A new SNP in cyOsPPDK gene is associated with floury endosperm in Suweon 542.

Pyruvate orthophosphate dikinase (PPDK) is a component of glycolysis to mediate endosperm energy charge by adjusting the ratio of ATP to ADP and AMP that proposed to balance the flow of carbon into starch, protein, fatty acid and amino acid biosynthesis. However, these were inconsistent with the first report of a T-DNA insertional knockout mutant of the rice PPDK gene (flo4) showed that rice with inactivated PPDK gene failed to produce a opaque seeds. Therefore, the PPDK might have multifaceted functions in grain filling stage, which in some ways might depend on the direction of the reversible catalysis. Suweon 542 is a rice (Oryza sativa L.) mutant developed from Oryza sativa ssp. japonica cv. Namil. Suweon 542 has a milky-white floury endosperm suitable for dry filling, with low starch damage, low grain hardness, and fine flour particle size. The mutant locus on chromosome 5 controls the floury endosperm phenotype of Suweon 542. Fine mapping of this locus is required for efficient breeding of rice germplasm suitable for dry milling. In this study, whole genome of Suweon 542 and Milyang 23 were re-sequenced using Illumina HiSeq 2500. Co-segregation analysis of F3:4 family populations derived from Suweon 542/Milyang 23 was performed using eight CAPS markers and phenotypic evaluation of the endosperm. The target region was mapped to a 33 kb region and identified to encode cytosolic pyruvate orthophosphate dikinase protein (cyOsPPDK). A G→A SNP in exon 8 of cyOsPPDK resulting in a missense mutation from Gly to Asp at amino acid position 404 was responsible for the floury endosperm of Suweon 542. qRT-PCR experiments revealed that FLO4-4 was expressed to a considerably higher level in Suweon 542 than in Namil during the grain filling stage. Overall, fine mapping of FLO4-4 and candidate gene analysis provided further insight into the floury endosperm of rice, and reveal a novel SNP in cyOsPPDK gene can affect the floury endosperm phenotype through active PPDK gene during grain filling stage.

OsMPK3 is a TEY-type rice MAPK in Group C and phosphorylates OsbHLH65, a transcription factor binding to the E-box element.

KEY MESSAGE: OsMPK3 is a TEY-type rice MAPK belonging to Group C and directly phosphorylates OsbHLH65 in the nucleus. OsMPK3 and OsbHLH65 are induced by biotic stress and defense-related hormones. Mitogen-activated protein kinases (MAPKs) are involved in the majority of signaling pathways that regulate plant development and stress tolerance via the phosphorylation of target molecules. Plant MAPKs are classified into two subtypes, TEY and TDY, according to the TxY (x = E or D) motif in their activation loop, and the TDY motif is unique to plant MAPKs. In rice, 17 MAPKs have been classified into six groups. To date, the functions of many TDY-type rice MAPKs have been characterized, but little is known of the TEY-type MAPKs in Group C and their possible target substrates. In the study reported here, we determined that a TEY-type rice MAPK belonging to subgroup C, named OsMPK3, phosphorylates its substrate OsbHLH65 in the nucleus. Our electrophoresis mobility shift assay results revealed that OsbHLH65 specifically binds to the E-box cis-element, but not to the G-box. Both OsMPK3 and OsbHLH65 were induced by treatments with rice blast (Magnaporthe grisea), brown planthopper (Nilaparvata lugens), and defense-related hormones, such as methyl jasmonic acid and salicylic acid. Our results suggest the possibility that OsMPK3 contributes to the defense signal transduction by phosphorylating the basic helix-loop-helix transcription factor.

Effects of allelic variations in starch synthesis-related genes on grain quality traits of Korean nonglutinous rice varieties under different temperature conditions.

Influences of allelic variations in starch synthesis-related genes (SSRGs) on rice grain quality were examined. A total of 187 nonglutinous Korean rice varieties, consisting of 170 Japonica and 17 Tongil-type varieties, were grown in the field and in two greenhouse conditions. The percentages of head rice and chalky grains, amylose content, alkali digestion value, and rapid visco-analysis characteristics were evaluated in the three different environments. Among the 10 previously reported SSRG markers used in this study, seven were polymorphic, and four of those showed subspecies-specific allele distributions. Six out of the seven polymorphic SSRG markers were significantly associated with at least one grain quality trait (R (2) > 0.1) across the three different environments. However, the association level and significance were markedly lower when the analysis was repeated using only the 170 Japonica varieties. Similarly, the significant associations between SSRG allelic variations and changes in grain quality traits under increased temperature were largely attributable to the biased allele frequency between the two subpopulations. Our results suggest that within Korean Japonica varieties, these 10 major SSRG loci have been highly fixed during breeding history and variations in grain quality traits might be influenced by other genetic factors.

Allelic combinations of Hd1, Hd16, and Ghd7 exhibit pleiotropic effects on agronomic traits in rice.

Heading date is a critical agronomic trait that significantly affects grain yield and quality in rice. As early heading is typically associated with reduced yield due to shorter growth duration, it is essential to harness optimum heading date genes and their allelic combinations to promote heading while minimizing yield penalties. In this study, we identified quantitative trait loci (QTLs) for heading date and other major agronomic traits in a recombinant inbred line (RIL) population derived from a cross between Koshihikari and Baegilmi. Analyses on 3 major QTLs for heading date and their underlying genes (Hd1, Hd16, and Ghd7) revealed their pleiotropic effects on culm length, panicle length, and head rice percentage. Additionally, Ghd7 exhibited pleiotropic effects on panicle number and grain size. Among 8 different types of allelic combinations of the 3 heading date genes, RILs carrying a single nonfunctional hd16 or ghd7 under the functional background of the other 2 genes (Hd1hd16Ghd7 and Hd1Hd16ghd7) showed potential for maintaining yield and quality-related traits while accelerating heading. These results provide valuable insights for fine-tuning heading dates in rice breeding programs.

RicePilaf: a post-GWAS/QTL dashboard to integrate pangenomic, coexpression, regulatory, epigenomic, ontology, pathway, and text-mining information to provide functional insights into rice QTLs and GWAS loci.

BACKGROUND: As the number of genome-wide association study (GWAS) and quantitative trait locus (QTL) mappings in rice continues to grow, so does the already long list of genomic loci associated with important agronomic traits. Typically, loci implicated by GWAS/QTL analysis contain tens to hundreds to thousands of single-nucleotide polmorphisms (SNPs)/genes, not all of which are causal and many of which are in noncoding regions. Unraveling the biological mechanisms that tie the GWAS regions and QTLs to the trait of interest is challenging, especially since it requires collating functional genomics information about the loci from multiple, disparate data sources. RESULTS: We present RicePilaf, a web app for post-GWAS/QTL analysis, that performs a slew of novel bioinformatics analyses to cross-reference GWAS results and QTL mappings with a host of publicly available rice databases. In particular, it integrates (i) pangenomic information from high-quality genome builds of multiple rice varieties, (ii) coexpression information from genome-scale coexpression networks, (iii) ontology and pathway information, (iv) regulatory information from rice transcription factor databases, (v) epigenomic information from multiple high-throughput epigenetic experiments, and (vi) text-mining information extracted from scientific abstracts linking genes and traits. We demonstrate the utility of RicePilaf by applying it to analyze GWAS peaks of preharvest sprouting and genes underlying yield-under-drought QTLs. CONCLUSIONS: RicePilaf enables rice scientists and breeders to shed functional light on their GWAS regions and QTLs, and it provides them with a means to prioritize SNPs/genes for further experiments. The source code, a Docker image, and a demo version of RicePilaf are publicly available at https://github.com/bioinfodlsu/rice-pilaf.

GWAS analysis reveals the genetic basis of blast resistance associated with heading date in rice.

Rice blast is a destructive fungal disease affecting rice plants at various growth stages, significantly threatening global yield stability. Development of resistant rice cultivars stands as a practical means of disease control. Generally, association mapping with a diversity panel powerfully identifies new alleles controlling trait of interest. On the other hand, utilization of a breeding panel has its advantage that can be directly applied in a breeding program. In this study, we conducted a genome-wide association study (GWAS) for blast resistance using 296 commercial rice cultivars with low population structure but large phenotypic diversity. We attempt to answer the genetic basis behind rice blast resistance among early maturing cultivars by subdividing the population based on its Heading date 1 (Hd1) functionality. Subpopulation-specific GWAS using the mixed linear model (MLM) based on blast nursery screening conducted in three years revealed a total of 26 significant signals, including three nucleotide-binding site leucine-rich repeat (NBS-LRR) genes (Os06g0286500, Os06g0286700, and Os06g0287500) located at Piz locus on chromosome 6, and one at the Pi-ta locus (Os12g0281300) on chromosome 12. Haplotype analysis revealed blast resistance associated with Piz locus was exclusively specific to Type 14 hd1 among japonica rice. Our findings provide valuable insights for breeding blast resistant rice and highlight the applicability of our elite cultivar panel to detect superior alleles associated with important agronomic traits.

A cystathionine-ß-synthase domain-containing protein, CBSX2, regulates endothecial secondary cell wall thickening in anther development.

Anther formation and dehiscence are complex pivotal processes in reproductive development. The secondary wall thickening in endothecial cells of the anther is a known prerequisite for successful anther dehiscence. However, many gaps remain in our understanding of the regulatory mechanisms underlying anther dehiscence in planta, including a possible role for jasmonic acid (JA) and H(2)O(2) in secondary wall thickening of endothecial cells. Here, we report that the cystathionine ß-synthase domain-containing protein CBSX2 located in the chloroplast plays a critical role in thickening of the secondary cell walls of the endothecium during anther dehiscence in Arabidopsis. A T-DNA insertion mutant of CBSX2 (cbsx2) showed increased secondary wall thickening of endothecial cells and early anther dehiscence. Consistently, overexpression of CBSX2 resulted in anther indehiscence. Exogenous JA application induced secondary wall thickening and caused flower infertility in the cbsx2 mutant, whereas it partially restored fertility in the CBSX2-overexpressing lines lacking the wall thickening. CBSX2 directly modulated thioredoxin (Trx) in chloroplasts, which affected the level of H(2)O(2) and, consequently, expression of the genes involved in secondary cell wall thickening. Our findings have revealed that CBSX2 modulates the H(2)O(2) status, which is linked to the JA response and in turn controls secondary wall thickening of the endothecial cells in anthers for dehiscence to occur.

A stress-responsive caleosin-like protein, AtCLO4, acts as a negative regulator of ABA responses in Arabidopsis.

Caleosins or related sequences have been found in a wide range of higher plants. In Arabidopsis, seed-specific caleosins are viewed as oil-body (OB)-associated proteins that possess Ca(2+)-dependent peroxygenase activity and are involved in processes of lipid degradation. Recent experimental evidence suggests that one of the Arabidopsis non-seed caleosins, AtCLO3, is involved in controlling stomatal aperture during the drought response; the roles of the other caleosin-like proteins in Arabidopsis remain largely uncharacterized. We have demonstrated that a novel stress-responsive and OB-associated Ca(2+)-binding caleosin-like protein, AtCLO4, is expressed in non-seed tissues of Arabidopsis, including guard cells, and down-regulated following exposure to exogenous ABA and salt stress. At the seed germination stage, a loss-of-function mutant (atclo4) was hypersensitive to ABA, salt and mannitol stresses, whereas AtCLO4-overexpressing (Ox) lines were more hyposensitive to those stresses than the wild type. In adult stage, atclo4 mutant and AtCLO4-Ox plants showed enhanced and decreased drought tolerance, respectively. Following exposure to exogenous ABA, the expression of key ABA-dependent regulatory genes, such as ABF3 and ABF4, was up-regulated in the atclo4 mutant, while it was down-regulated in AtCLO4-Ox lines. Based on these results, we propose that the OB-associated Ca(2+)-binding AtCLO4 protein acts as a negative regulator of ABA responses in Arabidopsis.

Novel bifunctional nucleases, OmBBD and AtBBD1, are involved in abscisic acid-mediated callose deposition in Arabidopsis.

Screening of the expressed sequence tag library of the wild rice species Oryza minuta revealed an unknown gene that was rapidly and strongly induced in response to attack by a rice fungal pathogen (Magnaporthe oryzae) and an insect (Nilaparvata lugens) and by wounding, abscisic acid (ABA), and methyl jasmonate treatments. Its recombinant protein was identified as a bifunctional nuclease with both RNase and DNase activities in vitro. This gene was designated OmBBD (for O. minuta bifunctional nuclease in basal defense response). Overexpression of OmBBD in an Arabidopsis (Arabidopsis thaliana) model system caused the constitutive expression of the PDF1.2, ABA1, and AtSAC1 genes, which are involved in priming ABA-mediated callose deposition. This activation of defense responses led to an increased resistance against Botrytis cinerea. atbbd1, the knockout mutant of the Arabidopsis ortholog AtBBD1, was susceptible to attack by B. cinerea and had deficient callose deposition. Overexpression of either OmBBD or AtBBD1 in atbbd1 plants complemented the susceptible phenotype of atbbd1 against B. cinerea as well as the deficiency of callose deposition. We suggest that OmBBD and AtBBD1 have a novel regulatory role in ABA-mediated callose deposition.

Novel Sources of Pre-Harvest Sprouting Resistance for Japonica Rice Improvement.

Pre-harvest sprouting (PHS), induced by unexpected weather events, such as typhoons, at the late seed maturity stage, is becoming a serious threat to rice production, especially in the state of California, USA, Japan, and the Republic of Korea, where japonica varieties (mostly susceptible to PHS) are mainly cultivated. A projected economic loss by severe PHS in these three countries could range between 8-10 billion USD per year during the next 10 years. Here, we present promising rice germplasm with strong resistance to PHS that were selected from a diverse rice panel of accessions held in the International Rice Genebank (IRG) at the International Rice Research Institute (IRRI). To induce PHS, three panicle samples per accession were harvested at 20 and 30 days after flowering (DAF), respectively, and incubated at 100% relative humidity (RH), 30 °C in a growth chamber for 15 days. A genome-wide association (GWA) analysis using a 4.8 million single nucleotide polymorphisms (SNP) marker set was performed to identify loci and candidate genes conferring PHS resistance. Interestingly, two tropical japonica and four temperate japonica accessions showed outstanding PHS resistance as compared to tolerant indica accessions. Two major loci on chromosomes 1 and 4 were associated with PHS resistance. A priori candidate genes interactions with rice gene networks, which are based on the gene ontology (GO), co-expression, and other evidence, suggested that a key resistance mechanism is related to abscisic acid (ABA), gibberellic acid (GA), and auxin mediated signaling pathways.

Hd1 Allele Types and Their Associations with Major Agronomic Traits in Korean Rice Cultivars.

Optimizing flowering time in crop plants is critical for maximizing yield and quality under target environments. While there is a wide range of heading date variation in Korean rice cultivars, the underlying gene mechanisms are unclear. Here, we sequenced the protein coding regions of Hd1, the major rice heading date gene, from 293 Korean rice cultivars and investigated the associations between Hd1 allele types and major agronomic traits under four different environments. There were four functional Hd1 and five nonfunctional hd1 alleles distributed among the 293 Korean rice cultivars. The effects of the Hd1 allele types were highly significant for days to heading in all four environments, explaining 51.4-65.8% of the phenotypic variation. On average, cultivars carrying nonfunctional hd1 headed 13.7 days earlier than those carrying functional Hd1. While the Hd1 allele types exhibited highly significant effects on culm length and protein content under all four environments, the differences between cultivars carrying Hd1 and hd1 were minimal. The effects of the Hd1 allele types on amylose content were significant in only one of the four environments. Our results provide useful information for fine-tuning rice heading dates by utilizing different Hd1 alleles in rice breeding programs.

Construction of genomic DNA library of Korean ginseng (Panax ginseng C. A. MEYER) and development of sequence-tagged sites.

This study describes an efficient approach for developing sequence tagged sites (STS) for Panax ginseng C.A. MEYER, and their applications for line discrimination. By using the methylation filtering (MF) technique, a genomic library was constructed, in which clone inserts were derived from the hypomethylated regions of ginseng genome. A methylation unfiltered genomic library was also constructed and the clone inserts were compared to those from the MF library in terms of sequence characteristics. Sequence analysis revealed that MF efficiently enriched the protein coding region of P. ginseng, for which the repetitive DNA appeared to be as little as 2.5 fold lower than clones in the unfiltered library, and also indicated that the P. ginseng genome may contain a large fraction of methylated repetitive DNA elements. A total of 99 and 100 highly stringent STS primer sets were designed from the filtered and unfiltered library, respectively. Amplification products were tested for latent polymorphism across six cultivars of P. ginseng and other 2 Panax species using six endonucleases recognizing four-bases. STS primer sets described here will be useful for marker-assisted selection, genome mapping and line discrimination of P. ginseng or its cultivars from other Panax species.

Characterization of QTLs and Candidate Genes for Days to Heading in Rice Recombinant Inbred Lines.

Understanding the gene mechanisms controlling days to heading (DH) is important in rice breeding for adaption in the target environment. Using a recombinant inbred line population derived from the cross between two japonica rice cultivars, Koshihikari and Baegilmi, we identified three consistent quantitative trait loci (QTLs) for DH for two years, qDH3, qDH6, and qDH7 on chromosomes 3, 6, and 7, respectively. While Baegilmi contributed the allele for early heading at qDH6 and qDH7 with the additive effect of five days each, Koshihikari contributed the allele for early heading at qDH3 with the additive effect of three days. Notably, pyramiding two or more alleles for early heading at these QTLs accelerated heading effectively. Sequencing of Hd16, Hd1, and Ghd7, the previously known heading date genes underlying qDH3, qDH6, and qDH7, respectively, revealed that Baegilmi and Koshihikari carry different alleles at the three genes. Molecular markers were developed to screen the allelic compositions of the three genes among 295 Korean commercial rice cultivars. The results showed that few cultivars carry alleles for early heading at the three genes, highlighting that DH can be further accelerated and fine-tuned in breeding programs by combining the desirable alleles of Hd16, Hd1, and Ghd7.

A New SNP in Rice Gene Encoding Pyruvate Phosphate Dikinase (PPDK) Associated with Floury Endosperm.

Rice varieties with suitable flour-making qualities are required to promote the rice processed-food industry and to boost rice consumption. A rice mutation, Namil(SA)-flo1, produces grains with floury endosperm. Overall, grains with low grain hardness, low starch damage, and fine particle size are more suitable for use in flour processing grains with waxy, dull endosperm with normal grain hardness and a high amylose content. In this study, fine mapping found a C to T single nucleotide polymorphism (SNP) in exon 2 of the gene encoding cytosolic pyruvate phosphate dikinase (cyOsPPDK). The SNP resulted in a change of serine to phenylalanine acid at amino acid position 101. The gene was named FLOURY ENDOSPERM 4-5 (FLO4-5). Co-segregation analysis with the developed cleaved amplified polymorphic sequence (CAPS) markers revealed co-segregation between the floury phenotype and the flo4-5. This CAPS marker could be applied directly for marker-assisted selection. Real-time RT-PCR experiments revealed that PPDK was expressed at considerably higher levels in the flo4-5 mutant than in the wild type during the grain filling stage. Plastid ADP-glucose pyrophosphorylase small subunit (AGPS2a and AGPS2b) and soluble starch synthase (SSIIb and SSIIc) also exhibited enhanced expression in the flo4-5 mutant.

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta.

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F(2) population derived from a cross between an introgression line, 'IR71033-121-15', from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, 'Junambyeo'. Resistance to BPH (biotype 1) was evaluated using 190 F(3) families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.

Development of near-isogenic Japonica rice lines with enhanced resistance to Magnaporthe grisea.

Thirteen near-isogenic lines (NILs) of japonica rice were developed via a backcross method using the recurrent parent Chucheong, which is of good eating quality but is susceptible to Magnaporthe grisea, and three blast resistant japonica donors, Seolak, Daeseong and Bongkwang. The agro-morphological traits of these NILs, such as heading date, culm length, and panicle length, were similar to those of Chucheong. In a genome-wide scan using 158 SSR markers, chromosome segments of Chucheong were identified in most polymorphic regions of the 13 NIL plants, and only a few chromosome segments were found to have been substituted by donor alleles. The genetic similarities of the 13 NILs to the recurrent parent Chucheong averaged 0.961, with a range of 0.932-0.984. Analysis of 13 major blast resistance (R) genes in these lines using specific DNA markers showed that each NIL appeared to contain some combination of the four R genes, Pib, Pii, Pik-m and Pita-2, with the first three genes being present in each line. Screening of nine M. grisea isolates revealed that one NIL M7 was resistant to all nine isolates; the remaining NILs were each resistant to between three and seven isolates, except for NIL M106, which was resistant to only two isolates. In a blast nursery experiment, all the NILs proved to be more resistant than Chucheong. These newly developed NILs have potential as commercial rice varieties because of their increased resistance to M. grisea combined with the desirable agronomic traits of Chucheong. They also provide material for studying the genetic basis of blast resistance.

Identification of putative MAPK kinases in Oryza minuta and O. sativa responsive to biotic stresses.

The mitogen-activated protein kinase (MAPK) signaling cascade is critical for regulating plant defense systems against various kinds of pathogen and environmental stresses. One component of this cascade, the MAP kinase kinases (MAPKK), has not yet been shown to be induced in plants following biotic attacks, such as those by insects and fungi. We describe here a gene coding for a blast (Magnaporthe grisea)- and insect (Nilaparvata lugens)-responsive putative MAPK kinase, OmMKK1 (Oryza minuta MAPKK 1), which was identified in a library of O. minuta expressed sequence tags (ESTs). Two copies of OmMKK1 are present in the O. minuta genome. They encode a predicted protein with molecular mass 39 kDa and pI of 6.2. Transcript patterns following imbibition of plant hormones such as methyl jasmonic acid (MeJA), ethephone, salicylic acid (SA) and abscisic acid (ABA), as well as exposure to methyl viologen (MV), revealed that the expression of OmMKK1 is related to defense response signaling pathways. A comparative analysis of OmMKK1 and its O. sativa ortholog OsMKK1 showed that both were induced by stress-related hormones and biotic stresses, but that the kinetics of their responses differed despite their high amino acid sequence identity (96%).

Identification of QTLs for some agronomic traits in rice using an introgression line from Oryza minuta.

Wild progenitor species provide potential gene sources for complex traits such as yield and multiple resistances to biotic and abiotic stresses, and thus are expected to contribute to sustainable food supplies. An introgression line 'IR71033-121-15' was derived from a wild species Oryza minuta (2n = 48, BBCC, Acc No. 101141) at IRRI. Introgression analysis using 530 SSR and STS markers revealed that at least 14 chromosomal segments distributed over 12 chromosomes had been introgressed from O. minuta. An F2:3 population from the cross between IR71033 and Junambyeo (a Korean japonica cultivar) consisting of 146 lines was used for quantitative trait loci (QTL) analysis of 16 agronomic traits. A total of 36 single-locus QTLs (S-QTLs) and 45 digenic epistasis (E-QTLs) were identified. In spite of it's inferiority of O. minuta for most of the traits studied, its alleles contributed positively to 57% of the QTLs. The other QTLs originated from either parent, IR71033 or Junambyeo. QTLs for phenotypically correlated traits were mostly detected on introgressed segments. Fourteen QTLs corresponded to QTLs reported earlier, indicating that these QTLs are stable across genetic backgrounds. Twenty-two QTLs controlling yield and its components had not been detected in previous QTL studies. Of these, thirteen consisted of potentially novel alleles from O. minuta. QTLs from O. minuta introgression could be new sources of natural variation for the genetic improvement of rice.

Characterization of Ty3-gypsy-like elements in Hibiscus syriacus.

Southern blot analysis revealed a ubiquitous distribu-tion and high copy number of Ty3-gypsy-like elements in the genome of Hibiscus syriacus. Comparative phylogenetic analysis of the large subunit of Rubisco and the integrase region of Ty3-gypsy elements in various plant species indicated that the retrotransposon-like sequences have different evolutionary histories and their own unique polymorphism in the H. syriacus population. Sequence-tagged site-restriction fragment length polymorphisms (STS-RFLP) analysis also indicated great variability in the numbers and sequences of Ty3-gypsy-like elements within H. syriacus varieties. Ty3-gypsy-like elements may still be active within H. syriacus, since Northern analysis of wounded leaves of H. syriacus variety Saehan with a probe for the integrase domain gave strong hybridization signals. The sequence heterogeneity and ubiquity of the Ty3-gypsy-like elements in H. syriacus genomes could provide reliable DNA markers for line identification as well for the analysis of genetic diversity in H. syriacus.

A partial-complementary adapter for an improved and simplified ligation-mediated suppression PCR technique.

Ligation-mediated suppression PCR (LMS-PCR) is a powerful tool for walking in unknown genomic DNA regions from known adjacent sequences. This approach has made it feasible to obtain promoter sequences and to enable researchers to identify full-length gene sequences or isoforms of multigene families. However, the advantages of LMS-PCR can be obviated by the presence of incomplete base modifications on the suppression adapters. We propose here that a 'partial-complementary adapter' is a more reliable suppression adapter, demanding only 5'-end phosphorylation. We also describe a simplified procedure for the easier preparation of PCR templates with very small quantities of DNA and a fast and direct characterization of the suppression-PCR products. A set of practical guidelines is proposed for pre-checking the efficiency of the adapter modification using two model systems: bacteriophage lambda (lambda) and Arabidopsis.