Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.

A CLAVATA3-like Gene Acts as a Gynoecium Suppression Function in White Campion.

How do separate sexes originate and evolve? Plants provide many opportunities to address this question as they have diverse mating systems and separate sexes (dioecy) that evolved many times independently. The classic "two-factor" model for evolution of separate sexes proposes that males and females can evolve from hermaphrodites via the spread of male and female sterility mutations that turn hermaphrodites into females and males, respectively. This widely accepted model was inspired by early genetic work in dioecious white campion (Silene latifolia) that revealed the presence of two sex-determining factors on the Y-chromosome, though the actual genes remained unknown. Here, we report identification and functional analysis of the putative sex-determining gene in S. latifolia, corresponding to the gynoecium suppression factor (GSF). We demonstrate that GSF likely corresponds to a Y-linked CLV3-like gene that is specifically expressed in early male flower buds and encodes the protein that suppresses gynoecium development in S. latifolia. Interestingly, GSFY has a dysfunctional X-linked homolog (GSFX) and their synonymous divergence (dS = 17.9%) is consistent with the age of sex chromosomes in this species. We propose that female development in S. latifolia is controlled via the WUSCHEL-CLAVATA feedback loop, with the X-linked WUSCHEL-like and Y-linked CLV3-like genes, respectively. Evolution of dioecy in the S. latifolia ancestor likely involved inclusion of ancestral GSFY into the nonrecombining region on the nascent Y-chromosome and GSFX loss of function, which resulted in disbalance of the WUSCHEL-CLAVATA feedback loop between the sexes and ensured gynoecium suppression in males.

Development and chromosomal characterization of interspecific hybrids between common buckwheat (Fagopyrum esculentum) and a related perennial species (F. cymosum).

Common buckwheat (Fagopyrum esculentum) is an annual self-incompatible plant that is widely grown. The genus Fagopyrum comprises more than 20 species, including F. cymosum, a perennial that, unlike common buckwheat, is highly resistant to excess water. In this study, we developed interspecific hybrids between F. esculentum and F. cymosum via embryo rescue, to improve undesirable traits of common buckwheat, such as low tolerance to excess water. The interspecific hybrids were confirmed by genomic in situ hybridization (GISH). We also developed DNA markers to confirm the identity of the hybrids and if genes derived from each genome were inherited by the next generation. Observations of pollen indicated that the interspecific hybrids were essentially sterile. Unpaired chromosomes and abnormal segregation during meiosis were likely responsible for the pollen sterility of the hybrids. These findings could facilitate buckwheat breeding to produce lines that can withstand harsh environments with wild or related species in the genus Fagopyrum.

Targeted amplicon sequencing + next-generation sequencing-based bulked segregant analysis identified genetic loci associated with preharvest sprouting tolerance in common buckwheat (Fagopyrum esculentum).

BACKGROUND: Common buckwheat (2n = 2x = 16) is an outcrossing pseudocereal whose seeds contain abundant nutrients and potential antioxidants. As these beneficial compounds are damaged by preharvest sprouting (PHS) and PHS is likely to increase with global warming, it is important to find efficient ways to develop new PHS-tolerant lines. However, genetic loci and selection markers associated with PHS in buckwheat have not been reported. RESULTS: By next-generation sequencing (NGS) of whole-genome of parental lines, we developed a genome-wide set of 300 markers. By NGS- based bulked segregant analysis (NGS-BSA), we developed 100 markers linked to PHS tolerance. To confirm the effectiveness of marker development from NGS-BSA data, we developed 100 markers linked to the self-compatibility (SC) trait from previous NGS-BSA data. Using these markers, we developed genetic maps with AmpliSeq technology, which can quickly detect polymorphisms by amplicon-based multiplex targeted NGS, and performed quantitative trait locus (QTL) analysis for PHS tolerance in combination with NGS-BSA. QTL analysis detected two major and two minor QTLs for PHS tolerance in a segregating population developed from a cross between the PHS-tolerant 'Kyukei 29' and the self-compatible susceptible 'Kyukei SC7'. We found different major and minor QTLs in other segregating populations developed from the PHS-tolerant lines 'Kyukei 28' and 'NARO-FE-1'. Candidate markers linked to PHS developed by NGS-BSA were located near these QTL regions. We also investigated the effectiveness of markers linked to these QTLs for selection of PHS-tolerant lines among other segregating populations. CONCLUSIONS: We efficiently developed genetic maps using a method combined with AmpliSeq technology and NGS-BSA, and detected QTLs associated with preharvest sprouting tolerance in common buckwheat. This is the first report to identify QTLs for PHS tolerance in buckwheat. Our marker development system will accelerate genetic research and breeding in common buckwheat.

Genetic and genomic research for the development of an efficient breeding system in heterostylous self-incompatible common buckwheat (Fagopyrum esculentum).

Common buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is an annual crop that is cultivated widely around the world and contains an abundance of nutrients and bioactive compounds. However, the yield of buckwheat is low compared to that of other major crops, and it contains proteins that cause allergic reactions in some people. Much research has aimed to improve or eliminate these undesirable traits, and some major advances have recently been made. Here, we review recent advances in buckwheat breeding materials, tools, and methods, including the development of self-compatible lines, genetic maps, a buckwheat genome database, and an efficient breeding strategy. We also describe emerging breeding methods for high-value lines.

History of the progressive development of genetic marker systems for common buckwheat.

Genotyping is an essential procedure for identifying agronomically useful genes and analyzing population structure. Various types of genetic marker systems have been developed in common buckwheat (Fagopyrum esculentum Moench). In the 1980s, morphological and allozyme markers were used to construct linkage maps. Until the early 2000s, allozyme markers were widely used in population genetics studies. Such studies demonstrated that cultivated common buckwheat likely originated in the Sanjiang area of China. In the late 1990s and early 2000s, advances in PCR technology led to the development of various DNA marker systems for use in linkage mapping. However, PCR-based markers did not completely cover the genome, making genetic analysis of buckwheat challenging. The subsequent development of next generation sequencing, a game-changing technology, has allowed genome-wide analysis to be performed for many species. Indeed, 8,884 markers spanning 756 loci were recently mapped onto eight linkage groups of common buckwheat; these markers were successfully used for genomic selection to increase yield. Furthermore, draft genome sequences are now available in the Buckwheat Genome DataBase (BGDB). In this review, I summarize advances in the breeding and genetic analysis of common buckwheat based on contemporary genetic marker systems.

Buckwheat heteromorphic self-incompatibility: genetics, genomics and application to breeding.

Common buckwheat (Fagopyrum esculentum Moench 2n = 2x = 16) is an outcrossing crop with heteromorphic self-incompatibility due to its distylous flowers, called pin and thrum. In pin plants, a long style is combined with short stamens and small pollen grains; in thrum plants, a short style is combined with long stamens and large pollen grains. Both the intra-morph self-incompatibility and flower morphology are controlled by a single genetic locus named the S locus; thrum plants are heterozygous (Ss) and pin plants are homozygous recessive (ss) at this locus. Self-incompatibility is an obstacle for establishing pure lines and fixation of agronomically useful genes. Elucidation of the molecular mechanism of heterostylous self-incompatibility of common buckwheat has continued for a quarter of a century. Recent advances in genomic and transcriptomic analyses using next-generation sequencing have made it possible to determine the genomic region harboring the buckwheat S locus and to identify novel genes at this locus. In this review, we summarize the current knowledge on buckwheat heterostyly gained from conventional and molecular genetics and genomics. We also discuss the application of these studies to breeding of common buckwheat.

Development of co-dominant markers linked to a hemizygous region that is related to the self-compatibility locus (S) in buckwheat (Fagopyrum esculentum).

Common buckwheat (Fagopyrum esculentum) is a heterostylous self-incompatible (SI) species with two different flower morphologies, pin and thrum. The SI trait is controlled by a single gene complex locus, S. Self-compatible (SC) lines were developed by crossing F. esculentum and F. homotropicum; these lines have an SC gene, Sh , which is dominant over the s allele and recessive to the S allele. S-ELF3 has been identified as a candidate gene in the S locus and is present in the S and Sh but not s alleles. A single-nucleotide deletion in the S-ELF3 gene of the Sh allele results in a frame shift. To develop co-dominant markers to distinguish between ShSh and Shs plants, we performed a next-generation sequencing analysis in combination with bulked-segregant analysis. We developed four co-dominant markers linked to the S locus. We investigated the polymorphism frequency between a self-compatible line and leading Japanese buckwheat cultivars. Linkage between a developed sequence-tagged-site marker and flower morphology was confirmed using more than 1000 segregating plants and showed no recombination. The developed markers would be useful for buckwheat breeding and also to produce lines for genetic analysis such as recombinant inbred lines.

Gene flow signature in the S-allele region of cultivated buckwheat.

BACKGROUND: Buckwheat (Fagopyrum esculentum Moench.) is an annual crop that originated in southern China. The nutritious seeds are used in cooking much like cereal grains. Buckwheat is an outcrossing species with heteromorphic self-incompatibility due to its dimorphic (i.e., short- and long-styled) flowers and intra-morph infertility. The floral morphology and intra-morph incompatibility are both determined by a single S locus. Plants with short-styled flowers are heterozygous (S/s) and plants with long-styled flowers are homozygous recessive (s/s) at this locus, and the S/S genotype is not found. Recently, we built a draft genome assembly of buckwheat and identified the 5.4-Mb-long S-allele region harbored by short-styled plants. In this study, the first report on the genome-wide diversity of buckwheat, we used a genotyping-by-sequencing (GBS) dataset to evaluate the genome-wide nucleotide diversity within cultivated buckwheat landraces worldwide. We also investigated the utility of the S-allele region for phylogenetic analysis of buckwheat. RESULTS: Buckwheat showed high nucleotide diversity (0.0065), comparable to that of other outcrossing plants, based on a genome-wide simple nucleotide polymorphism (SNP) analysis. Phylogenetic analyses based on genome-wide SNPs showed that cultivated buckwheat comprises two groups, Asian and European, and revealed lower nucleotide diversity in the European group (0.0055) and low differentiation between the Asian and European groups. The nucleotide diversity (0.0039) estimated from SNPs in the S-allele region is lower than that in genome-wide SNPs. Phylogenetic analysis based on this region detected three diverged groups, S-1, S-2, and S-3. CONCLUSION: The SNPs detected using the GBS dataset were effective for elucidating the evolutionary history of buckwheat, and led to the following conclusions: (1) the low nucleotide diversity of the entire genome in the European group and low differentiation between the Asian and European groups suggested genetic bottlenecks associated with dispersion from Asia to Europe, and/or recent intensified cultivation and selection in Europe; and (2) the high diversification in the S-allele region was indicative of gene flows from wild to cultivated buckwheat, suggesting that cultivated buckwheat may have multiple origins.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

BACKGROUND: Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. RESULTS: By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. CONCLUSIONS: We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Rapid genotyping with DNA micro-arrays for high-density linkage mapping and QTL mapping in common buckwheat (Fagopyrum esculentum Moench).

For genetic studies and genomics-assisted breeding, particularly of minor crops, a genotyping system that does not require a priori genomic information is preferable. Here, we demonstrated the potential of a novel array-based genotyping system for the rapid construction of high-density linkage map and quantitative trait loci (QTL) mapping. By using the system, we successfully constructed an accurate, high-density linkage map for common buckwheat (Fagopyrum esculentum Moench); the map was composed of 756 loci and included 8,884 markers. The number of linkage groups converged to eight, which is the basic number of chromosomes in common buckwheat. The sizes of the linkage groups of the P1 and P2 maps were 773.8 and 800.4 cM, respectively. The average interval between adjacent loci was 2.13 cM. The linkage map constructed here will be useful for the analysis of other common buckwheat populations. We also performed QTL mapping for main stem length and detected four QTL. It took 37 days to process 178 samples from DNA extraction to genotyping, indicating the system enables genotyping of genome-wide markers for a few hundred buckwheat plants before the plants mature. The novel system will be useful for genomics-assisted breeding in minor crops without a priori genomic information.

Chromosome-scale genome assembly of a Japanese chili pepper landrace, Capsicum annuum 'Takanotsume'.

Here, we report the genome sequence of a popular Japanese chili pepper landrace, Capsicum annuum 'Takanotsume'. We used long-read sequencing and optical mapping, together with the genetic mapping technique, to obtain the chromosome-scale genome assembly of 'Takanotsume'. The assembly consists of 12 pseudomolecules, which corresponds to the basic chromosome number of C. annuum, and is 3,058.5 Mb in size, spanning 97.0% of the estimated genome size. A total of 34,324 high-confidence genes were predicted in the genome, and 83.4% of the genome assembly was occupied by repetitive sequences. Comparative genomics of linked-read sequencing-derived de novo genome assemblies of two Capsicum chinense lines and whole-genome resequencing analysis of Capsicum species revealed not only nucleotide sequence variations but also genome structure variations (i.e. chromosomal rearrangements and transposon-insertion polymorphisms) between 'Takanotsume' and its relatives. Overall, the genome sequence data generated in this study will accelerate the pan-genomics and breeding of Capsicum, and facilitate the dissection of genetic mechanisms underlying the agronomically important traits of 'Takanotsume'.

Phosphate starvation response precedes abscisic acid response under progressive mild drought in plants.

Drought severely damages crop production, even under conditions so mild that the leaves show no signs of wilting. However, it is unclear how field-grown plants respond to mild drought. Here, we show through six years of field trials that ridges are a useful experimental tool to mimic mild drought stress in the field. Mild drought reduces inorganic phosphate levels in the leaves to activate the phosphate starvation response (PSR) in soybean plants in the field. Using Arabidopsis thaliana and its mutant plants grown in pots under controlled environments, we demonstrate that PSR occurs before abscisic acid response under progressive mild drought and that PSR plays a crucial role in plant growth under mild drought. Our observations in the field and laboratory using model crop and experimental plants provide insight into the molecular response to mild drought in field-grown plants and the relationship between nutrition and drought stress response.

Genome sequencing reveals the genetic architecture of heterostyly and domestication history of common buckwheat.

Common buckwheat, Fagopyrum esculentum, is an orphan crop domesticated in southwest China that exhibits heterostylous self-incompatibility. Here we present chromosome-scale assemblies of a self-compatible F. esculentum accession and a self-compatible wild relative, Fagopyrum homotropicum, together with the resequencing of 104 wild and cultivated F. esculentum accessions. Using these genomic data, we report the roles of transposable elements and whole-genome duplications in the evolution of Fagopyrum. In addition, we show that (1) the breakdown of heterostyly occurs through the disruption of a hemizygous gene jointly regulating the style length and female compatibility and (2) southeast Tibet was involved in common buckwheat domestication. Moreover, we obtained mutants conferring the waxy phenotype for the first time in buckwheat. These findings demonstrate the utility of our F. esculentum assembly as a reference genome and promise to accelerate buckwheat research and breeding.

Phylogenetic analysis of AGAMOUS sequences reveals the origin of the diploid and tetraploid forms of self-pollinating wild buckwheat, Fagopyrum homotropicum Ohnishi.

Fagopyrum homotropicum Ohnishi is a self-pollinating wild buckwheat species indigenous to eastern Tibet and the Yunnan and Sichuan Provinces of China. It is useful breeding material for shifting cultivated buckwheat (F. esculentum ssp. esculentum Moench) from out-crossing to self-pollinating. Despite its importance as a genetic resource in buckwheat breeding, the genetic variation of F. homotropicum is poorly understood. In this study, we investigated the genetic variation and phylogenetic relationships of the diploid and tetraploid forms of F. homotropicum based on the nucleotide sequences of a nuclear gene, AGAMOUS (AG). Neighbor-joining analysis revealed that representative individuals clustered into three large groups (Group I, II and III). Each group contained diploid and tetraploid forms of F. homotropicum. We identified tetraploid plants that had two diverged AG sequences; one belonging to Group I and the other belonging to Group II, or one belonging to Group II and the other belonging to Group III. These results suggest that the tetraploid form originated from at least two hybridization events between deeply differentiated diploids. The results also imply that the genetic diversity contributed by tetraploidization of differentiated diploids may have allowed the distribution range of F. homotropicum to expand to the northern areas of China.

MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.).

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3,000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat.

QTL analysis of heterostyly in Primula sieboldii and its application for morph identification in wild populations.

BACKGROUND AND AIMS: Primula sieboldii is a perennial clonal herb that is distributed around the Sea of Japan and is endangered in Japan. Its breeding system is characterized by heteromorphic self-incompatibility, and the morph ratio within a population is very important for reproductive success. The aims of this study were to construct a linkage map, map the S locus as a qualitative trait and quantitative trait loci (QTLs) for floral morphological traits related to heterostyly, and predict the morph type in wild populations by using molecular markers for devising a conservation strategy. METHODS: A linkage map was constructed with 126 markers. The QTLs for four floral traits and the S locus were mapped. Using the genotypes of loci that were located near both the S locus and the QTLs with large effects, morphs of 59 wild genets were predicted. KEY RESULTS: The linkage map consisted of 14 linkage groups (LGs). The S locus was mapped to LG 7. Major QTLs for stigma and anther heights were detected in the same region as the S locus. These QTLs exhibited high logarithm of the odds scores and explained a high percentage of the phenotypic variance (>85 %). By analysing these two traits within each morph, additional QTLs for each trait were detected. Using the four loci linked to the S locus, the morphs of 43 genets in three wild populations could be predicted. CONCLUSIONS: This is the first report of a linkage map and QTL analysis for floral morphology related to heterostyly in P. sieboldii. Floral morphologies related to heterostyly are controlled by the S locus in LG 7 and by several QTLs in other LGs. Additionally, this study showed that molecular markers are effective tools for investigating morph ratios in a population containing the non-flowering individuals or during the non-flowering seasons.

Virus-Mediated Transient Expression Techniques Enable Functional Genomics Studies and Modulations of Betalain Biosynthesis and Plant Height in Quinoa.

Quinoa (Chenopodium quinoa), native to the Andean region of South America, has been recognized as a potentially important crop in terms of global food and nutrition security since it can thrive in harsh environments and has an excellent nutritional profile. Even though challenges of analyzing the complex and heterogeneous allotetraploid genome of quinoa have recently been overcome, with the whole genome-sequencing of quinoa and the creation of genotyped inbred lines, the lack of technology to analyze gene function in planta is a major limiting factor in quinoa research. Here, we demonstrate that two virus-mediated transient expression techniques, virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX), can be used in quinoa. We show that apple latent spherical virus (ALSV) can induce gene silencing of quinoa phytoene desaturase (CqPDS1) in a broad range of quinoa inbred lines derived from the northern and southern highland and lowland sub-populations. In addition, we show that ALSV can be used as a VOX vector in roots. Our data also indicate that silencing a quinoa 3,4-dihydroxyphenylalanine 4,5-dioxygenase gene (CqDODA1) or a cytochrome P450 enzyme gene (CqCYP76AD1) inhibits betalain production and that knockdown of a reduced-height gene homolog (CqRHT1) causes an overgrowth phenotype in quinoa. Moreover, we show that ALSV can be transmitted to the progeny of quinoa plants. Thus, our findings enable functional genomics in quinoa, ushering in a new era of quinoa research.

A novel WD40-repeat protein involved in formation of epidermal bladder cells in the halophyte quinoa.

Halophytes are plants that grow in high-salt environments and form characteristic epidermal bladder cells (EBCs) that are important for saline tolerance. To date, however, little has been revealed about the formation of these structures. To determine the genetic basis for their formation, we applied ethylmethanesulfonate mutagenesis and obtained two mutants with reduced levels of EBCs (rebc) and abnormal chloroplasts. In silico subtraction experiments revealed that the rebc phenotype was caused by mutation of REBC, which encodes a WD40 protein that localizes to the nucleus and chloroplasts. Phylogenetic and transformant analyses revealed that the REBC protein differs from TTG1, a WD40 protein involved in trichome formation. Furthermore, rebc mutants displayed damage to their shoot apices under abiotic stress, suggesting that EBCs may protect the shoot apex from such stress. These findings will help clarify the mechanisms underlying EBC formation and function.