Sequence analysis of cultivated strawberry (Fragaria × ananassa Duch.) using microdissected single somatic chromosomes
© The Author(s) 2017
Received: 18 May 2017
Accepted: 8 October 2017
Published: 30 October 2017
Cultivated strawberry (Fragaria × ananassa Duch.) has homoeologous chromosomes because of allo-octoploidy. For example, two homoeologous chromosomes that belong to different sub-genome of allopolyploids have similar base sequences. Thus, when conducting de novo assembly of DNA sequences, it is difficult to determine whether these sequences are derived from the same chromosome. To avoid the difficulties associated with homoeologous chromosomes and demonstrate the possibility of sequencing allopolyploids using single chromosomes, we conducted sequence analysis using microdissected single somatic chromosomes of cultivated strawberry.
Three hundred and ten somatic chromosomes of the Japanese octoploid strawberry ‘Reiko’ were individually selected under a light microscope using a microdissection system. DNA from 288 of the dissected chromosomes was successfully amplified using a DNA amplification kit. Using next-generation sequencing, we decoded the base sequences of the amplified DNA segments, and on the basis of mapping, we identified DNA sequences from 144 samples that were best matched to the reference genomes of the octoploid strawberry, F. × ananassa, and the diploid strawberry, F. vesca. The 144 samples were classified into seven pseudo-molecules of F. vesca. The coverage rates of the DNA sequences from the single chromosome onto all pseudo-molecular sequences varied from 3 to 29.9%.
We demonstrated an efficient method for sequence analysis of allopolyploid plants using microdissected single chromosomes. On the basis of our results, we believe that whole-genome analysis of allopolyploid plants can be enhanced using methodology that employs microdissected single chromosomes.
Cultivated strawberry (Fragaria × ananassa Duch.) is one of the most popular fruit crops worldwide and is grown across a wide range of regions from subarctic to tropical . Cytogenetic studies have determined that the chromosome number of somatic cells of cultivated strawberry is 56 [2–5]. In addition, cultivated strawberry is an allo-octoploid, having three complex genome compositions: AABBBBCC , AAA′A′BBBB , or AAA′A′BBB′B′ . In addition, Tennessen et al.  and Sargent et al.  have recently proposed updated models—AvAvB1B1B2B2BiBi and AA, bb, X–X, X–X, respectively. The amount of DNA within a haploid nucleus of cultivated strawberry has been estimated at 708–720 Mb . On the basis of this data, the average DNA size of a single chromosome can be calculated as approximately 25–27.8 Mb. In addition, the mean chromosome length of some wild octoploid strawberries has been determined to be approximately 1 µm . The size of a single chromosome in cultivated strawberry appears to be very small as likely as that in rice and Arabidopsis thaliana.
Allopolyploidy is a problem when conducting genetic analyses, because the presence of similar sub-genomes has led to multiple alleles and complex segregation ratios . Thus, theoretical genetic analysis and breeding in cultivated strawberry are extremely difficult based Mendel’s law of inheritance. To resolve this problem, the determination of accurate base sequences covering the entire genome of cultivated strawberry is needed to construct a high-density linkage map. If this can be achieved, many DNA markers that follow Mendel’s law of inheritance could be discovered, and theoretical breeding using genomic selection could be performed with high reliability. Hirakawa et al.  reported the draft genome sequences of a Japanese cultivated strawberry using next-generation sequencing (NGS). The base sequences of entire DNA segments from the strawberry ‘Reiko’ were determined, and approximately 70% of these sequences were assembled into larger DNA scaffolds. However, to date, the sequences have not been assigned to chromosomes. Moreover, 30% of the genome remains unsequenced, because of the existence of sub-genomes that have homoeologous chromosomes. For example, two homoeologous chromosomes that belong to different sub-genomes may have similar but slightly different base sequences. Thus, when performing standard genome assembly, it is difficult to accurately assign such sequences to an appropriate pseudo-molecular chromosome. To overcome this problem, it is necessary to develop an alternative method of sequence analysis for allopolyploids.
In the present study, in order to avoid difficulties arising from the occurrence of homoeologous chromosomes, we attempted to conduct sequence analysis using microdissected single somatic chromosomes. Although a technique for chromosome microdissection was developed in the 1980s , it has been unpopular for elucidating the base sequences of whole genomes. Scalenghe et al.  initially microdissected a small segment of the chromosome in Drosophila melanogaster, and demonstrated the possibility of directly generating DNA segments. As discussed by Zhou and Hu , many studies have been conducted using chromosome microdissection for human and animal cells, but a smaller number have been performed in plants because chromosome sample preparation is more difficult in plants. In higher plants, Sandery et al.  first reported microdissection and DNA generation of B-chromosomes in rye. Subsequently, chromosome microdissection has been used in several facets of genomic research, including (1) genetic linkage map and physical map construction, (2) generation of probes for chromosome painting, and (3) generation of chromosome-specific expressed sequence tag libraries . However, to date, few studies have used chromosome microdissection to determine base sequences of whole genomes in plants.
The purpose of the present study was to determine the effectiveness of sequence analysis using single chromosomes for a typical allopolyploid cultivated strawberry plant. Furthermore, we also examined the possibility of amplifying DNA from a very small single chromosome using a DNA amplification kit.
Results and discussion
Efficiency of the microdissection system
Confirmation of DNA amplification from a single chromosome of cultivated strawberry
Mapping onto pseudo-molecules of the diploid Fragaria vesca, one of the genome donors in cultivated strawberry
Coverage rate of the amplified DNA from single chromosomes of cultivated strawberry
The present study was conducted to determine the effectiveness of sequence analysis using single chromosomes for a typical allopolyploid species, cultivated strawberry. Then, the new efficient method to amplify DNA segments from a microdissected single somatic chromosome were exhibited. In addition, it confirmed that the amplified DNA segments were derived from the chromosome of strawberry plants by sequence analysis. The coverage rates of the DNA sequences from the single chromosome onto all pseudo-molecular sequences of the diploid F. vesca genome varied from 3 to 29.9%. On the basis of these results, we believe that whole-genome analysis of allopolyploid plants can be enhanced using methodology that employs microdissected single chromosomes.
Chromosome slide sample preparation
Some newly propagated plants of a Japanese octoploid strawberry ‘Reiko’ that was grown in a greenhouse condition were used for the experiment. Pretreatment and fixation of root tips were conducted using a modified version of the method described by Iwatsubo and Naruhashi [28, 29], Nathewet et al. [4, 5], and Yanagi and Noguchi . Root tips were collected at 17:00, pretreated with 2 mM 8-hydroxyquinoline solution for 1 h at approximately 20 °C, and subsequently maintained in the same solution at 4 °C for 15 h until 09:00 the following morning. The root tips were then fixed in a 1:3 (v:v) solution of acetic acid and ethanol for 40 min at room temperature. The fixed roots were trimmed to 2–3 mm from the tip and were softened using an enzyme cocktail, containing 4% cellulase Onozuka RS (Yakult Co. Ltd., Tokyo), 0.3% pectolyase Y-23 (Seishin Pharmaceutical Co. Ltd., Tokyo), 2.1% macerozyme R10 (Yakult Co. Ltd., Tokyo), and 1 mM EDTA pH 4.2 at 37 °C for 25 min. Subsequently, the roots were rinsed twice in distilled water. Then one root tip that was selected using a Pasteur pipette with distilled water and placed in the center of a glass slide. After eliminating the water, 10 µL 45% acetic acid was placed on the root tip, followed by incubation for 2 min and then maceration using forceps. A cover slip was placed on the preparation, tapped gently with a chopstick, heated using an alcohol lamp for a few seconds, and then pressed with a thumb. The glass slide was exposed to −80 °C for at least 5 min in an ultra-low temperature freezer, and then the cover slip was removed using a razor blade at room temperature. The slide samples were dipped in a 70% alcohol solution at −20 °C prior to microdissection.
Chromosome microdissection was conducted under a light microscope (BX51; Olympus Co.), which was equipped with a micromanipulation system (MN-4 and MMO-203; Narishige Co.) and a long focus objective lens (× 50 SLMPLN; Olympus Co.). A glass needle for picking up a single chromosome of the cultivated strawberry was fabricated using a glass puller device (PC-10; Narishige Co.). Chromosome microdissection was conducted in a clean room to avoid DNA contamination by atmospheric microorganisms. Single chromosomes on the sample slide were selected individually. After confirming the presence of a single chromosome at the tip of the glass needle under the light microscope, it was placed in a PCR tube containing 1 µL 1 × PBS buffer. The tip of the glass needle to which the microdissected single chromosome was adhered was then pressed against the bottom of the PCR tube and folded, and both were placed in the PCR tube. In total, 310 sample PCR tubes with single chromosomes were prepared from 10 somatic cells of cultivated strawberry.
DNA amplification and analysis by NGS
DNA amplification was conducted using an Illustra Single Cell GenomiPhi DNA Amplification kit (GE Healthcare Co.), according to the manufacturer’s protocol. After adding 1 µL lysis buffer, the PCR tube was heated at 65 °C for 10 min. To the PCR tube, 11 µL reaction buffer, 1 µL enzyme mix, 1 µL amplification mix, and 4 µL sterile water were then added. The PCR tube was incubated at 30 °C for 180 min, and subsequently heated at 65 °C for 10 min to inactivate the enzymes. Following amplification, the concentration of the DNA was measured using a fluorometer (Qubit® 3.0; Thermo Fisher Co.). The DNA was then fragmented using a DNA Shearing Tube g-TUBE (Covaris, Woburn, MA, USA) or NEBNext dsDNA Fragmentase (New England Biolabs, Hitchin, UK) into lengths of approximately 600 bp for sequencing library preparation using a TruSeq Nano DNA Sample Prep Kit (Illumina, San Diego, CA, USA). The nucleotide sequences of the libraries were determined using a MiSeq system (Illumina) in paired-end mode (301-base) or a NextSeq 500 system (Illumina) in paired-end mode (151-base). The sequence reads were submitted in the DDBJ Sequence Read Archive under the accession number DRA005991.
Data processing and mapping
Low-quality sequences were removed and adapters were trimmed using PRINSEQ  and fastx_clipper in the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit). Sequence similarity searches of 1000 randomly selected reads from each library were performed against the NCBI nt (non-redundant nucleotide sequences) database (http://www.ncbi.nlm.nih.gov), the F. vesca genome, v2.0.a1 , the cultivated strawberry genome, FAN_r1.1 , the F. vesca chloroplast genome (Accession number NC_015206), and the A. thaliana mitochondrion genome (Accession number NC_001284) using the BLASTN program with an E value cutoff of ≤ 1e −10 . Furthermore, all of the filtered reads were mapped onto the F. vesca genome (version v2.0.a1) and the cultivated strawberry genome (FAN_r1.1) as reference sequences using Bowtie 2 . The resulting sequence alignment/map format files were converted to binary sequence alignment/map format (BAM) files. Genome coverage was calculated from the BAM files using the BEDtools script genomeCoverageBed .
TY, KS, MT, and SI designed the study, performed experiments, and analyzed the data. MT microdissected the chromosomes. TY, KS, and SI wrote the manuscript. All authors read and approved the final manuscript.
We are grateful to Ms. F. Maeda (Chiba Prefectural Agriculture and Forestry Research Center, Japan) for providing the plant material, to Dr. H. Masumoto (Kazusa DNA Research Institute) for his kind support, and to Dr. T. Kornlawat (Kagawa University) and S. Sasamoto, C. Minami, and S. Nakayama (Kazusa DNA Research Institute) for their technical assistance.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Consent for publication
All authors have given consent for the manuscript.
This work was supported by the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food industry in Ministry of Agriculture, Forestry and Fisheries in Japan (27003A).
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