identified 41.8 M subgenome-specific DNA variants among resequenced wild and domesticated individuals. Strikingly, 98% of common alleles and 73% of total alleles were shared between wild and domesticated populations. Moreover, genome-wide estimates of nucleotide diversity were virtually identical in F. chiloensis,F. virginiana, and F. × ananassa (π = 0.0059–0.0060). We found, however, that nucleotide diversity and heterozygosity were significantly lower in modern F. × ananassa populations that have experienced significant genetic gains and have produced numerous agriculturally important cultivars.     

Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes

Background

The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes.

Results

We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58× coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons.

Conclusions

PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of “finished grade” because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-699) contains supplementary material, which is available to authorized users.     

Differential detection of transposable elements between Saccharum species

Cultivars of sugarcane (Saccharum) are hybrids between species S. officinarum (x = 10, 2n = 8x = 80) and S. spontaneum (x = 8, 2n = 5 – 16x = 40 – 128). These accessions have 100 to 130 chromosomes, 80–85% of which are derived from S. officinarum, 10–15% from S. spontaneum, and 5–10% are possible recombinants between the two genomes. The aim of this study was to analyze the repetition of DNA sequences in S. officinarum and S. spontaneum. For this purpose, genomic DNA from S. officinarum was digested with restriction enzymes and the fragments cloned. Sixty-eight fragments, approximately 500 bp, were cloned, sequenced and had their identity analyzed in NCBI, and in the rice, maize, and sorghum genome databases using BLAST. Twelve clones containing partial transposable elements, one single-copy control, one DNA repetitive clone control and two genome controls were analyzed by DNA hybridization on membrane, using genomic probes from S. officinarum and S. spontaneum. The hybridization experiment revealed that six TEs had a similar repetitive DNA pattern in the genomes of S. officinarum and S. spontaneum, while six TEs were more abundant in the genome of S. officinarum. We concluded that the species S. officinarum and S. spontaneum have differential accumulation LTR retrotransposon families, suggesting distinct insertion or modification patterns.     

Diverse retrotransposon families and an AT-rich satellite DNA revealed in giant genomes of Fritillaria lilies

Background and Aims

The genus Fritillaria (Liliaceae) comprises species with extremely large genomes (1C = 30 000–127 000 Mb) and a bicontinental distribution. Most North American species (subgenus Liliorhiza) differ from Eurasian Fritillaria species by their distinct phylogenetic position and increased amounts of heterochromatin. This study examined the contribution of major repetitive elements to the genome obesity found in Fritillaria and identified repeats contributing to the heterochromatin arrays in Liliorhiza species.

Methods

Two Fritillaria species of similar genome size were selected for detailed analysis, one from each phylogeographical clade: F. affinis (1C = 45·6 pg, North America) and F. imperialis (1C = 43·0 pg, Eurasia). Fosmid libraries were constructed from their genomic DNAs and used for identification, sequence characterization, quantification and chromosome localization of clones containing highly repeated sequences.

Key Results and Conclusions

Repeats corresponding to 6·7 and 4·7 % of the F. affinis and F. imperialis genome, respectively, were identified. Chromoviruses and the Tat lineage of Ty3/gypsy group long terminal repeat retrotransposons were identified as the predominant components of the highly repeated fractions in the F. affinis and F. imperialis genomes, respectively. In addition, a heterogeneous, extremely AT-rich satellite repeat was isolated from F. affinis. The FriSAT1 repeat localized in heterochromatic bands makes up approx. 26 % of the F. affinis genome and substantial genomic fractions in several other Liliorhiza species. However, no evidence of a relationship between heterochromatin content and genome size variation was observed. Also, this study was unable to reveal any predominant repeats which tracked the increasing/decreasing trends of genome size evolution in Fritillaria. Instead, the giant Fritillaria genomes seem to be composed of many diversified families of transposable elements. We hypothesize that the genome obesity may be partly determined by the failure of removal mechanisms to counterbalance effectively the retrotransposon amplification.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.