Isogenic Japonica Rice Koshihikari Integrated with Late Flowering Gene Hd16 and Semidwarfing Gene sd1 to Prevent High Temperature Maturation and Lodging by Typhoon.

We developed semidwarf and late-maturing isogenics of Koshihikari to stabilize high yield and avoid high temperature maturation. Whole-genome analysis (WGS) was conducted to examine the transitional changes in the entire genome, the size of DNA fragments integrated with the target gene, and genes accompanying the target gene owing to the progress of backcrossing. In both Koshihikari Hd16 (BC7F4) and Koshihikari sd1Hd16 (BC8F2), an SNP from adenine to guanine was detected in Hd16 at 32,996,608 bp on chromosome 3, which is known to be a causative mutation of Hd16 in Nipponbare. In Koshihikari sd1Hd16 (BC8F2), an SNP from thymine to guanine was detected in sd1 at 38,267,510 bp on chromosome 1. From BC7 to BC8, the size of the DNA fragment integrated with Hd16 decreased by 5871 bp. Koshihikari sd1Hd16 flowered 12.1 days later than Koshishikari or Koshihikari sd1 did and was 14.2 cm (15%) shorter than Koshihikari. The yield in Koshishikari sd1Hd16 (63.2 kg/a) was 7.0% higher than that of Koshihikari. This is a new germplasm designed to avoid heat damage at ripening during high-temperature summer periods by late maturation owing to Hd16 as well as to avoid lodging by autumn typhoons by semidwarfness owing to sd1.

Whole-Genome Sequencing Revealed a Late-Maturing Isogenic Rice Koshihikari Integrated with Hd16 Gene Derived from an Ise Shrine Mutant.

We identified the key genes controlling the late maturation of the Japonica cultivar Isehikari, which was found at Ise Jingu Shrine and matures 6 days later than Koshihikari. We conducted a genetics-based approach through this study. First, the latest mature plants, which flowered later than Isehikari, were segregated in the F2 and F3 generations of Koshihikari×Isehikari. Next, the linkage relationship of a single late-maturing gene with the SSR markers on the long arm of chromosome 3 was inferred by using late-maturing homozygous F2 segregants. Moreover, genetic analyses of late maturity were conducted through the process of six times of continuous backcross with Koshihikari as a recurrent parent by using the late-maturing homozygous F3 line as a nonrecurrent parent, thus developing a late-maturing isogenic Koshihikari (BC6F2). As a result, we elucidated a single late-maturing gene with incomplete dominance that caused the 14-day maturation delay of Koshihikari. The whole-genome sequencing was conducted on both of Koshihikari and the late-maturing isogenic Koshihikari. Then, the SNP call was conducted as the reference genome of Koshihikari. Finally, a single SNP was identified in the key gene Hd16 of the late-maturing isogenic Koshihikari.

Year-round flowering gene e1, a mutation at the E1 locus on rice chromosome 7 and its combination with green revolution gene sd1 in an isogenic cell line.

Genetic analysis on the year-round flowering gene e1, which was derived from Kanto No. 79, an induced mutant by Koshihikari gamma-ray irradiated, was conducted through the backcross process to combine e1 and sd1 in the genetic background of Koshihikari. e1 strongly forwarded flowering 14 days earlier than the original variety Koshihikairi. Isogenic Koshihiakri combining e1 and sd1 was developed by four times of backcross with either Koshihikari or Koshihikari sd1 as recurrent parents by using the e1sd1 homozygous F3 plant in Koshihikari sd1 × Kanto No. 79 as a non-recurrent parent. As a result, "Koshihikari e1sd1" maturing 14 days earlier was approximately 30 cm shorter than Koshihikari. e1 was linked with DNA markers, which were near to Ghd7 on the short arm of chromosome 7. The whole genome sequencing revealed a single candidate SNP, which is specific to Koshihikari e1sd1, in the Xa21-like sequence, at 35213 bp downstream from Ghd7 on chromosome 7. Koshihikari e1sd1-specific SNP beside Ghd7 is expected to downregulate with Ghd7.

Gene structure of three kinds of vacuolar-type Na+/H+ antiporters including TaNHX2 transcribed in bread wheat.

The vacuolar-type sodium/proton antiporter is considered to play an important role in withstanding salt stress by transporting sodium ions into vacuoles. In this study, the gene structures of three kinds of vacuolar-type antiporters transcribed in bread wheat under salt stress were analyzed. After spraying 0.5 M NaCl to seedlings of wheat cultivar Chinese Spring, 1,392~1,400 bp cDNA fragments were isolated by RT-PCR using primers designed from common regions in rice OsNHX1 and Atriplex subcordata AgNHX1. Next, the entire structure of the genomic DNA and cDNA were determined via CapFishing-5' Rapid Amplification of cDNA Ends (RACE), 3'RACE, and genomic PCR cloning. As a result, 3 kinds of vacuolar-type Na+/H+ antiporter genes, TaNHXa (genome DNA 4,255 bp, cDNA 2,414 bp, 539 a.a.), TaNHXb (gDNA 4,167 bp, cDNA 1,898 bp, 539 a.a.) and TaNHXc (gDNA 4,966 bp, cDNA 1,928 bp, 547 a.a.), were identified. They encode 12 transmembrane domains containing third domain's amyloid binding sites (FFIYLLPP), characteristic of the vacuolar-type Na+/H+ antiporter, binding to the cell vacuolar membrane. TaNHXa, b and c consisting of 14 exons and 13 introns were 22~55 % longer than A. thaliana AtNHX1 in total length. TaNHXa (TaNHX2) showed homogeneity with OsNHX1, while TaNHXb and c were phylogenetically independent.

Clustered and dispersed chromosomal distribution of the two classes of Revolver transposon family in rye (Secale cereale).

The chromosomal locations of a new class of Revolver transposon-like elements were analyzed by using FISH method on the metaphase chromosome in somatic cell division of the rye cultivar Petkus. First, the Revolver standard element probe λ2 was weakly hybridized throughout the rye chromosome, and comparatively large interstitial signals spotted with a dot shape were detected together with several telomeric regions. The dot shape interstitial signal was stably detected at one site on Chromosome (Chr) 1R (middle part of the interstitial region of the short arm), three sites on Chr 2R (distal part of the interstitial region and adjacent to the centromere on the short arm, middle part of the interstitial region of the long arm), and two sites on Chr 5R (middle part of the interstitial region and adjacent to the centromere on the long arm). The Revolver λ2 probe was effective for identification of 1R, 2R, and 5R chromosomes. On the other hand, Revolver nonautonomous element-specific L626-BARE-100 probe was strongly distributed throughout the rye chromosomes, and considerable numbers and diverse lengths of transcripts were detected by RT-PCR. Although the standard elements were found in localized clusters, the nonautonomous elements tended to be dispersed throughout the genome. Clustered nature of Revolver is a significantly rare case in genomics.

The Gametic Non-Lethal Gene Gal on Chromosome 5 Is Indispensable for the Transmission of the Co-Induced Semidwarfing Gene d60 in Rice.

The gametic lethal gene gal in combination with the semidwarfing gene d60 causes complementary lethality in rice. Here, we attempted to ascertain the existence of gal and clarify male gamete abortion caused by d60 and gal. Through the F2 to F4 generations derived from the cross between D60gal-homozygous and d60Gal-homozygous, progenies of the partial sterile plants (D60d60Galgal) were segregated in a ratio of 1 semidwarf (1 d60d60GalGal):2 tall and quarter sterile (2 D60d60Galgal):6 tall (2 D60d60GalGal:1 D60D60GalGal:2 D60D60Galgal:1 D60D60galgal), which is skewed from the Mendelian ratio of 1 semidwarf:3 tall. However, the F4 generation was derived from fertile and tall heterozygous F2 plants (D60d60GalGal), which were segregated in the Mendelian ratio of 1[semidwarf (d60d60GalGal)]:2[1 semidwarf:3 tall (D60d60GalGal)]:1[tall (D60D60GalGal)]. The backcrossing of D60Gal-homozygous tall F4 plants with Hokuriku 100 resulted in fertile BCF1 and BCF2 segregated in a ratio of 1 semidwarf:3 tall, proving that d60 is inherited as a single recessive gene in the D60d60GalGal genetic background (i.e., in the absence of gal). Further, gal was localized on chromosome 5, which is evident from the deviated segregation of d1 as 1:8 and linkage with simple sequence repeat (SSR) markers. Next-generation sequencing identified the candidate SNP responsible for Gal. In F1 and sterile F2, at the binucleate stage, partial pollen discontinued development. Degraded pollen lost vegetative nuclei, but second pollen mitosis raising two generative nuclei was observed. Thus, our study describes a novel genetic model for a reproductive barrier. This is the first report on such a complementary lethal gene, whose mutation allows the transmission of a co-induced valuable semidwarfing gene d60.

Semidwarf Gene d60 Affected by Ubiquitous Gamete Lethal Gene gal Produced Rare Double Dwarf with d30 via Recombination Breaking Repulsion-Phase Linkage on Rice Chromosome 2.

The genotype of gal and d60 were investigated in 33 rice varieties chosen from representative semidwarf and dwarf rice varieties. These were crossed with three tester lines, the d60Gal line (genotype d60d60GalGal), the D60gal line (Koshihikari, D60D60galgal), and the D60Gal line (D60D60GalGal). Each F1 plant was measured for culm length, and seed fertility. As a result, all F1 lines with the d60Gal line showed tallness and partial sterility, reduced by 25% in average from those with the D60gal line (Koshihikari) and the D60Gal line. These data indicated that the genotype of the 33 varieties is D60D60galgal and that the d60 locus is not allelic to those of sd1, d1, d2, d6, d18k, d29, d30, d35, d49, d50, and qCL1 involved in the 33 varieties. In addition, the gal gene is not complementarily activated with the semidwarf and dwarf genes described above, other than d60. The Gal gene will be ubiquitously distributed in rice. It is emphasized that Gal is a rare and valuable mutant gene essential to the transmission of d60. The double dwarf genotype of homozygous d30d60 was rarely gained in the F3 of the d30 line × d60 line by breaking their repulsion d60-D30 linkage on chromosome 2.

Identification of Rice Large Grain Gene GW2 by Whole-Genome Sequencing of a Large Grain-Isogenic Line Integrated with Japonica Native Gene and Its Linkage Relationship with the Co-integrated Semidwarf Gene d60 on Chromosome 2.

Genetic analysis of "InochinoIchi," an exceptionally large grain rice variety, was conducted through five continuous backcrosses with Koshihikari as a recurrent parent using the large grain F3 plant in Koshihikari × Inochinoichi as a nonrecurrent parent. Thorough the F2 and all BCnF2 generations, large, medium, and small grain segregated in a 1:2:1 ratio, indicating that the large grain is controlled by a single allele. Mapping by using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers with small grain homozygous segregants in the F2 of Nipponbare × Inochinoichi, revealed linkage with around 7.7 Mb markers from the distal end of the short arm of chromosome 2. Whole-genome sequencing on a large grain isogenic Koshihikari (BC4F2) using next-generation sequencing (NGS) identified a single nucleotide deletion in GW2 gene, which is located 8.1 Mb from the end of chromosome 2, encoding a RING protein with E3 ubiquitin ligase activity. The GW2-integrated isogenic Koshihikari showed a 34% increase in thousand kernel weight compared to Koshihikari, while retaining a taste score of 80. We further developed a large grain/semi-dwarf isogenic Koshihikari integrated with GW2 and the semidwarfing gene d60, which was found to be localized on chromosome 2. The combined genotype secured high yielding while providing robustness to withstand climate change, which can contribute to the New Green Revolution.

Rice Novel Semidwarfing Gene d60 Can Be as Effective as Green Revolution Gene sd1.

Gene effects on the yield performance were compared among promising semidwarf genes, namely, novel gene d60, representative gene sd1 with different two source IR8 and Jukkoku, and double dwarf combinations of d60 with each sd1 allele, in a Koshihikari background. Compared with the culm length of variety Koshihikari (mean, 88.8 cm), that of the semidwarf or double dwarf lines carrying Jukkoku_sd1, IR8_sd1, d60, Jukkoku_sd1 plus d60, or IR8_sd1 plus d60 was shortened to 71.8 cm, 68.5 cm, 65.7 cm, 48.6 cm, and 50.3 cm, respectively. Compared with the yield of Koshihikari (mean, 665.3 g/m2), that of the line carrying Jukkoku_sd1 allele showed the highest value (772.6 g/m2, 16.1% higher than Koshihikari), while that of IR8_sd1, d60 and IR8_sd1 plus d60, was slightly decreased by 7.1%, 5.5%, and 9.7% respectively. The line carrying Jukkoku_sd1 also showed the highest value in number of panicles and florets/panicle, 16.2% and 11.1% higher than in Koshihikari, respectively, and these effects were responsible for the increases in yield. The 1000-grain weight was equivalent among all genetic lines. Except for the semidwarf line carrying Jukkoku_sd1, semidwarf line carrying d60 was equivalent to line carrying IR8_sd1in the yield of unpolished rice, and yield components such as panicle length, panicle number, floret number /panicle. Therefore, the semidwarfing gene d60 is one of the best possible choices in practical breeding.

Genetic Performance of the Semidwarfing Allele sd1 Derived from a Japonica Rice Cultivar and Minimum Requirements to Detect Its Single-Nucleotide Polymorphism by MiSeq Whole-Genome Sequencing.

The influence of the semidwarfing gene sd1 derived from the rice cultivar Jukkoku (Jukkoku_sd1) and IR8 (IR8_sd1), which contributed to the Green Revolution, d60 from Hokuriku 100, as well as the combination of sd1 and d60 (Jukkoku_sd1 plus d60 and IR8_sd1 plus d60), was investigated using isogenic lines raised by backcrossing with the cultivar Koshihikari. The isogenic lines carrying Jukkoku_sd1, IR8_sd1, d60, Jukkoku_sd1 plus d60, and IR8_sd1 plus d60 had considerably shorter culm lengths than Koshihikari by 19.2%, 22.8%, 26.0%, 45.1%, and 43.4%, respectively. The sd1 plus d60 lines showed additively reduced culms, indicating that the function of d60 was different from sd1. In contrast to the culm reduction, Jukkoku_sd1 showed productive merit with a panicle length of 2.5% greater than the origin. MiSeq next-generation sequencer was used to optimize a minimum scale to detect Jukkoku_sd1 in practical breeding. Mapping with the reference genome of Nipponbare gained the average depths of Koshihikari Jukkoku_sd1 and Koshihikari being 9.17 and 7.29, respectively. Comparing the vcf files of the entire genomes of Koshihikari Jukkoku_sd1 and the virtual Koshihikari revealed a G to T SNP at position 38,382,746 in the sd1 locus on chromosome 1 of Koshihikari, causing a loss-of-function mutation of GA20-oxidase.

Thinopyrum ponticum chromatin-integrated wheat genome shows salt-tolerance at germination stage.

A wild wheatgrass, Thinopyrum ponticum (2n = 10x = 70), which exhibits substantially higher levels of salt tolerance than cultivated wheat, was employed to transfer its salt tolerance to common wheat by means of wide hybridization. A highly salt-tolerant wheat line S148 (2n = 42) was obtained from the BC3F2 progenies between Triticum aestivum (2n = 42) and Th. ponticum. In the cross of S148 × salt-sensitive wheat variety Chinese Spring, the BC4F2 seeds at germination stage segregated into a ratio of 3 salt tolerant to 1 salt sensitive, indicating that the salt tolerance was conferred by a dominant gene block. Genomic in situ hybridization analysis revealed that S148 had a single pair of Th. ponticum-T. aestivum translocated chromosomes bearing the salt-tolerance. This is an initial step of molecular breeding for salt-tolerant wheat.

Identification of an isogenic semidwarf rice cultivar carrying the Green Revolution sd1 gene by multiplex codominant ASP-PCR and SSR markers.

The rice cultivar Hikarishinseiki, a semidwarf isogenotype of Koshihikari carrying the Green Revolution sd1 gene, is increasingly grown in both Japan and the United States. Here, we report DNA diagnosis for Hikarishinseiki targeting its Jukkoku-type sd1 locus, which codes for a defective gibberellin 20-oxidase, with a 1 bp substitution in exon 1 (Jukkoku-type GA20ox-2 mutant allele: Jukkoku_GA20ox-2). An allele-specific primer (ASP)-polymerase chain reaction (PCR) with primers SD1F3 and SD1JR gave a PCR product specific to Jukkoku_GA20ox-2. In addition, ASP-PCR with primers SD1F3 and SD1NRM (which contains a mismatch at the third nucleotide from the 3'-terminus of SD1NR) gave a PCR product specific to non-Jukkoku_GA20ox-2. Multiplex ASP-PCR using SD1F3, VIC dye-labeled SD1JR, and FAM dye-labeled SD1NRM enabled simultaneous codominant detection of Jukkoku_GA20ox-2 and non-Jukkoku_GA20ox-2 among 188 cultivars. Also, Hikarishinseiki is identifiable by RM253 polymorphism from 11 cultivars carrying Jukkoku_GA20ox-2. Taken together, our results establish a methodology for distinguishing Hikarishinseiki.

Rye chromosome-specific polymerase chain reaction products developed by primers designed from the EcoO109I recognition site.

From our analysis of repeat sequences in the rye genome, the presence of multiple restriction sites of EcoO109I (5'-PuGGNCCPy-3') across the genome has been predicted. By first using primers designed to contain EcoO109I sites in polymerase chain reaction (PCR), polymorphic DNA markers were effectively obtained. A total of 43 types of 10-mer primers containing EcoO109I sites were applied for PCR by using genomic DNA of Secale cereale self-fertile line IR27 and Triticum aestivum 'Chinese Spring' (CS) as the template. Twenty two primers detected polymorphisms between wheat and rye, and they were applied for PCR using a series of CS wheat--'Imperial' rye chromosome addition lines as templates. Nine chromosome-specific amplification fragments identified on five chromosomes were collected from gels and hybridized with nylon membrane-transferred PCR products from the wheat-rye chromosome addition lines. The gel blot was only observed between the collected fragments; therefore, these fragments were confirmed to be chromosome-specific. These fragments were sequenced and converted to sequence-tagged site (STS) primers. We therefore introduce a new method for building chromosome-specific DNA markers: (i) multiple polymorphic fragments can be obtained from EcoO109I primers and (ii) the addition of three nucleotides to the EcoO109I site restricts the amplification region to generate chromosome-specific fragments.

Combining two semidwarfing genes d60 and sd1 for reduced height in 'Minihikari', a new rice germplasm in the 'Koshihikari' genetic background.

Dwarfing in rice has dramatically improved and stabilized rice yields worldwide, often controlled by a single dwarf gene, sd1. A novel semidwarf gene d60 complements the gametic lethal gene gal, such that the F(1) between 'Hokuriku 100' (genotype d60d60GalGal, Gal: mutant non-lethal allele) and 'Koshihikari' (D60D60galgal, D60: tall allele) would show 25% sterility due to deterioration of gametes bearing both gal and d60. The F(2) would segregate as one semidwarf (1 d60d60GalGal) : two tall and 25% sterile (2 D60d60Galgal) : six tall (2 D60d60GalGal : 1 D60D60GalGal : 2 D60D60Galgal : 1 D60D60galgal), skewed from a Mendelian segregation ratio of one semidwarf : three tall for a single recessive gene. To pyramid d60 and sd1, into the Japanese super-variety 'Koshihikari', the F(1) (D60d60Galgal) of 'Koshihikari' × 'Hokuriku 100' was first backcrossed with 'Koshihikari', and the BCF(1) segregated into a ratio of one tall and 25% sterile (D60d60Galgal) : two tall (1 D60D60Galgal : 1 D60D60galgal). Tall, 25% sterile BC(1)F(1) plants (D60d60Galgal) were then selected for pollen sterility and backcrossed with 'Koshihikari' as the recurrent parent. It is unnecessary to grow out and select a semidwarf from the BC(n)F(2) if a pollen parent with ~70% pollen fertility is chosen from the BC(n)F(1) to backcross with the recurrent parent. Semidwarfing genes d60 and sd1 were successfully pyramided into the 'Koshihikari' genome by crossing isogenic lines 'Koshihikari d60' and 'Koshihikari sd1', to produce 'Minihikari', a new parental source of both d60 and sd1. 'Minihikari' displayed super-short stature due to the combination of sd1 and d60, which are genetically and functionally independent.

Genomic, RNA, and ecological divergences of the Revolver transposon-like multi-gene family in Triticeae.

BACKGROUND: Revolver is a newly discovered multi-gene family of transposable elements in the Triticeae genome. Revolver encompasses 2929 to 3041 bp, has 20 bp of terminal inverted repeated sequences at both ends, and contains a transcriptionally active gene encoding a DNA-binding-like protein. A putative TATA box is located at base 221, with a cap site at base 261 and a possible polyadenylation signal AATAAA at base 2918. Revolver shows considerable quantitative variation in wheat and its relatives. RESULTS: Revolver cDNAs varied between 395 and 2,182 bp in length. The first exon exhibited length variation, but the second and third exons were almost identical. These variants in the Revolver family shared the downstream region of the second intron, but varied structurally at the 5' first exon. There were 58 clones, which showed partial homology to Revolver, among 440,000 expressed sequence tagged (EST) clones sourced from Triticeae. In these Revolver homologues with lengths of 360-744 bp, the portion after the 2nd exon was conserved (65-79% homology), but the 1st exon sequences had mutually low homology, with mutations classified into 12 types, and did not have EST sequences with open reading frames (ORFs). By PCR with the 3'-flanking region of a typical genomic clone of Revolver-2 used as a single primer, rye chromosomes 1R and 5R could be simultaneously identified. Extensive eco-geographic diversity and divergence was observed among 161 genotypes of the single species Triticum dicoccoides collected from 18 populations in Israel with varying exposures to abiotic and biotic stresses (soil, temperature, altitude, water availability, and pathogens). CONCLUSIONS: On the base of existing differences between Revolver variants, the molecular markers that can distinguish different rye chromosomes were developed. Eco-geographic diversification of wild emmer T. dicoccoides in Israel and high Revolver copy numbers are associated with higher rainfall and biotic stresses. The remarkable quantitative differences among copy numbers of Revolver in the same species from different ecosystems suggest strong amplification activity within the last 10,000 years. It is the interesting finding because the majority of Triticeae high-copy transposable elements seem to be inactive at the recent time except for BARE-1 element in Hordeum and the fact might be interesting to perceive the processes of plant adaptive evolution.

Long-culm mutations with dominant genes are induced by mPing transposon in rice.

Development of semidwarf rice cultivars contributed to the epoch of high yielding crops called the 'Green Revolution'. However, over-reliance on semidwarf rice also has intrinsic limitations to supply food for an ever expanding world population. As a solution to the food supply problem, we propose the development of 'tall dwarf' rice cultivars that are characterized by increased biomass with long culms or large grains. However, genetic studies on the elongation of rice culms have remained scarce. This study seeks to analyze mutant genes involved in culm elongation in long-culm mutants induced by the MITE transposon mPing, which has been shown to be active in the japonica cultivar Gimbozu. Through analysis of the experimental results, we have confirmed that the three mutant long-culm genes exhibit genetic dominance. These represent rare cases of artificially induced dominant mutations. It is very likely that the mPing transposons played an important role in inducing the dominant mutations and also play an evolutionary interesting role.

Revolver and superior: novel transposon-like gene families of the plant kingdom.

High-throughput sequencing of eukaryotic genomes has revived interest in the structure and function of repetitive genomic sequences, previously referred to as junk DNA. Repetitive sequences, including transposable elements, are now believed to play a significant role in genomic differentiation and evolution. Some are also expressed as regulatory noncoding RNAs. Vast DNA databases exist for higher eukaryotes; however, with the exception of homologues of known repetitive-sequence-families and transposable elements, most repetitive elements still need to be annotated. Revolver and Superior, both discovered in the Triticeae, are novel classes of transposon-like genes and major components of large cereal genomes. Revolver was isolated from rye via genome subtraction of sequences common to rye and wheat. Superior was isolated from rye by cleavage with EcoO109I, the recognition sites of which consist of a 5'- PuGGNCCPy-3' multi-sequence. Revolver is 2929-3041 bp long with an inverted repeat sequence on each end. The Superior family elements are 1292-1432 bp in length, with divergent 5' regions, indicating the presence of considerable structural diversity. Revolver and Superior are transcriptionally active elements; Revolver harbors a single gene consisting of three exons and two introns, encoding a protein of 139 amino acid residues. Revolver variants range in size from 2665 bp to 4269 bp, with some variants lacking the 5' region, indicating structural diversity around the first exon. Revolver and Superior are dispersed across all seven chromosomes of rye. Revolver has existed since the diploid progenitor of wheat, and has been amplified or lost in several species during the evolution of the Triticeae. This article reviews the recently discovered Revolver and Superior families of plant transposons, which do not share identity with any known autonomous transposable elements or repetitive elements from any living species.

Effective isolation of retrotransposons and repetitive DNA families from the wheat genome.

New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cleavage with EcoO109I, the recognition sites of which consisted of 5'-PuGGNCCPy-3' multi-sequences. Three novel repetitive sequences pTm6, pTm69 and pTm58 derived from the A genome were screened and tested for high copy number using a blotting approach. pTm6 showed identity with integrase domains of the barley Ty1-Copia-retrotransposon BARE-1 and pTm58 showed similarity to the barley Ty3-gypsy-like retrotransposon Romani. pTm69, however, constituted a tandem array with useful genomic specificities, but did not share any identity with known repetitive elements. This study also sought to isolate wheat D-genome-specific repetitive elements regardless of the level of methylation, by genomic subtraction. Total genomic DNA of Aegilops tauschii was cleaved into short fragments with a methylation-insensitive 4 bp cutter, MboI, and then common DNA sequences between Ae. tauschii and Triticum turgidum were subtracted by annealing with excess T. turgidum genomic DNA. The D genome repetitive sequence pAt1 was isolated and used to identify an additional novel repetitive sequence family from wheat bacterial artificial chromosomes with a size range of 1 395-1 850 bp. The methods successfully led pathfinding of two unique repetitive families.

Centromeric distribution of 350-family in Dasypyrum villosum and its application to identifying Dasypyrum chromatin in the wheat genome.

Variants of the 350-family sequences were isolated from Dasypyrum villosum genomic DNA. Although the consensus sequence shared 86% homology to p380 and 84% homology to pHvNAU62 of D. villosum, a 63 bp long region including variable microsatellite repeat (TG)(7-9) was found to be absent in pHvNAU62 or much different from p380. This sequence also shared 67% homology to the 350-family of rye. The representative clone pDvTU383 was localized on the D. villosum chromosomes by using the sequential C-banding and fluorescence in situ hybridization (FISH). Combining with the probes of pTa71 and pTa794 containing 18S-5.8S-28S rDNA (NOR) and 5S rDNA multigene families, respectively, the pDvTU383 probe allowed identification of all seven pairs of D. villosum chromosomes. The pair of the 1V chromosomes fluoresced strong in situ hybridization signals of 18S-5.8S-28S rRNA genes in the NORs of 1VS, and the pair of the 5V chromosomes had weak signals of 5S rRNA genes at the end of 5VS. The pair of the 4V chromosomes showed sub-telomeric signals of pDvTU383 probe on the both arms, however, the signal of the short arm is weaker than that of the long arm. The pair of the 7V chromosomes showed no signals of pDvTU383 on the both sub-telomeric regions. The pair of the 6V chromosomes showed a characteristic interstitial signal of pDvTU383 on the short arm. The pairs of the 2V and 3V chromosomes were distinguished by the difference of interstitial signals of pDvTU383 on their long arms. Furthermore, hybridization signals of pDvTU383 were also visualized on centromeres of all chromosomes. Based on the above D. villosum chromosomal patterns made by FISH, one pair of the 4V chromosomes originated from D. villosum were found in a trigeneric hybrid line involving wheat, Dasypyrum and Thinopyrum. The sequence pDvTU383 provides an effective probe for further analysis of the D. villosum genome.

Genomic subtraction recovers rye-specific DNA elements enriched in the rye genome.

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3-1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.