Whole-genome sequence analysis of mutations in rice plants regenerated from zygotes, mature embryos, and immature embryos.

Somaclonal variation was studied by whole-genome sequencing in rice plants (Oryza sativa L., 'Nipponbare') regenerated from the zygotes, mature embryos, and immature embryos of a single mother plant. The mother plant and its seed-propagated progeny were also sequenced. A total of 338 variants of the mother plant sequence were detected in the progeny, and mean values ranged from 9.0 of the seed-propagated plants to 37.4 of regenerants from mature embryos. The natural mutation rate of 1.2 × 10-8 calculated using the variants in the seed-propagated plants was consistent with the values reported previously. The ratio of single nucleotide variants (SNVs) among the variants in the seed-propagated plants was 91.1%, which is higher than 56.1% previously reported, and not significantly different from those in the regenerants. Overall, the ratio of transitions to transversions of SNVs was lower in the regenerants as shown previously. Plants regenerated from mature embryos had significantly more variants than different progeny types. Therefore, using zygotes and immature embryos can reduce somaclonal variation during the genetic manipulation of rice.

Plant GARDEN: a portal website for cross-searching between different types of genomic and genetic resources in a wide variety of plant species.

BACKGROUND: Plant genome information is fundamental to plant research and development. Along with the increase in the number of published plant genomes, there is a need for an efficient system to retrieve various kinds of genome-related information from many plant species across plant kingdoms. Various plant databases have been developed, but no public database covers both genomic and genetic resources over a wide range of plant species. MAIN BODY: We have developed a plant genome portal site, Plant GARDEN (Genome And Resource Database Entry: https://plantgarden.jp/en/index ), to provide diverse information related to plant genomics and genetics in divergent plant species. Elasticsearch is used as a search engine, and cross-keyword search across species is available. Web-based user interfaces (WUI) for PCs and tablet computers were independently developed to make data searches more convenient. Several types of data are stored in Plant GARDEN: reference genomes, gene sequences, PCR-based DNA markers, trait-linked DNA markers identified in genetic studies, SNPs, and in/dels on publicly available sequence read archives (SRAs). The data registered in Plant GARDEN as of March 2023 included 304 assembled genome sequences, 11,331,614 gene sequences, 419,132 DNA markers, 8,225 QTLs, and 5,934 SNP lists (gvcf files). In addition, we have re-annotated all the genes registered in Plant GARDEN by using a functional annotation tool, Hayai-Annotation, to compare the orthologous relationships among genes. CONCLUSION: The aim of Plant GARDEN is to provide plant genome information for use in the fields of plant science as well as for plant-based industries, education, and other relevant areas. Therefore, we have designed a WUI that allows a diverse range of users to access such information in an easy-to-understand manner. Plant GARDEN will eventually include a wide range of plant species for which genome sequences are assembled, and thus the number of plant species in the database will continue to expand. We anticipate that Plant GARDEN will promote the understanding of genomes and gene diversity by facilitating comparisons of the registered sequences.

An improved reference genome for Trifolium subterraneum L. provides insight into molecular diversity and intra-specific phylogeny.

Subterranean clover (Trifolium subterraneum L., Ts) is a geocarpic, self-fertile annual forage legume with a compact diploid genome (n = x = 8, 544 Mb/1C). Its resilience and climate adaptivity have made it an economically important species in Mediterranean and temperate zones. Using the cultivar Daliak, we generated higher resolution sequence data, created a new genome assembly TSUd_3.0, and conducted molecular diversity analysis for copy number variant (CNV) and single-nucleotide polymorphism (SNP) among 36 cultivars. TSUd_3.0 substantively improves prior genome assemblies with new Hi-C and long-read sequence data, covering 531 Mb, containing 41,979 annotated genes and generating a 94.4% BUSCO score. Comparative genomic analysis among select members of the tribe Trifolieae indicated TSUd 3.0 corrects six assembly-error inversion/duplications and confirmed phylogenetic relationships. Its synteny with T. pratense, T. repens, Medicago truncatula and Lotus japonicus genomes were assessed, with the more distantly related T. repens and M. truncatula showing higher levels of co-linearity with Ts than between Ts and its close relative T. pratense. Resequencing of 36 cultivars discovered 7,789,537 SNPs subsequently used for genomic diversity assessment and sequence-based clustering. Heterozygosity estimates ranged from 1% to 21% within the 36 cultivars and may be influenced by admixture. Phylogenetic analysis supported subspecific genetic structure, although it indicates four or five groups, rather than the three recognized subspecies. Furthermore, there were incidences where cultivars characterized as belonging to a particular subspecies clustered with another subspecies when using genomic data. These outcomes suggest that further investigation of Ts sub-specific classification using molecular and morpho-physiological data is needed to clarify these relationships. This upgraded reference genome, complemented with comprehensive sequence diversity analysis of 36 cultivars, provides a platform for future gene functional analysis of key traits, and genome-based breeding strategies for climate adaptation and agronomic performance. Pangenome analysis, more in-depth intra-specific phylogenomic analysis using the Ts core collection, and functional genetic and genomic studies are needed to further augment knowledge of Trifolium genomes.

Chromosome-scale genome assembly of Eustoma grandiflorum, the first complete genome sequence in the genus Eustoma.

Eustoma grandiflorum (Raf.) Shinn. is an annual herbaceous plant native to the southern United States, Mexico, and the Greater Antilles. It has a large flower with a variety of colors and is an important flower crop. In this study, we established a chromosome-scale de novo assembly of E. grandiflorum genome sequences by integrating four genomic and genetic approaches: (1) Pacific Biosciences (PacBio) Sequel deep sequencing, (2) error correction of the assembly by Illumina short reads, (3) scaffolding by chromatin conformation capture sequencing (Hi-C), and (4) genetic linkage maps derived from an F2 mapping population. Thirty-six pseudomolecules and 64 unplaced scaffolds were created, with a total length of 1,324.8 Mb. A total of 36,619 genes were predicted on the genome as high-confidence genes. A comparison of genome structure between E. grandiflorum and C. canephora or O. pumila suggested whole-genome duplication after the divergence between the families Gentianaceae and Rubiaceae. Phylogenetic analysis with single-copy genes suggested that the divergence time between Gentianaceae and Rubiaceae was 74.94 MYA. Genetic diversity analysis was performed for nine commercial E. grandiflorum varieties bred in Japan, from which 254,205 variants were identified. This first report on the construction of a reference genome sequence in the genus Eustoma is expected to contribute to genetic and genomic studies in this genus and in the family Gentianaceae.

De novo whole-genome assembly in an interspecific hybrid table grape, 'Shine Muscat'.

The first genome sequence of an interspecific grape hybrid (Vitis labruscana × Vitis vinifera), 'Shine Muscat', an elite table grape cultivar bred in Japan, is presented. The resultant genome assemblies included two types of sequences: a haplotype-phased sequence of the highly heterozygous genomes and an unphased sequence representing a 'pseudo-haploid' genome. The unphased sequences, assembled to the chromosome level with Hi-C reads, spanned 488.97 Mb in length, 99.1% of the estimated genome size, with 4,595 scaffold sequences and a 23.9-Mb N50 length. The phased sequences had 15,650 scaffolds spanning 1.0 Gb and a 4.2-Mb N50 length. 32,827 high-confidence genes were predicted on the unphased genomes. Clustering analysis of the 'Shine Muscat' gene sequences with three other Vitis species and Arabidopsis indicated that 11,279 orthologous gene clusters were common to Vitis spp. and Arabidopsis, 4,385 were Vitis specific, and 234 were 'Shine Muscat' specific. Whole-genome resequencing was also performed for the parental lines of 'Shine Muscat', Akitsu-21 and 'Hakunan', and parental-specific copy number variations were identified. The obtained genome resources provide new insights that could assist in cultivation and breeding strategies to produce high-quality table grapes.

DNA marker for resistance to Puccinia horiana in chrysanthemum (Chrysanthemum morifolium Ramat.) "Southern Pegasus".

White rust caused by Puccinia horiana Henn. adversely affects chrysanthemum (Chrysanthemum morifolium 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 P. horiana. 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 F1 biparental population. These F1 individuals segregated in a 1:1 ratio of resistant to susceptible. Twenty-one simplex SNPs were significantly associated with P. horiana 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 P. horiana 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 P. horiana resistance in chrysanthemum.

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

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.

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

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.

Climate Clever Clovers: New Paradigm to Reduce the Environmental Footprint of Ruminants by Breeding Low Methanogenic Forages Utilizing Haplotype Variation.

Mitigating methane production by ruminants is a significant challenge to global livestock production. This research offers a new paradigm to reduce methane emissions from ruminants by breeding climate-clever clovers. We demonstrate wide genetic diversity for the trait methanogenic potential in Australia's key pasture legume, subterranean clover (Trifolium subterraneum L.). In a bi-parental population the broadsense heritability in methanogenic potential was moderate (H2 = 0.4) and allelic variation in a region of Chr 8 accounted for 7.8% of phenotypic variation. In a genome-wide association study we identified four loci controlling methanogenic potential assessed by an in vitro fermentation system. Significantly, the discovery of a single nucleotide polymorphism (SNP) on Chr 5 in a defined haplotype block with an upstream putative candidate gene from a plant peroxidase-like superfamily (TSub_g18548) and a downstream lectin receptor protein kinase (TSub_g18549) provides valuable candidates for an assay for this complex trait. In this way haplotype variation can be tracked to breed pastures with reduced methanogenic potential. Of the quantitative trait loci candidates, the DNA-damage-repair/toleration DRT100-like protein (TSub_g26967), linked to avoid the severity of DNA damage induced by secondary metabolites, is considered central to enteric methane production, as are disease resistance (TSub_g26971, TSub_g26972, and TSub_g18549) and ribonuclease proteins (TSub_g26974, TSub_g26975). These proteins are good pointers to elucidate the genetic basis of in vitro microbial fermentability and enteric methanogenic potential in subterranean clover. The genes identified allow the design of a suite of markers for marker-assisted selection to reduce rumen methane emission in selected pasture legumes. We demonstrate the feasibility of a plant breeding approach without compromising animal productivity to mitigate enteric methane emissions, which is one of the most significant challenges to global livestock production.

Discrimination of candidate subgenome-specific loci by linkage map construction with an S1 population of octoploid strawberry (Fragaria × ananassa).

BACKGROUND: The strawberry, Fragaria × ananassa, is an allo-octoploid (2n = 8x = 56) and outcrossing species. Although it is the most widely consumed berry crop in the world, its complex genome structure has hindered its genetic and genomic analysis, and thus discrimination of subgenome-specific loci among the homoeologous chromosomes is needed. In the present study, we identified candidate subgenome-specific single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) loci, and constructed a linkage map using an S1 mapping population of the cultivar 'Reikou' with an IStraw90 Axiom® SNP array and previously published SSR markers. RESULTS: The 'Reikou' linkage map consisted of 11,574 loci (11,002 SNPs and 572 SSR loci) spanning 2816.5 cM of 31 linkage groups. The 11,574 loci were located on 4738 unique positions (bin) on the linkage map. Of the mapped loci, 8999 (8588 SNPs and 411 SSR loci) showed a 1:2:1 segregation ratio of AA:AB:BB allele, which suggested the possibility of deriving loci from candidate subgenome-specific sequences. In addition, 2575 loci (2414 SNPs and 161 SSR loci) showed a 3:1 segregation of AB:BB allele, indicating they were derived from homoeologous genomic sequences. Comparative analysis of the homoeologous linkage groups revealed differences in genome structure among the subgenomes. CONCLUSIONS: Our results suggest that candidate subgenome-specific loci are randomly located across the genomes, and that there are small- to large-scale structural variations among the subgenomes. The mapped SNPs and SSR loci on the linkage map are expected to be seed points for the construction of pseudomolecules in the octoploid strawberry.

Draft genome sequence of subterranean clover, a reference for genus Trifolium.

Clovers (genus Trifolium) are widely cultivated across the world as forage legumes and make a large contribution to livestock feed production and soil improvement. Subterranean clover (T. subterraneum L.) is well suited for genomic and genetic studies as a reference species in the Trifolium genus, because it is an annual with a simple genome structure (autogamous and diploid), unlike the other economically important perennial forage clovers, red clover (T. pratense) and white clover (T. repens). This report represents the first draft genome sequence of subterranean clover. The 471.8 Mb assembled sequence covers 85.4% of the subterranean clover genome and contains 42,706 genes. Eight pseudomolecules of 401.1 Mb in length were constructed, based on a linkage map consisting of 35,341 SNPs. The comparative genomic analysis revealed that different clover chromosomes showed different degrees of conservation with other Papilionoideae species. These results provide a reference for genetic and genomic analyses in the genus Trifolium and new insights into evolutionary divergence in Papilionoideae species.

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.

Dissection of the octoploid strawberry genome by deep sequencing of the genomes of Fragaria species.

Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species.

Construction of an integrated high density simple sequence repeat linkage map in cultivated strawberry (Fragaria × ananassa) and its applicability.

The cultivated strawberry (Fragaria × ananassa) is an octoploid (2n = 8x = 56) of the Rosaceae family whose genomic architecture is still controversial. Several recent studies support the AAA'A'BBB'B' model, but its complexity has hindered genetic and genomic analysis of this important crop. To overcome this difficulty and to assist genome-wide analysis of F. × ananassa, we constructed an integrated linkage map by organizing a total of 4474 of simple sequence repeat (SSR) markers collected from published Fragaria sequences, including 3746 SSR markers [Fragaria vesca expressed sequence tag (EST)-derived SSR markers] derived from F. vesca ESTs, 603 markers (F. × ananassa EST-derived SSR markers) from F. × ananassa ESTs, and 125 markers (F. × ananassa transcriptome-derived SSR markers) from F. × ananassa transcripts. Along with the previously published SSR markers, these markers were mapped onto five parent-specific linkage maps derived from three mapping populations, which were then assembled into an integrated linkage map. The constructed map consists of 1856 loci in 28 linkage groups (LGs) that total 2364.1 cM in length. Macrosynteny at the chromosome level was observed between the LGs of F. × ananassa and the genome of F. vesca. Variety distinction on 129 F. × ananassa lines was demonstrated using 45 selected SSR markers.

Comparative Genetic Mapping and Discovery of Linkage Disequilibrium Across Linkage Groups in White Clover (Trifolium repens L.).

White clover (Trifolium repens L.) is an allotetraploid species (2n = 4X = 32) that is widely distributed in temperate regions and cultivated as a forage legume. In this study, we developed expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers, constructed linkage maps, and performed comparative mapping with other legume species. A total of 7982 ESTs that could be assembled into 5400 contigs and 2582 singletons were generated. Using the EST sequences that were obtained, 1973 primer pairs to amplify EST-derived SSR markers were designed and used for linkage analysis of 188 F(1) progenies, which were generated by a cross between two Japanese plants, '273-7' and 'T17-349,' with previously published SSR markers. An integrated linkage map was constructed by combining parental-specific maps, which consisted of 1743 SSR loci on 16 homeologous linkage groups with a total length of 2511 cM. The primer sequences of the developed EST-SSR markers and their map positions are available on http://clovergarden.jp/. Linkage disequilibrium (LD) was observed on 9 of 16 linkage groups of a parental-specific map. The genome structures were compared among white clover, red clover (T. pratense L.), Medicago truncatula, and Lotus japonicus. Macrosynteny was observed across the four legume species. Surprisingly, the comparative genome structure between white clover and M. truncatula had a higher degree of conservation than that of the two clover species.

Will genomic selection be a practical method for plant breeding?

BACKGROUND: Genomic selection or genome-wide selection (GS) has been highlighted as a new approach for marker-assisted selection (MAS) in recent years. GS is a form of MAS that selects favourable individuals based on genomic estimated breeding values. Previous studies have suggested the utility of GS, especially for capturing small-effect quantitative trait loci, but GS has not become a popular methodology in the field of plant breeding, possibly because there is insufficient information available on GS for practical use. SCOPE: In this review, GS is discussed from a practical breeding viewpoint. Statistical approaches employed in GS are briefly described, before the recent progress in GS studies is surveyed. GS practices in plant breeding are then reviewed before future prospects are discussed. CONCLUSIONS: Statistical concepts used in GS are discussed with genetic models and variance decomposition, heritability, breeding value and linear model. Recent progress in GS studies is reviewed with a focus on empirical studies. For the practice of GS in plant breeding, several specific points are discussed including linkage disequilibrium, feature of populations and genotyped markers and breeding scheme. Currently, GS is not perfect, but it is a potent, attractive and valuable approach for plant breeding. This method will be integrated into many practical breeding programmes in the near future with further advances and the maturing of its theory.