Publications by authors named "Sachiko Isobe"

91 Publications

Genome features of common vetch () in natural habitats.

Plant Direct 2021 Oct 7;5(10):e352. Epub 2021 Oct 7.

Kazusa DNA Research Institute Kisarazu Japan.

Wild plants are often tolerant to biotic and abiotic stresses in their natural environments, whereas domesticated plants such as crops frequently lack such resilience. This difference is thought to be due to the high levels of genome heterozygosity in wild plant populations and the low levels of heterozygosity in domesticated crop species. In this study, common vetch () was used as a model to examine this hypothesis. The common vetch genome (2n = 14) was estimated as 1.8 Gb in size. Genome sequencing produced a reference assembly that spanned 1.5 Gb, from which 31,146 genes were predicted. Using this sequence as a reference, 24,118 single nucleotide polymorphisms were discovered in 1243 plants from 12 natural common vetch populations in Japan. Common vetch genomes exhibited high heterozygosity at the population level, with lower levels of heterozygosity observed at specific genome regions. Such patterns of heterozygosity are thought to be essential for adaptation to different environments. The resources generated in this study will provide insights into de novo domestication of wild plants and agricultural enhancement.
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http://dx.doi.org/10.1002/pld3.352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8496506PMC
October 2021

A chromosome-level genome sequence of Chrysanthemum seticuspe, a model species for hexaploid cultivated chrysanthemum.

Commun Biol 2021 Oct 7;4(1):1167. Epub 2021 Oct 7.

Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.

Chrysanthemums are one of the most industrially important cut flowers worldwide. However, their segmental allopolyploidy and self-incompatibility have prevented the application of genetic analysis and modern breeding strategies. We thus developed a model strain, Gojo-0 (Chrysanthemum seticuspe), which is a diploid and self-compatible pure line. Here, we present the 3.05 Gb chromosome-level reference genome sequence, which covered 97% of the C. seticuspe genome. The genome contained more than 80% interspersed repeats, of which retrotransposons accounted for 72%. We identified recent segmental duplication and retrotransposon expansion in C. seticuspe, contributing to arelatively large genome size. Furthermore, we identified a retrotransposon family, SbdRT, which was enriched in gene-dense genome regions and had experienced a very recent transposition burst. We also demonstrated that the chromosome-level genome sequence facilitates positional cloning in C. seticuspe. The genome sequence obtained here can greatly contribute as a reference for chrysanthemum in front-line breeding including genome editing.
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http://dx.doi.org/10.1038/s42003-021-02704-yDOI Listing
October 2021

Chromosome-level genome assembly of Japanese chestnut (Castanea crenata Sieb. et Zucc.) reveals conserved chromosomal segments in woody rosids.

DNA Res 2021 Sep;28(5)

Kazusa DNA Research Institute, Chiba 292-0818, Japan.

Japanese chestnut (Castanea crenata Sieb. et Zucc.), unlike other Castanea species, is resistant to most diseases and wasps. However, genomic data of Japanese chestnut that could be used to determine its biotic stress resistance mechanisms have not been reported to date. In this study, we employed long-read sequencing and genetic mapping to generate genome sequences of Japanese chestnut at the chromosome level. Long reads (47.7 Gb; 71.6× genome coverage) were assembled into 781 contigs, with a total length of 721.2 Mb and a contig N50 length of 1.6 Mb. Genome sequences were anchored to the chestnut genetic map, comprising 14,973 single nucleotide polymorphisms (SNPs) and covering 1,807.8 cM map distance, to establish a chromosome-level genome assembly (683.8 Mb), with 69,980 potential protein-encoding genes and 425.5 Mb repetitive sequences. Furthermore, comparative genome structure analysis revealed that Japanese chestnut shares conserved chromosomal segments with woody plants, but not with herbaceous plants, of rosids. Overall, the genome sequence data of Japanese chestnut generated in this study is expected to enhance not only its genetics and genomics but also the evolutionary genomics of woody rosids.
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http://dx.doi.org/10.1093/dnares/dsab016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8435554PMC
September 2021

DNA marker for resistance to in chrysanthemum ( Ramat.) "Southern Pegasus".

Breed Sci 2021 Apr 25;71(2):261-267. Epub 2021 Mar 25.

Institute of Fruit Tree and Tea Science, NARO, Tsukuba, Ibaraki 305-8605, Japan.

White rust caused by Henn. adversely affects chrysanthemum ( Ramat.) production. The breeding of resistant varieties is effective in controlling the disease. Here we aimed to develop DNA markers for the strong resistance to . We conducted a linkage analysis based on the genome-wide association study (GWAS) method. We employed a biparental population for the GWAS, wherein the single nucleotide polymorphism (SNP) allele frequency could be predicted. The population was derived from crosses between a strong resistant "Southern Pegasus" and a susceptible line. The GWAS used simplex and double-simplex SNP markers selected out of SNP candidates mined from ddRAD-Seq data of an F biparental population. These F individuals segregated in a 1:1 ratio of resistant to susceptible. Twenty-one simplex SNPs were significantly associated with resistance in "Southern Pegasus" and generated one linkage group. These results show the presence of a single resistance gene in "Southern Pegasus". We identified the nearest SNP marker located 2.2 cM from resistance locus and demonstrated this SNP marker-resistance link using an independent population. This is the first report of an effective DNA marker linked to a gene for resistance in chrysanthemum.
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http://dx.doi.org/10.1270/jsbbs.20063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329880PMC
April 2021

Development of PCR-based DNA marker for detection of white carrot contamination caused by locus.

Breed Sci 2021 Apr 30;71(2):201-207. Epub 2021 Mar 30.

Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.

In carrot ( L.), the taproot colors orange, yellow and white are determined mostly by the , , and loci. One of the most severe issues in carrot seed production is contamination by wild white carrot. To evaluate the contamination ratio, easily detectable DNA markers for white carrot are desired. To develop PCR-based DNA markers for the locus, we have re-sequenced two orange-colored carrot cultivars at our company (Fujii Seed, Japan), as well as six white- and one light-orange-colored carrots that contaminated our seed products. Within the candidate region previously reported for the locus, only one DNA marker, Y2_7, clearly distinguished white carrots from orange ones in the re-sequenced samples. The Y2_7 marker was further examined in 12 of the most popular hybrid orange cultivars in Japan, as well as 'Nantes' and 'Chantenay Red Cored 2'. The Y2_7 marker showed that all of the orange cultivars examined had the orange allele except for 'Beta-441'. False white was detected in the orange-colored 'Beta-441'. The Y2_7 marker detected white root carrot contamination in an old open-pollinated Japanese cultivar, 'Nakamura Senkou Futo'. This marker would be a useful tool in a carrot seed quality control for some cultivars.
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http://dx.doi.org/10.1270/jsbbs.20120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329877PMC
April 2021

Strawberry fruit shape: quantification by image analysis and QTL detection by genome-wide association analysis.

Breed Sci 2021 Apr 25;71(2):167-175. Epub 2021 Feb 25.

Fukuoka Agriculture and Forestry Research Center, 587 Yoshiki, Chikushino, Fukuoka 818-8549, Japan.

Fruit shape of cultivated strawberry ( × Duch.) is an important breeding target. To detect genomic regions associated with this trait, its quantitative evaluation is needed. Previously we created a multi-parent advanced-generation inter-cross (MAGIC) strawberry population derived from six founder parents. In this study, we used this population to quantify fruit shape. Elliptic Fourier descriptors (EFDs) were generated from 2 969 two-dimensional binarized fruit images, and principal component (PC) scores were calculated on the basis of the EFD coefficients. PC1-PC3 explained 96% of variation in shape and thus adequately quantified it. In genome-wide association study, the PC scores were used as phenotypes. Genome wide association study using mixed linear models revealed 2 quantitative trait loci (QTLs) for fruit shape. Our results provide a novel and effective method to analyze strawberry fruit morphology; the detected QTLs and presented method can support marker-assisted selection in practical breeding programs to improve fruit shape.
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http://dx.doi.org/10.1270/jsbbs.19106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329875PMC
April 2021

Chromosome-level genome assemblies of over 100 plant species.

Breed Sci 2021 Apr 6;71(2):117-124. Epub 2021 Apr 6.

ICAR-National Institute for Plant Biotechnology, Pusa, New Delhi 110012, India.

Genome sequence analysis in higher plants began with the whole-genome sequencing of . Owing to the great advances in sequencing technologies, also known as next-generation sequencing (NGS) technologies, genomes of more than 400 plant species have been sequenced to date. Long-read sequencing technologies, together with sequence scaffolding methods, have enabled the synthesis of chromosome-level genome sequence assemblies, which has further allowed comparative analysis of the structural features of multiple plant genomes, thus elucidating the evolutionary history of plants. However, the quality of the assembled chromosome-level sequences varies among plant species. In this review, we summarize the status of chromosome-level assemblies of 114 plant species, with genome sizes ranging from 125 Mb to 16.9 Gb. While the average genome coverage of the assembled sequences reached up to 89.1%, the average coverage of chromosome-level pseudomolecules was 73.3%. Thus, further improvements in sequencing technologies and scaffolding, and data analysis methods, are required to establish gap-free telomere-to-telomere genome sequence assemblies. With the forthcoming new technologies, we are going to enter into a new genomics era where pan-genomics and the >1,000 or >1 million genomes' project will be routine in higher plants.
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http://dx.doi.org/10.1270/jsbbs.20146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329882PMC
April 2021

Whole-genome sequencing and analysis of two azaleas, Rhododendron ripense and Rhododendron kiyosumense.

DNA Res 2021 Sep;28(5)

Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.

To enhance the genomics and genetics of azalea, the whole-genome sequences of two species of Rhododendron were determined and analysed in this study: Rhododendron ripense, the cytoplasmic donor and ancestral species of large-flowered and evergreen azalea cultivars; and Rhododendron kiyosumense, a native of Chiba prefecture (Japan) seldomly bred and cultivated. A chromosome-level genome sequence assembly of R. ripense was constructed by single-molecule real-time sequencing and genetic mapping, while the genome sequence of R. kiyosumense was assembled using the single-tube long fragment read sequencing technology. The R. ripense genome assembly contained 319 contigs (506.7 Mb; N50 length: 2.5 Mb) and was assigned to the genetic map to establish 13 pseudomolecule sequences. On the other hand, the genome of R. kiyosumense was assembled into 32,308 contigs (601.9 Mb; N50 length: 245.7 kb). A total of 34,606 genes were predicted in the R. ripense genome, while 35,785 flower and 48,041 leaf transcript isoforms were identified in R. kiyosumense through Iso-Seq analysis. Overall, the genome sequence information generated in this study enhances our understanding of genome evolution in the Ericales and reveals the phylogenetic relationship of closely related species. This information will also facilitate the development of phenotypically attractive azalea cultivars.
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http://dx.doi.org/10.1093/dnares/dsab010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8435550PMC
September 2021

Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar 'Fielder'.

DNA Res 2021 Jun;28(3)

Kazusa DNA Research Institute, Kisarazu, 292-0818, Japan.

We have established a high-quality, chromosome-level genome assembly for the hexaploid common wheat cultivar 'Fielder', an American, soft, white, pastry-type wheat released in 1974 and known for its amenability to Agrobacterium tumefaciens-mediated transformation and genome editing. Accurate, long-read sequences were obtained using PacBio circular consensus sequencing with the HiFi approach. Sequence reads from 16 SMRT cells assembled using the hifiasm assembler produced assemblies with N50 greater than 20 Mb. We used the Omni-C chromosome conformation capture technique to order contigs into chromosome-level assemblies, resulting in 21 pseudomolecules with a cumulative size of 14.7 and 0.3 Gb of unanchored contigs. Mapping of published short reads from a transgenic wheat plant with an edited seed-dormancy gene, TaQsd1, identified four positions of transgene insertion into wheat chromosomes. Detection of guide RNA sequences in pseudomolecules provided candidates for off-target mutation induction. These results demonstrate the efficiency of chromosome-scale assembly using PacBio HiFi reads and their application in wheat genome-editing studies.
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http://dx.doi.org/10.1093/dnares/dsab008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8320877PMC
June 2021

Genomic Selection for F Hybrid Breeding in Strawberry ( × ).

Front Plant Sci 2021 4;12:645111. Epub 2021 Mar 4.

Department of Frontier Research and Development, Kazusa DNA Research Institute, Kisarazu, Japan.

Cultivated strawberry is the most widely consumed fruit crop in the world, and therefore, many breeding programs are underway to improve its agronomic traits such as fruit quality. Strawberry cultivars were vegetatively propagated through runners and carried a high risk of infection with viruses and insects. To solve this problem, the development of F hybrid seeds has been proposed as an alternative breeding strategy in strawberry. In this study, we conducted a potential assessment of genomic selection (GS) in strawberry F hybrid breeding. A total of 105 inbred lines were developed as candidate parents of strawberry F hybrids. In addition, 275 parental combinations were randomly selected from the 105 inbred lines and crossed to develop test F hybrids for GS model training. These populations were phenotyped for petiole length, leaf area, Brix, fruit hardness, and pericarp color. Whole-genome shotgun sequencing of the 105 inbred lines detected 20,811 single nucleotide polymorphism sites that were provided for subsequent GS analyses. In a GS model construction, inclusion of dominant effects showed a slight advantage in GS accuracy. In the across population prediction analysis, GS models using the inbred lines showed predictability for the test F hybrids and vice versa, except for Brix. Finally, the GS models were used for phenotype prediction of 5,460 possible F hybrids from 105 inbred lines to select F hybrids with high fruit hardness or high pericarp color. These F hybrids were developed and phenotyped to evaluate the efficacy of the GS. As expected, F hybrids that were predicted to have high fruit hardness or high pericarp color expressed higher observed phenotypic values than the F hybrids that were selected for other objectives. Through the analyses in this study, we demonstrated that GS can be applied for strawberry F hybrid breeding.
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http://dx.doi.org/10.3389/fpls.2021.645111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969887PMC
March 2021

Genomic region associated with pod color variation in pea (Pisum sativum).

G3 (Bethesda) 2021 05;11(5)

Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.

Pea (Pisum sativum) was chosen as the research material by Gregor Mendel to discover the laws of inheritance. Out of seven traits studied by Mendel, genes controlling three traits including pod shape, pod color, and flower position have not been identified to date. With the aim of identifying the genomic region controlling pod color, we determined the genome sequence of a pea line with yellow pods. Genome sequence reads obtained using a Nanopore sequencing technology were assembled into 117,981 contigs (3.3 Gb), with an N50 value of 51.2 kb. A total of 531,242 potential protein-coding genes were predicted, of which 519,349 (2.8 Gb) were located within repetitive sequences (2.8 Gb). The assembled sequences were ordered using a reference as a guide to build pseudomolecules. Subsequent genetic and association analyses led to the identification of a genomic region that controls pea pod color. DNA sequences at this genomic location and transcriptome profiles of green and yellow pod lines were analyzed, and genes encoding 3' exoribonucleases were selected as potential candidates controlling pod color. The results presented in this study are expected to accelerate pan-genome studies in pea and facilitate the identification of the gene controlling one of the traits studied by Mendel.
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http://dx.doi.org/10.1093/g3journal/jkab081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104947PMC
May 2021

Chromosome-scale genome assembly of Japanese pear (Pyrus pyrifolia) variety 'Nijisseiki'.

DNA Res 2021 May;28(2)

Department of Frontier Research and Development, Kazusa DNA Research Institute, Chiba, Japan.

We analyzed the genome sequence of a Japanese pear (Pyrus pyrifolia) to facilitate its genetics and genomics as well as breeding programs, in which a variety 'Nijisseiki' with superior flesh texture has been used as a parent for most Japanese pear cultivars. De novo assembly of long sequence reads covered 136× of the Japanese pear genome and generated 503.9 Mb contigs consisting of 114 sequences with an N50 value of 7.6 Mb. Contigs were assigned to Japanese pear genetic maps to establish 17 chromosome-scale sequences. In total, 44,876 high-confidence protein-encoding genes were predicted, 84.3% of which were supported by predicted genes and transcriptome data from Japanese pear relatives. As expected, evidence of genome-wide duplication was observed, consistent with related species. This is the first chromosome-scale genome sequence analysis reported for Japanese pear, and this resource will support breeding programs and provide new insights into the physiology and evolutionary history of Japanese pear.
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http://dx.doi.org/10.1093/dnares/dsab001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092371PMC
May 2021

Editorial: Achieving Nutritional Security and Food Safety Through Genomics-Based Breeding of Crops.

Front Nutr 2021 4;8:638845. Epub 2021 Feb 4.

Department of Frontier Research and Development, Kazusa DNA Research Institute, Kisarazu, Japan.

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http://dx.doi.org/10.3389/fnut.2021.638845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890118PMC
February 2021

Whole-genome sequence diversity and association analysis of 198 soybean accessions in mini-core collections.

DNA Res 2021 Jan;28(1)

Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.

We performed whole-genome Illumina resequencing of 198 accessions to examine the genetic diversity and facilitate the use of soybean genetic resources and identified 10 million single nucleotide polymorphisms and 2.8 million small indels. Furthermore, PacBio resequencing of 10 accessions was performed, and a total of 2,033 structure variants were identified. Genetic diversity and structure analysis congregated the 198 accessions into three subgroups (Primitive, World, and Japan) and showed the possibility of a long and relatively isolated history of cultivated soybean in Japan. Additionally, the skewed regional distribution of variants in the genome, such as higher structural variations on the R gene clusters in the Japan group, suggested the possibility of selective sweeps during domestication or breeding. A genome-wide association study identified both known and novel causal variants on the genes controlling the flowering period. Novel candidate causal variants were also found on genes related to the seed coat colour by aligning together with Illumina and PacBio reads. The genomic sequences and variants obtained in this study have immense potential to provide information for soybean breeding and genetic studies that may uncover novel alleles or genes involved in agronomically important traits.
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http://dx.doi.org/10.1093/dnares/dsaa032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934572PMC
January 2021

Genome sequence of Hydrangea macrophylla and its application in analysis of the double flower phenotype.

DNA Res 2021 Jan;28(1)

Tochigi Prefectural Agricultural Experimental Station, Utsunomiya, Tochigi 320-0002, Japan.

Owing to its high ornamental value, the double flower phenotype of hydrangea (Hydrangea macrophylla) is one of its most important traits. In this study, genome sequence information was obtained to explore effective DNA markers and the causative genes for double flower production in hydrangea. Single-molecule real-time sequencing data followed by a Hi-C analysis were employed. Two haplotype-phased sequences were obtained from the heterozygous genome of hydrangea. One assembly consisted of 3,779 scaffolds (2.256 Gb in length and N50 of 1.5 Mb), the other also contained 3,779 scaffolds (2.227 Gb in length, and N50 of 1.4 Mb). A total of 36,930 genes were predicted in the sequences, of which 32,205 and 32,222 were found in each haplotype. A pair of 18 pseudomolecules was constructed along with a high-density single-nucleotide polymorphism (SNP) genetic linkage map. Using the genome sequence data, and two F2 populations, the SNPs linked to double flower loci (djo and dsu) were discovered. DNA markers linked to djo and dsu were developed, and these could distinguish the recessive double flower allele for each locus, respectively. The LEAFY gene is a very likely candidate as the causative gene for dsu, since frameshift was specifically observed in the double flower accession with dsu.
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http://dx.doi.org/10.1093/dnares/dsaa026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934569PMC
January 2021

QTL analysis and GWAS of agronomic traits in sweetpotato ( L.) using genome wide SNPs.

Breed Sci 2020 Jun 19;70(3):283-291. Epub 2020 May 19.

Kyushu Okinawa Agricultural Research Center, NARO, 6651-2 Yokoichi-cho, Miyakonojo, Miyazaki 885-0091, Japan.

While sweetpotato ( L.) improvement has generally been done by field-based selection, molecular genetic studies on traits of interest, i.e., molecular markers are needed for enhancing the breeding program of this world's 7 most important crop, as such markers facilitate marker-assisted selection. Here, we performed a combined approach of QTLs analyses and GWAS of storage root β-carotene content (BC), dry-matter (DM) and starch content (SC) using the genetic linkage maps constructed with 5,952 and 5,640 SNPs obtained from F progenies between cultivars 'J-Red' and 'Choshu'. BC was negatively correlated with DM (r = -0.45) and SC (r = -0.51), while DM was positively correlated with SC (r = 0.94). In both parental maps, a total of five, two and five QTL regions on linkage groups 7 and 8 were associated with BC, DM and SC, respectively. In GWAS of BC, one strong signal ( = 1.04 × 10) was observed on linkage group 8, which co-located with one of the above QTL regions. The SNPs markers found here, particularly for β-carotene, would be useful base resources for future marker-assisted selection program with this trait.
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http://dx.doi.org/10.1270/jsbbs.19099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372034PMC
June 2020

Genetic Mapping in Autohexaploid Sweet Potato with Low-Coverage NGS-Based Genotyping Data.

G3 (Bethesda) 2020 08 5;10(8):2661-2670. Epub 2020 Aug 5.

Kazusa DNA Research Institute, Japan.

Next-generation sequencing (NGS)-based genotyping methods can generate numerous genetic markers in a single experiment and have contributed to plant genetic mapping. However, for high precision genetic analysis, the complicated genetic segregation mode in polyploid organisms requires high-coverage NGS data and elaborate analytical algorithms. In the present study, we propose a simple strategy for the genetic mapping of polyploids using low-coverage NGS data. The validity of the strategy was investigated using simulated data. Previous studies indicated that accurate allele dosage estimation from low-coverage NGS data (read depth < 40) is difficult. Therefore, we used allele dosage probabilities calculated from read counts in association analyses to detect loci associated with phenotypic variations. The allele dosage probabilities showed significant detection power, although higher allele dosage estimation accuracy resulted in higher detection power. On the contrary, differences in the segregation patterns between the marker and causal genes resulted in a drastic decrease in detection power even if the marker and casual genes were in complete linkage and the allele dosage estimation was accurate. These results indicated that the use of a larger number of markers is advantageous, even if the accuracy of allele dosage estimation is low. Finally, we applied the strategy for the genetic mapping of autohexaploid sweet potato () populations to detect loci associated with agronomic traits. Our strategy could constitute a cost-effective approach for preliminary experiments done performed to large-scale studies.
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http://dx.doi.org/10.1534/g3.120.401433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407471PMC
August 2020

Genome sequence and analysis of a Japanese radish (Raphanus sativus) cultivar named 'Sakurajima Daikon' possessing giant root.

DNA Res 2020 Apr;27(2)

Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.

Aim: The complex genome of a Japanese radish (Raphanus sativus) cultivar named 'Okute-Sakurajima' with an extremely large edible round root was analysed to explore its genomic characteristics.

Methods And Results: Single-molecule real-time technology was used to obtain long sequence reads to cover 60× of the genome. De novo assembly generated 504.5 Mb contigs consisting of 1,437 sequences with the N50 value of 1.2 Mb and included 94.1% of the core eukaryotic genes. Nine pseudomolecules, comprising 69.3% of the assembled contigs, were generated along with a high-density SNP genetic map. The sequence data thus established revealed the presence of structural variations and rearrangements in the Brassicaceae genomes.

Conclusion And Perspective: A total of 89,915 genes were identified in the 'Okute-Sakurajima' genome, 30,033 of which were newly found in this study. The genome information reported here will not only contribute to the establishment of a new resource for the radish genomics but also provide insights into the molecular mechanisms underlying formation of the giant root.
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http://dx.doi.org/10.1093/dnares/dsaa010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334891PMC
April 2020

The Ficus erecta genome aids Ceratocystis canker resistance breeding in common fig (F. carica).

Plant J 2020 06 24;102(6):1313-1322. Epub 2020 Feb 24.

Kazusa DNA Research Institute, Kisarazu, Japan.

Ficus erecta, a wild relative of the common fig (F. carica), is a donor of Ceratocystis canker resistance in fig breeding programmes. Interspecific hybridization followed by recurrent backcrossing is an effective method to transfer the resistance trait from wild to cultivated fig. However, this process is time consuming and labour intensive for trees, especially for gynodioecious plants such as fig. In this study, genome resources were developed for F. erecta to facilitate fig breeding programmes. The genome sequence of F. erecta was determined using single-molecule real-time sequencing technology. The resultant assembly spanned 331.6 Mb with 538 contigs and an N50 length of 1.9 Mb, from which 51 806 high-confidence genes were predicted. Pseudomolecule sequences corresponding to the chromosomes of F. erecta were established with a genetic map based on single nucleotide polymorphisms from double-digest restriction-site-associated DNA sequencing. Subsequent linkage analysis and whole-genome resequencing identified a candidate gene for the Ceratocystis canker resistance trait. Genome-wide genotyping analysis enabled the selection of female lines that possessed resistance and effective elimination of the donor genome from the progeny. The genome resources provided in this study will accelerate and enhance disease-resistance breeding programmes in fig.
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http://dx.doi.org/10.1111/tpj.14703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317799PMC
June 2020

Genome-wide association study overcomes the genome complexity in autohexaploid chrysanthemum and tags SNP markers onto the flower color genes.

Sci Rep 2019 09 26;9(1):13947. Epub 2019 Sep 26.

Institute of Vegetable and Floriculture Sciences, NARO, Tsukuba, Ibaraki, 305-0852, Japan.

The use of DNA markers has revolutionized selection in crop breeding by linkage mapping and QTL analysis, but major problems still remain for polyploid species where marker-assisted selection lags behind the situation in diploids because of its high genome complexity. To overcome the complex genetic mode in the polyploids, we investigated the development of a strategy of genome-wide association study (GWAS) using single-dose SNPs, which simplify the segregation patterns associated polyploids, with respect to the development of DNA markers. In addition, we employed biparental populations for the GWAS, wherein the SNP allele frequency could be predicted. The research investigated whether the method could be used to effectively develop DNA markers for petal color in autohexaploid chrysanthemum (Chrysanthemum morifolium; 2n = 6x = 54). The causal gene for this trait is already-known CmCCD4a encoding a dioxygenase which cleaves carotenoids in petals. We selected 9,219 single-dose SNPs, out of total 52,489 SNPs identified by dd-RAD-Seq, showing simplex (1 × 0) and double-simplex (1 × 1) inheritance pattern according to alternative allele frequency with respect to the SNP loci in the F population. GWAS, using these single-dose SNPs, discovered highly reproducible SNP markers tightly linked to the causal genes. This is the first report of a straightforward GWAS-based marker developing system for use in autohexaploid species.
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http://dx.doi.org/10.1038/s41598-019-50028-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763435PMC
September 2019

A pure line derived from a self-compatible Chrysanthemum seticuspe mutant as a model strain in the genus Chrysanthemum.

Plant Sci 2019 Oct 28;287:110174. Epub 2019 Jun 28.

Graduate school of Integrated Sciences for Life, Hiroshima University, 1-4-3, Kagamiyama, Higashi-Hiroshima, 739-8526, Japan. Electronic address:

Asteraceae is the largest family of angiosperms, comprising approximately 24,000 species. Molecular genetic studies of Asteraceae are essential for understanding plant diversity. Chrysanthemum morifolium is the most industrially important ornamental species in Asteraceae. Most cultivars of C. morifolium are autohexaploid and self-incompatible. These properties are major obstacles to the genetic analysis and modern breeding of C. morifolium. Furthermore, high genome heterogeneity complicates molecular biological analyses. In this study, we developed a model strain in the genus Chrysanthemum. C. seticuspe is a diploid species with a similar flowering property and morphology to C. morifolium and can be subjected to Agrobacterium-mediated transformation. We isolated a natural self-compatible mutant of C. seticuspe and established a pure line through repeated selfing and selection. The resultant strain, named Gojo-0, was favorable for genetic analyses, including isolation of natural and induced mutants, and facilitated molecular biological analysis, including whole genome sequencing, owing to the simplicity and homogeneity of its genome. Interspecific hybridization with Chrysanthemum species was possible, enabling molecular genetic analysis of natural interspecific variations. The accumulation of research results and resources using Gojo-0 as a platform is expected to promote molecular genetic studies on the genus Chrysanthemum and the genetic improvement of chrysanthemum cultivars.
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http://dx.doi.org/10.1016/j.plantsci.2019.110174DOI Listing
October 2019

Current status in whole genome sequencing and analysis of Ipomoea spp.

Plant Cell Rep 2019 Nov 29;38(11):1365-1371. Epub 2019 Aug 29.

Kazusa DNA Research Institute, Kazusa-Kamatari 2-6-7, Kisarazu, Chiba, Japan.

The recent advances of next-generation sequencing have made it possible to construct reference genome sequences in divergent species. However, de novo assembly at the chromosome level remains challenging in polyploid species, due to the existence of more than two pairs of homoeologous chromosomes in one nucleus. Cultivated sweet potato (Ipomoea batatas (L.) Lam) is a hexaploid species with 90 chromosomes (2n = 6X = 90). Although the origin of sweet potato is also still under discussion, diploid relative species, I. trifida and I. triloba have been considered as one of the most possible progenitors. In this manuscript, we review the recent results and activities of whole-genome sequencing in the genus Ipomoea series Batatas, I. trifida, I. triloba and sweet potato (I. batatas). Most of the results of genome assembly suggest that the genomes of sweet potato consist of two pairs and four pairs of subgenomes, i.e., B1B1B2B2B2B2. The results also revealed the relation between sweet potato and other Ipomoea species. Together with the development of bioinformatics approaches, the large-scale publicly available genome and transcript sequence resources and international genome sequencing streams are expected to promote the genome sequence dissection in sweet potato.
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http://dx.doi.org/10.1007/s00299-019-02464-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797701PMC
November 2019

Development of molecular markers associated with resistance to Meloidogyne incognita by performing quantitative trait locus analysis and genome-wide association study in sweetpotato.

DNA Res 2019 Oct;26(5):399-409

Graduate School of Environmental and Life Science, Okayama University, Okayama, Okayama, Japan.

The southern root-knot nematode, Meloidogyne incognita, is a pest that decreases yield and the quality of sweetpotato [Ipomoea batatas (L.) Lam.]. There is a demand to produce resistant cultivars and develop DNA markers to select this trait. However, sweetpotato is hexaploid, highly heterozygous, and has an enormous genome (∼3 Gb), which makes genetic linkage analysis difficult. In this study, a high-density linkage map was constructed based on retrotransposon insertion polymorphism, simple sequence repeat, and single nucleotide polymorphism markers. The markers were developed using F1 progeny between J-Red, which exhibits resistance to multiple races of M. incognita, and Choshu, which is susceptible to multiple races of such pest. Quantitative trait locus (QTL) analysis and a genome-wide association study detected highly effective QTLs for resistance against three races, namely, SP1, SP4, and SP6-1, in the Ib01-6 J-Red linkage group. A polymerase chain reaction marker that can identify genotypes based on single nucleotide polymorphisms located in this QTL region can discriminate resistance from susceptibility in the F1 progeny at a rate of 70%. Thus, this marker could be helpful in selecting sweetpotato cultivars that are resistant to multiple races of M. incognita.
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http://dx.doi.org/10.1093/dnares/dsz018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796513PMC
October 2019

Genome-Wide Association Studies (GWAS) for Yield and Weevil Resistance in Sweet potato (Ipomoea batatas (L.) Lam).

Plant Cell Rep 2019 Nov 24;38(11):1383-1392. Epub 2019 Jul 24.

Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushimanaka Kitaku, Okayama, Okayama, 700-8530, Japan.

Key Message: We apply the GWAS to sweet potato genome, and identified the SNPs associated with yield and weevil resistance. The sweet potato (Ipomoea batatas (L.) Lam) is a highly heterozygous, outcrossing, polyploid species, which presents challenges for genetic analysis. Therefore, we considered that genome-wide association studies (GWAS) may be applied to the study of the sweet potato genome. The yield of two sweet potato varieties [Purple Sweet Lord (PSL) and 90IDN-47] was assessed at two locations (Kumamoto and Okinawa prefectures) in Japan in 2013 and the yield scores were used for GWAS. The results showed that there were several single nucleotide polymorphisms (SNP) above the significance thresholds in PSL; two peaks were detected in Kumamoto and Okinawa on the Ib03-3 and Ib01-4 linkage groups of PSL, respectively. As for 90IDN-47, one relatively high peak was detected in Kumamoto on the Ib13-8 linkage group. Interestingly, although high peaks above significance thresholds were detected in Kumamoto and Okinawa in PSL, the peaks were located in different linkage groups. This result suggests that the genetic regions controlling yield may change in response to environmental conditions. Additionally, we investigated the degree of weevil damage to the plants, which is the greatest problem in sweet potato cultivation in Okinawa. In this experiment, no SNPs were identified above the significance thresholds. However, one relatively high peak was found in the 90IDN-47 genotype, which showed resistance to weevils. On the other hand, one relatively high peak was also detected in the PSL genotype, which showed susceptibility to weevils. These results suggest that two regions could affect weevil resistance and may contain the gene(s) controlling weevil resistance.
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http://dx.doi.org/10.1007/s00299-019-02445-7DOI Listing
November 2019

Phased genome sequence of an interspecific hybrid flowering cherry, 'Somei-Yoshino' (Cerasus × yedoensis).

DNA Res 2019 Oct;26(5):379-389

Kazusa DNA Research Institute, Japan.

We report the phased genome sequence of an interspecific hybrid, the flowering cherry 'Somei-Yoshino' (Cerasus × yedoensis). The sequence data were obtained by single-molecule real-time sequencing technology, split into two subsets based on genome information of the two probable ancestors, and assembled to obtain two haplotype phased genome sequences of the interspecific hybrid. The resultant genome assembly consisting of the two haplotype sequences spanned 690.1 Mb with 4,552 contigs and an N50 length of 1.0 Mb. We predicted 95,076 high-confidence genes, including 94.9% of the core eukaryotic genes. Based on a high-density genetic map, we established a pair of eight pseudomolecule sequences, with highly conserved structures between the two haplotype sequences with 2.4 million sequence variants. A whole genome resequencing analysis of flowering cherries suggested that 'Somei-Yoshino' might be derived from a cross between C. spachiana and either C. speciosa or its relatives. A time-course transcriptome analysis of floral buds and flowers suggested comprehensive changes in gene expression in floral bud development towards flowering. These genome and transcriptome data are expected to provide insights into the evolution and cultivation of flowering cherry and the molecular mechanism underlying flowering.
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http://dx.doi.org/10.1093/dnares/dsz016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796508PMC
October 2019

Hayai-Annotation Plants: an ultra-fast and comprehensive functional gene annotation system in plants.

Bioinformatics 2019 11;35(21):4427-4429

Laboratory of Plant Genetics and Genomics, Department of Frontier Research and Development, Kazusa DNA Research Institute, Kazusa-kamatari, Chiba, Japan.

Summary: Hayai-Annotation Plants is a browser-based interface for an ultra-fast and accurate functional gene annotation system for plant species using R. The pipeline combines the sequence-similarity searches, using USEARCH against UniProtKB (taxonomy Embryophyta), with a functional annotation step. Hayai-Annotation Plants provides five layers of annotation: i) protein name; ii) gene ontology terms consisting of its three main domains (Biological Process, Molecular Function and Cellular Component); iii) enzyme commission number; iv) protein existence level; and v) evidence type. It implements a new algorithm that gives priority to protein existence level to propagate GO and EC information and annotated Arabidopsis thaliana representative peptide sequences (Araport11) within 5 min at the PC level.

Availability And Implementation: The software is implemented in R and runs on Macintosh and Linux systems. It is freely available at https://github.com/kdri-genomics/Hayai-Annotation-Plants under the GPLv3 license.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821316PMC
November 2019

A reference-grade wild soybean genome.

Nat Commun 2019 03 14;10(1):1216. Epub 2019 Mar 14.

Centre for Soybean Research of the State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region, China.

Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, with a final assembled genome size of 1013.2 Mb and a contig N50 of 3.3 Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz trypsin inhibitor (KTI) genes. Such findings illustrate the power of this assembly in the analysis of large structural variations in soybean germplasm collections. The wild soybean genome assembly has wide applications in comparative genomic and evolutionary studies, as well as in crop breeding and improvement programs.
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http://dx.doi.org/10.1038/s41467-019-09142-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418295PMC
March 2019

De novo whole-genome assembly in Chrysanthemum seticuspe, a model species of Chrysanthemums, and its application to genetic and gene discovery analysis.

DNA Res 2019 Jun;26(3):195-203

Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.

Cultivated chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most economically important ornamental crops grown worldwide. It has a complex hexaploid genome (2n = 6x = 54) and large genome size. The diploid Chrysanthemum seticuspe is often used as a model of cultivated chrysanthemum, since the two species are closely related. To expand our knowledge of the cultivated chrysanthemum, we here performed de novo whole-genome assembly in C. seticuspe using the Illumina sequencing platform. XMRS10, a C. seticuspe accession developed by five generations of self-crossing from a self-compatible strain, AEV2, was used for genome sequencing. The 2.72 Gb of assembled sequences (CSE_r1.0), consisting of 354,212 scaffolds, covered 89.0% of the 3.06 Gb C. seticuspe genome estimated by k-mer analysis. The N50 length of scaffolds was 44,741 bp. For protein-encoding genes, 71,057 annotated genes were deduced (CSE_r1.1_cds). Next, based on the assembled genome sequences, we performed linkage map construction, gene discovery and comparative analyses for C. seticuspe and cultivated chrysanthemum. The generated C. seticuspe linkage map revealed skewed regions in segregation on the AEV2 genome. In gene discovery analysis, candidate flowering-related genes were newly found in CSE_r1.1_cds. Moreover, single nucleotide polymorphism identification and annotation on the C. × morifolium genome showed that the C. seticuspe genome was applicable to genetic analysis in cultivated chrysanthemums. The genome sequences assembled herein are expected to contribute to future chrysanthemum studies. In addition, our approach demonstrated the usefulness of short-read genome assembly and the importance of choosing an appropriate next genome sequencing technology based on the purpose of the post-genome analysis.
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http://dx.doi.org/10.1093/dnares/dsy048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589549PMC
June 2019

Mapping of a novel clubroot resistance QTL using ddRAD-seq in Chinese cabbage (Brassica rapa L.).

BMC Plant Biol 2019 Jan 8;19(1):13. Epub 2019 Jan 8.

Department of Horticulture, Sunchon National University, Suncheon, 57922, Republic of Korea.

Background: Plasmodiophora brassicae is a soil-borne plant pathogen that causes clubroot disease, which results in crop yield loss in cultivated Brassica species. Here, we investigated whether a quantitative trait locus (QTL) in B. rapa might confer resistance to a Korean P. brassicae pathotype isolate, Seosan. We crossed resistant and susceptible parental lines and analyzed the segregation pattern in a F population of 348 lines. We identified and mapped a novel clubroot resistance QTL using the same mapping population that included susceptible Chinese cabbage and resistant turnip lines. Forty-five resistant and 45 susceptible F lines along with their parental lines were used for double digest restriction site-associated DNA sequencing (ddRAD-seq). High resolution melting (HRM)-based validation of SNP positions was conducted to confirm the novel locus.

Results: A 3:1 ratio was observed for resistant: susceptible genotypes, which is in accordance with Mendelian segregation. ddRAD-seq identified a new locus, CRs, on chromosome A08 that was different from the clubroot resistance (CR) locus, Crr1. HRM analysis validated SNP positions and constricted CRs region. Four out of seventeen single nucleotide polymorphisms (SNPs) positions were within a 0.8-Mb region that included three NBS-LRR candidate genes but not Crr1.

Conclusion: The newly identified CRs locus is a novel clubroot resistance locus, as the cultivar Akimeki bears the previously known Crr1 locus but remains susceptible to the Seosan isolate. These results could be exploited to develop molecular markers to detect Seosan-resistant genotypes and develop resistant Chinese cabbage cultivars.
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http://dx.doi.org/10.1186/s12870-018-1615-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325862PMC
January 2019

SNP discovery of Korean short day onion inbred lines using double digest restriction site-associated DNA sequencing.

PLoS One 2018 7;13(8):e0201229. Epub 2018 Aug 7.

Department of Horticulture, Sunchon National University, Suncheon, Jeonnam, South Korea.

Onion (Allium cepa L.) is an economically important vegetable crop around the world. Genetic and genomic research into various onion accessions will provide insights into the onion genome to enhance breeding strategies and improve crops. However, the onion's large genome size means that studies of molecular markers are limited in onion. This study aimed to discover high quality single nucleotide polymorphisms (SNPs) from 192 onion inbred lines relating to short-day cultivation in Korea. Paired-end (PE) double digested restriction site-associated DNA sequencing (ddRAD-seq) was used to discover SNPs in onion. A total of 538,973,706 reads (25.9 GB), with an average of 2,658,491 high-quality reads, were generated using ddRAD-seq. With stringent filtering, 1904 SNPs were discovered based on onion reference scaffolds. Further, population structure and genetic relationship studies suggested that two well-differentiated sub-populations exist in onion lines. SNP-associated flanking sequences were also compared with a public non-redundant database for gene ontology and pathway analysis. To our knowledge, this is the first report to identify high-quality SNPs in onion based on reference sequences using the ddRAD-seq platform. The SNP markers identified will be useful for breeders and the research community to deepen their understanding, enhance breeding programs, and support the management of onion genomic resources.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080773PMC
February 2019
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