Abstract
BackgroundCultivated 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.ResultsThree 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%.ConclusionWe 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.
Highlights
Cultivated strawberry (Fragaria × ananassa Duch.) has homoeologous chromosomes because of allooctoploidy
The maximum matched value of 983 reads was recorded for sample FaMD-4-A10. These results clearly demonstrated that the Illustra Single Cell GenomiPhi DNA Amplification kit could amplify the DNA sequence of the strawberry chromosome by following the manufacturer’s protocol, DNA in approximately half
The assignment of repeat sequences might be problematic, our results indicate that a large percentage of the DNA reads derived from each single chromosome of cultivated strawberry could correspond to a specific pseudo-molecule of F. vesca
Summary
Cultivated strawberry (Fragaria × ananassa Duch.) has homoeologous chromosomes because of allooctoploidy. 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. Cytogenetic studies have determined that the chromosome number of somatic cells of cultivated strawberry is 56 [2,3,4,5]. Cultivated strawberry is an allo-octoploid, having three complex genome compositions: AABBBBCC [6], AAA′A′BBBB [7], or AAA′A′BBB′B′ [8]. 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 [12]. The size of a single chromosome in cultivated strawberry appears to be very small as likely as that in rice and Arabidopsis thaliana
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