菊池 真司

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.

Floral morphology varies considerably between dicots and monocots. The ABCDE model explaining how floral organ development is controlled was formulated using core eudicots and applied to grass crops. Barley (Hordeum. vulgare) has unique floral morphogenesis. Wild barley (H. vulgare ssp. spontaneum), which is the immediate ancestor of cultivated barley (H. vulgare ssp. vulgare), contains a rich reservoir of genetic diversity. However, the wild barley genes involved in floral organ development are still relatively uncharacterized. In this study, we generated an organ-specific transcriptome atlas for wild barley floral organs. Genome-wide transcription profiles indicated that 22 838 protein-coding genes were expressed in at least one organ. These genes were grouped into seven clusters according to the similarities in their expression patterns. Moreover, 5619 genes exhibited organ-enriched expression, 677 of which were members of 47 transcription factor families. Gene ontology analyses suggested that the functions of the genes with organ-enriched expression influence the biological processes in floral organs. The co-expression regulatory network showed that the expression of 690 genes targeted by MADS-box proteins was highly positively correlated with the expression of ABCDE model genes during floral morphogenesis. Furthermore, the expression of 138 genes was specific to the wild barley OUH602 genome and not the Morex genome; most of these genes were highly expressed in the glume, awn, lemma, and palea. This study revealed the global gene expression patterns underlying floral morphogenesis in wild barley. On the basis of the study findings, a molecular mechanism controlling floral morphology in barley was proposed.

KEY MESSAGE: Epidendrum produces 2n gametes with high frequency. This paper is the first to report on multiple pathways for forming 2n gametes, meiotic defeats, and pre-meiotic chromosome doubling. Unreduced 2n reproductive cells are predominantly involved in pathways that lead to polyploid plants. Although one of the most common pathways for inducing 2n gametes is through meiotic defects, a small set of isolated species alternatively generates 2n gametes from tetraploid pollen mother cells in the pre-meiotic phase. Hence, determining the mechanisms underlying 2n gamete formation is critical to improving breeding programmes and understanding plant evolution. We investigated sporads to reveal the pathway(s) accounting for the formation and frequencies of 2n gametes in wild species and interspecific hybrids in the genus Epidendrum. We investigated different types of sporads with varying frequencies, sizes, and viability in the wild species and hybrids of the genus Epidendrum. Large tetrad-estimated pre-meiotic chromosome doubling was observed in wild species. The Epidendrum is unique in that it forms 2n pollens via two pathways, namely, meiotic defects and pre-meiotic chromosome doubling. These two pathways of 2n pollen formation could influence the high diversity generation of polyploidy with different degrees of heterozygosity and genetic backgrounds in the genus Epidendrum. Therefore, these findings are proposed to influence polyploid breeding of Epidendrum via 2n pollen, helping us understand evolution and speciation via unreduced 2n gamete formation in Orchidaceae.

Cytological studies using fluorescence in situ hybridization (FISH) technique provides phylogenetical information in closely related taxa and have been widely applied for karyotyping and studying chromosomal organization and evolution in plant species. In the present study, FISH using a microsatellite sequence of (AAG)7 as the probe was performed in order to discriminate the chromosomes in four Lactuca species, i.e., L. sativa, L. serriola, L. saligna, and L. virosa. The experiment was carried out in April to September 2018 at Laboratory of Genetic and Plant Breeding of Breeding of Graduate School of Horticulture, Chiba University, Japan. Different distribution patterns of (AAG)n signals were shown on the chromosomes in the four Lactuca species studied, In L. sativa and L. serriola, FISH with (AAG)7 sequences revealed dispersed distribution patterns with one pair of bright signals, respectively. While in L. saligna and L. virosa, distinct signals with different intensities were observed in two pairs of chromosomes of L. saligna and five pairs of chromosomes of L. virosa. In conclusion, the AAG repeat signals could be used as cytogenetic landmarks for chromosome identification in Lactuca species.

Chromosome numbers of wild species in the genus Epidendrum have previously been reported in the range of 2n= 24-240. However, the ploidy levels of cultivars in the given genus originating from interspecific hybridization among several wild species have never been reported. To elucidate their ploidy levels, we analyzed the chromosome number of cultivars and related wild species. Four wild species showed a new record of chromosome number in the present study, namely, E. radicans 2n= 38, 80, E. secundum 2n=60, and E. cinnabarinum 2n= 64. Six cultivars examined were revealed to have more chromosomes than the wild species, with a range of 2n= 84-164, suggesting that the cultivars have high polyploid levels. A significant correlation between the nuclear DNA amount and the chromosome number in wild species as well as cultivars was observed. Those cultivars originated from hybridization. Thus, the results from the present study suggest that the polyploid cultivars in this genus could have resulted from pollination involving unreduced gametes during breeding.

Background and Aims The brittle rachis trait is a feature of many wild grasses, particularly within the tribe Triticeae. Wild Hordeum and Triticum species form a disarticulation layer above the rachis node, resulting in the production of wedge-type dispersal units. In Aegilops longissima, only one or two of the nodes in the central portion of its rachis are brittle. In Triticeae species, the formation of a disarticulation layer above the rachis node requires the co-transcription of the two dominant and complementary genes Btr1 and Btr2. This study aims to establish whether homologues of Btr1 and/or Btr2 underlie the unusual brittle rachis phenotype observed in Ae. longissima.Methods Scanning electron microscopy was used to examine the disarticulation surfaces. Quantitative RT-PCR and RNA in situ hybridization experiments were used to identify gene expression in the immature inflorescence.Key Results Analysis based on scanning electron microscopy was able to demonstrate that the disarticulation surfaces formed in the Ae. longissima rachis are morphologically indistinguishable from those formed in the rachises of wild Hordeum and Triticum species. RNA in situ hybridization showed that in the immature Ae. longissima inflorescence, the intensity of Btr1 transcription varied from high at the rachis base to low at its apex, while that of Btr2 was limited to the nodes in the central to distal portion of the rachis.Conclusions The disarticulation pattern shown by Ae. longissima results from the limitation of Btr1 and Btr2 co-expression to nodes lying in the centre of the rachis.

Plant genomes remain highly fragmented and are often characterized by hundreds to thousands of assembly gaps. Here, we report chromosome-level reference and phased genome assembly of Ophiorrhiza pumila, a camptothecin-producing medicinal plant, through an ordered multi-scaffolding and experimental validation approach. With 21 assembly gaps and a contig N50 of 18.49Mb, Ophiorrhiza genome is one of the most complete plant genomes assembled to date. We also report 273 nitrogen-containing metabolites, including diverse monoterpene indole alkaloids (MIAs). A comparative genomics approach identifies strictosidine biogenesis as the origin of MIA evolution. The emergence of strictosidine biosynthesis-catalyzing enzymes precede downstream enzymes' evolution post gamma whole-genome triplication, which occurred approximately 110 Mya in O. pumila, and before the whole-genome duplication in Camptotheca acuminata identified here. Combining comparative genome analysis, multi-omics analysis, and metabolic gene-cluster analysis, we propose a working model for MIA evolution, and a pangenome for MIA biosynthesis, which will help in establishing a sustainable supply of camptothecin.Ophiorrhiza pumila is a medicinal plant that can produce the anti-cancer monoterpene indole alkaloid (MIA) camptothecin. Here, the authors report its genome assembly and propose a working model for MIA evolution and biosynthesis through comparative genomics, synteny, and metabolic gene cluster analyses.

Mobile elements are major regulators of genome evolution through their effects on genome size and chromosome structure in higher organisms. Non-long terminal repeat (non-LTR) retrotransposons, one of the subclasses of transposons, are specifically inserted into repetitive DNA sequences. While studies on the insertion of non-LTR retrotransposons into ribosomal RNA genes and other repetitive DNA sequences have been reported in the animal kingdom, studies in the plant kingdom are limited. Here, using FISH, we confirmed that Menolird18, a member of LINE (long interspersed nuclear element) in non-LTR retrotransposons and found in Cucumis melo, was inserted into ITS and ETS (internal and external transcribed spacers) regions of 18S rDNA in melon and cucumber. Beside the 18S rDNA regions, Menolird18 was also detected in all centromeric regions of melon, while it was located at pericentromeric and sub-telomeric regions in cucumber. The fact that FISH signals of Menolird18 were found in centromeric and rDNA regions of mitotic chromosomes suggests that Menolird18 is a rDNA and centromere-specific non-LTR retrotransposon in melon. Our findings are the first report on a non-LTR retrotransposon that is highly conserved in 2 different plant species, melon and cucumber. The clear distinction of chromosomal localization of Menolird18 in melon and cucumber implies that it might have been involved in the evolutionary processes of the melon (C. melo) and cucumber (C. sativus) genomes.

Seed dispersal among wild species belonging to the tribe Triticeae is typically achieved by the formation of a brittle rachis. The trait relies on the development of a disarticulation layer, most frequently above the rachis node (resulting in wedge type dispersal units), but in some species below the rachis node (resulting in barrel type dispersal units). The genes responsible for the former type are the complementary pair Btr1 and Btr2, while the genetic basis of the latter type has yet to be determined. Aegilops tauschii forms barrel type dispersal units and previous study showed this species lacked an intact copy of Btr1. Here it has been demonstrated that Ae. tauschii carries two of Btr2; and that Btr2 transcript is present in a region below the rachis node where the abscission zone forms. The implication is that in this species, the Btr2 product is involved in the formation of barrel type dispersal units.

In many non-cultivated angiosperm species, seed dispersal is facilitated by the shattering of the seed head at maturity; in the Triticeae tribe, to which several of the world's most important cereals belong, shattering takes the form of a disarticulation of the rachis. The products of the genes Btr1 and Btr2 are both required for disarticulation to occur above the rachis nodes within the genera Hordeum (barley) and Triticum/Aegilops (wheat). Here, it has been shown that both Btr1 and Btr2 are specific to the Triticeae tribe, although likely paralogs (Btr1-like and Btr2-like) are carried by the family Poaceae including Triticeae. Aegilops tauschii (the donor of the bread wheat D genome) lacks a copy of Btr1 and disarticulation in this species occurs below, rather than above the rachis node; thus, the product of Btr1 appears to be required for disarticulation to occur above the rachis node.

Lactuca sativa L. is one of the most important reference species for genome research in Compositae. In the present study, chromosome image analysis system IV (CHIAS IV) and fluorescence in situ hybridization (FISH) were applied to obtain detailed information of the characteristics of lettuce chromosomes. One lettuce cultivar of L. sativa and three wild species of L. serriola, L. saligna and L. virosa were used to analyze chromosome lengths, arm ratios and degrees of chromosomal condensation at prometaphase. Locations of 45S and 5S rRNA genes were determined by FISH technique. Our results showed that nine pairs of homologous chromosomes could be discriminated for all four species by their morphological characteristics, condensation patterns (CPs) and localization of rRNA loci. Statistical analysis showed that the total length of L. saligna chromosomes was significantly the shortest among the species tested. The present study is the first for the physical mapping of condensed regions and rDNAs using CPs and FISH of prometaphase chromosomes in lettuce. The information provided here would be valuable for practical breeding and genetics of lettuce.

Centromeres are prerequisite for accurate segregation and are landmarks of primary constrictions of metaphase chromosomes in eukaryotes. In melon, high-copy-number satellite DNAs (SatDNAs) were found at various chromosomal locations such as centromeric, peri-centromeric, and subtelomeric regions. In the present study, utilizing the published draft genome sequence of melon, two new SatDNAs (CmSat162 and CmSat189) of melon were identified and their chromosomal distributions were confirmed using fluorescence in situ hybridization. DNA probes prepared from these SatDNAs were successfully hybridized to melon somatic and meiotic chromosomes. CmSat162 was located on 12 pairs of melon chromosomes and co-localized with the centromeric repeat, Cmcent, at the centromeric regions. In contrast, CmSat189 was found to be located not only on centromeric regions but also on specific regions of the chromosomes, allowing the characterization of individual chromosomes of melon. It was also shown that these SatDNAs were transcribed in melon. These results suggest that CmSat162 and CmSat189 might have some functions at the centromeric regions.

Jatropha (Jatropha curcas) is an oil-bearing plant used for biodiesel production. Construction of its standard karyotype and identification of the euchromatin/heterochromatin distribution associated with gene expression and meiotic recombination are essential to fully characterize its genome. Here, we developed a J. curcas karyotype based on meiotic pachytene chromosomes. In addition, a karyotype of J. integerrima, a useful species for jatropha breeding, was also constructed. Five out of eleven J. curcas chromosomes were metacentric, but only two were metacentric in J. integerrima. Almost all of the heterochromatin was distributed around the pericentric regions. The interstitial and distal regions were euchromatic without heterochromatic knobs, except for small heterochromatin regions associated with the subtelomeric repeat sequence JcSat1. These pericentric heterochromatin distribution patterns, together with chromosome structure data and the results of FISH probing with rDNA and JcSat1, allowed us to classify all chromosomes of both species. The two species had two 35S rDNA loci and one 5S rDNA locus; one 35S rDNA locus in J. integerrima was located on the interstitial region of the short arms. In addition, JcSat1 was found at only the heterochromatic ends of the J. curcas chromosome, not the J. integerrima chromosome. Despite the same chromosome number, the two pachytene chromosome-based karyotypes suggest variation in chromosome structure and distribution of repetitive DNAs in these two species.

Background: Detailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase chromosomes. However, suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated.Results: In this study, a modified Carnoy's solution II (MC II) [6:3:1 (v/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase chromosomes. We demonstrated that the prometaphase chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene chromosome preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase chromosomes of melon and Abelia x grandiflora generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that Abelia x grandiflora has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase chromosomes.Conclusion: The MC II and ADI method are simple and effective for acquiring prometaphase and pachytene chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants.

To analyze the karyotypes of five Jatropha species (J. cinerea, J. curcas, J. integerrima, J. multfida, and J. podagrica), fluorescence in situ hybridization (FISH) with a 35S ribosomal RNA gene (rDNA) probe was conducted. FISH analysis of mitotic metaphase chromosomes showed that the numbers of chromosomes and rDNA foci in all species analyzed were conserved, although signal intensities and their locations varied. The conserved karyotypes may be advantageous for the use of J. curcas genome data and for breeding in the progeny of interspecific crosses.

To analyze the genetic relationship between Torenia baillonii (2n=2x=16) and Torenia fournieri (2n=2x=18), an interspecific diploid hybrid (2n=2x=17) and a triploid hybrid (2n=3x=26) were produced. The behavior of meiotic chromosomes in the hybrids was examined by fluorescence in situ hybridization (FISH) with species-specific repeats. The mean pairing configuration of the diploid hybrid was 1.95I+6.75II+0.48III and that of the triploid hybrid was 5.6I+6.74II+2.44III. Genome analysis in the triploid hybrid using FISH revealed that the affinity between the genomes was x=0.79. The relatively low value indicates considerable conservation between homoeologous chromosomes of T. baillonii and T. fournieri, even though the two species have different chromosome numbers.

Verticillium dahliae infecting tomato can be differentiated into races 1 and 2 based on differential pathogenicity on tomato cultivars carrying resistance gene Ve1. Although no commercial cultivars resistant to race 2 are available, race 2-resistant rootstock cultivars Aibou and Ganbarune-Karis have been bred in Japan. Nevertheless, the resistance of these rootstocks appears to be unstable in commercial tomato fields. Pathogenicity assays conducted under controlled conditions revealed that these rootstock cultivars are resistant to some isolates of race 2; this resistance is controlled by a single dominant locus, denoted by V2, based on segregation of resistance in F2 populations from selfed rootstock cultivars. However, some other isolates of race 2 can overcome this resistance. Therefore it is proposed that the current race 2 of V. dahliae should be divided into two races, i.e. 'race 2' (nonpathogenic on Aibou) and 'race 3' (pathogenic on Aibou). The distribution of these races was surveyed in 70 commercial tomato fields in Hida, Gifu Prefecture, Japan. Race 3 was found in 45 fields, indicating that race 3 had already spread throughout the region. On the other hand, 25 fields had only race 2, and thus race 2-resistant rootstocks would be effective for disease management in these fields. Races 2 and 3 could not be identified by genomic Southern hybridization probed with a telomere sequence, nor with previously reported race-specific PCR assays. Elucidation of race-determining mechanisms and development of methods for quick race identification should be made in future studies.

<p>アブラナ科アブラナ属コマツナ（B. rapa L. Perviridis Group）とキバナスズシロ属ルッコラ（E. sativa Mill.）を属間交雑し，胚珠培養により交雑個体を得た．得られた交雑個体の表現型のうち，花序や葉身は両種の中間型，胎座の配列はコマツナ型，茎生葉の基部の形状はルッコラ型を示した．GISH法によりコマツナおよびルッコラ由来の染色体が明確に識別されたことから，この交雑個体は両種の雑種であることが確認された．また，この交雑個体はルッコラ由来の機能性成分エルシン前駆体（4-methylthiobutyl-GSL: Glucoerucin）およびスルフォラファン前駆体（4-methylsulfinylbutyl-GSL: Glucoraphanin） も含有していたことから，今後，消費者ニーズに応えられる付加価値の高い新規軟弱野菜品種の育成につながるものと期待される．</p>

Genetic variation in Jatropha curcas, a prospective biodiesel plant, is limited, and interspecific hybridization needed for its genetic improvement. Progeny from interspecific crosses between J. curcas and Jatropha integerrima can be used to improve agronomic characters and to increase oil content and yield. However, these hybrids have not been characterized cytologically. The present study was aimed at the analysis of chromosome behavior during meiosis and chromosome composition of S-1 plants derived from an interspecific F-1 hybrid using genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). Bivalents that formed as a result of interspecific pairing were frequently observed, suggesting the presence of homoeologous chromosomes from the two species. Almost half of microspores were derived from the reduction division; GISH analysis indicated random transmission of the parent chromosomes to microspores. Male fertility measured as pollen staining with acetocarmine was 48.4%. In contrast, GISH analysis of Si plants revealed preferential transmission of J. curcas chromosomes. We also found segment exchange between chromosomes of the two species (interspecific translocation) by GISH and FISH analyses. Introgression of J integerrima chromosome segments into the J. curcas genome would help to improve Jatropha cultivars for mass production.

Somatic embryogenesis is one biotechnological method that can be used to produce genetically identical plants for use in plant breeding. In this research, we attempted to induce the formation of embryogenic callus from young leaves excised from in vitro Torenia spp. by culturing in the dark for 1, 2, 3 and 4 weeks on Murashige and Skoog (MS) medium with Picloram solution added at the concentrations of 0.5, 1, 1.5 and 2 mg(-l) and 2,4-D solution added at the concentrations of 0.5, 1, 1.5 and 2 mg(-l). The leaf explants from all the 2,4-D treatments turned brown and died, but the leaf explants cultured on media with Picloram added at every concentration tested formed soft, loosely aggregated callus tissue. Callus tissue was induced from leaves of diploid Torenia to the greatest degree (95% callus formation at 3 weeks) when cultured on MS medium containing Picloram at 1.5 mg(-l) in the dark, and callus tissue tended to turn brown with increasing Picloram concentration and increasing incubation time. For polyploid Torenia, the highest percentage of callus formation was observed on leaves cultured on MS medium containing Picloram, and callus tissue also tended to turn brown with increasing Picloram concentration and increasing incubation time. Following transfer of the embryogenic callus tissue to hormone-free MS medium under light conditions (16-h photoperiod), the greatest rate of somatic embryo formation was observed, in case of both diploid and polyploid Torenia accessions. (C) 2015 INT TRANS J ENG MANAG SCI TECH.

This research created greater genetic variation in Lindernia sp. by studying the most appropriate concentration and duration of colchicine treatment that could induce polyploidy in Lindernia sp. Leaf and node sections taken from 1-month old Lindernia plants grown in vitro were used as the explants to be immersed in colchicine solution at the concentrations of 0, 5, 10, 15 and 20 ppm for 1, 2 and 3 days. The experiment was a 5x3 Factorial in CRD with 10 replications and 2 explants per replication. The results showed that the plants regenerated from the colchicine-treated node and leaf sections had lower survival rate, shorter plant height, shorter internode length, fewer roots and shorter roots than the control group, with an increasing effect from increased concentrations of colchicine and increased exposure time; but the colchicine- treated plants had thicker stems, thicker roots and wider and longer leaves than the control. The treatment group exposed to colchicine at the concentration of 10 ppm for 3 days had the lowest survival rate (60% for node sections and 50% for leaf sections), but the highest polyploidy induction rate at 22.00% for node sections and 16.22% for leaf sections. The frequency of polyploidy induction was higher in node sections than in leaf sections, and the plants regenerated from node sections were also stronger and had a higher survival rate when planted out than those regenerated from leaf sections. (C) 2015 INT TRANS J ENG MANAG SCI TECH.

Using an F-2 population derived from cultivated and wild azuki bean, we previously detected a reciprocal translocation and a seed size QTL near the translocation site. To test the hypothesis that the translocation in the cultivated variety contributed to the larger seed size, we performed further linkage analyses with several cross combinations between cultivated and wild azuki beans. In addition, we visually confirmed the translocation by cytogenetic approach using 25 wild and cultivated accessions. As a result, we found the translocation-type chromosomes in none of the cultivated accessions, but in a number of the wild accessions. Interestingly, all the wild accessions with the translocation were originally collected from East Japan, while all the accessions with normal chromosomes were from West Japan or the Sea of Japan-side region. Such biased geographical distribution could be explained by the glacial refugium hypothesis, and supported narrowing down the domestication origin of cultivated azuki bean.

As the fruits of loquat (Eriobotiya japonica, 2n = 2x = 34) carry large seeds, the breeding of seedless loquat has long been a goal. The recent creation of triploid cultivars (2n = 3x = 51) and the application of gibberellins allow commercial production of seedless loquat, but the possibility of seed formation in triploid loquats has not been carefully investigated. Through crossing experiments and cytological observations of meiosis and pollen tube growth, we found that the triploid line 3N-N28 was essentially self-sterile, but developed seeds on pollination with pollen from diploid cultivars at rates of up to 5.5%. Almost half of the seedlings survived to 5 months, and carried diploid (2n = 34), tetraploid (2n = 68), or aneuploid chromosome numbers. Our results suggest that triploid loquat cultivars might retain the risk of seed formation. Protection from pollination by diploid cultivars or the development of new triploid cultivars will be necessary to ensure the production of seedless loquat fruits.

Gametophytic self-incompatibility (GSI) is controlled by a complex S locus containing the pistil determinant S-RNase and pollen determinant SFB/SLF. Tight linkage of the pistil and pollen determinants is necessary to guarantee the self-incompatibility (SI) function. However, multiple probable pollen determinants of apple and Japanese pear, SFBBs (S locus F-box brothers), exist in each S haplotype, and how these multiple genes maintain the SI function remains unclear. It is shown here by high-resolution fluorescence in situ hybridization (FISH) that SFBB genes of the apple S-9 haplotype are physically linked to the S-9-RNase gene, and the S locus is located in the subtelomeric region. FISH analyses also determined the relative order of SFBB genes and S-RNase in the S-9 haplotype, and showed that gene order differs between the S-9 and S-3 haplotypes. Furthermore, it is shown that the apple S locus is located in a knob-like large heterochromatin block where DNA is highly methylated. It is proposed that interhaplotypic heterogeneity and the heterochromatic nature of the S locus help to suppress recombination at the S locus in apple.

The presence of the centromere-specific histone H3 variant, CENH3, defines centromeric (CEN) chromatin, but poorly understood epigenetic mechanisms determine its establishment and maintenance. CEN chromatin is embedded within pericentromeric heterochromatin in most higher eukaryotes, but, interestingly, it can show euchromatic characteristics; for example, the euchromatic histone modification mark dimethylated H3 Lys 4 (H3K4me2) is uniquely associated with animal centromeres. To examine the histone marks and chromatin properties of plant centromeres, we developed a genomic tiling array for four fully sequenced rice (Oryza sativa) centromeres and used chromatin immunoprecipitation-chip to study the patterns of four euchromatic histone modification marks: H3K4me2, trimethylated H3 Lys 4, trimethylated H3 Lys 36, and acetylated H3 Lys 4, 9. The vast majority of the four histone marks were associated with genes located in the H3 subdomains within the centromere cores. We demonstrate that H3K4me2 is not a ubiquitous component of rice CEN chromatin, and the euchromatic characteristics of rice CEN chromatin are hallmarks of the transcribed sequences embedded in the centromeric H3 subdomains. We propose that the transcribed sequences located in rice centromeres may provide a barrier preventing loading of CENH3 into the H3 subdomains. The separation of CENH3 and H3 subdomains in the centromere core may be favorable for the formation of three-dimensional centromere structure and for rice centromere function.

Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a complex S locus that encodes separate proteins for pistil and pollen specificities, extracellular ribonucleases (S-RNases) and F-box proteins SFB/SLF, respectively. SFB/SLFs of Prunus (subfamily Prunoideae of Rosaceae), Solanaceae and Plantaginaceae are single copy in each S haplotype, while recently identified pollen S candidates SFBBs of subfamily Maloideae of Rosaceae, apple and Japanese pear, are multiple; two and three related SFBBs were isolated from each S haplotype of apple and Japanese pear, respectively. Here, we show that apple (Malus x domestica) SFBBs constitute a gene family that is much larger than initially thought. Twenty additional SFBB-like genes/alleles were isolated by screening of a BAC library derived from S (3) S (9) genotype, and tentatively named MdFBX1-20. All but one MdFBX showed S haplotype-specific polymorphisms. All the polymorphic MdFBXs were completely linked to S-RNase in 239 segregants. In addition, FISH revealed that the monomorphic gene MdFBX11 is also located near S-RNase, and the S locus is located in a subtelomeric region of a chromosome and is not close to the centromere. All MdFBXs were specifically expressed in pollen, except for a pseudogene MdFBX4 that showed no expression in any organs analyzed. Phylogenetic analysis revealed that the closest relatives of most MdFBXs were from a different S haplotype, suggesting that proliferation of MdSFBB/FBXs predates diversification of the S haplotypes.

P>We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3-associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.