Expressed Sequence Tag Markers Research Articles

Identification and Mapping of Nucleotide Binding Site–Leucine-rich Repeat Resistance Gene Analogs in Bermudagrass

Thirty-one partial bermudagrass (Cynodon spp.) disease-resistance gene analogs (BRGA) were cloned and sequenced from diploid, triploid, tetraploid, and hexaploid bermudagrass using degenerate primers to target the nucleotide binding site (NBS) of the NBS–leucine-rich repeat (LRR) resistance gene family. Alignment of deduced amino acid sequences revealed that the conserved motifs of the NBS are present and all sequences have non-Drosophila melanogaster Toll and mammalian interleukin-1 receptor (TIR) motifs. Using a neighbor-joining algorithm, a dendrogram was created and nine groups of deduced amino acid sequences from bermudagrass could be identified from those sequences that span the NBS. Four BRGA markers and 15 bermudagrass expressed sequence tags (ESTs) with similarity to resistance genes or resistance gene analogs were placed on a bermudagrass genetic map. Multiple BRGA and EST markers mapped on T89 linkage groups 1a and 5a and clusters were seen on T89 19 and two linkage groups previously unidentified. In addition, three primers made from BRGA groups and ESTs with similarity to NBS-LRR resistance genes amplify NBS-LRR analogs in zoysiagrass (Zoysia japonica or Z. matrella) or seashore paspalum (Paspalum vaginatum). This gives evidence of conservation of NBS-LRR analogs among the subfamilies Chloridoideae and Panicoideae. Once disease resistance genes are identified, these BRGA and EST markers may be useful in marker-assisted selection for the improvement of disease resistance in bermudagrass.

Identification and Molecular Mapping of Xa32(t), a Novel Resistance Gene for Bacterial Blight ( Xanthomonas oryzae pv. oryzae) in Rice

The rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae ( Xoo), is the most devastating bacterial disease of rice worldwide. Because of the rapid changes in the pathogenicity of the pathogen ( Xoo), novel BB-resistance genes are always needed for rice (Oryza sativa L.) breeding. In this article, a novel BB-resistance gene was identified from a wild rice ( Oryzae ustraliensis) introgression line, C4064, and its chromosomal location was reported. On the basis of the resistance spectrum analysis, C4064 presented resistance to Xoo strains P1 (PXO61), P4 (PXO71), P5 (PXO112), P6 (PXO99), P7 (PXO145), P8 (PXO280), P9 (PXO339), and KX085, but susceptible to P2 (PXO86) and P3 (PXO79). Therefore, the resistance gene harbored in C4064 was a novel BB-resistance gene and tentatively designated as Xa32(t). Furthermore, the chromosomal position of this gene was located using bulked segregant analysis with the help of simple sequence repeat and expressed sequence tag markers. Of the 299 markers tested, 6 markers, i.e., RM27256, RM27274, RM2064, ZCK24, RM6293, and RM5926 on the long arm of chromosome 11, were linked with Xa32(t) with genetic distances of 2.1, 1.0, 1.0, 0.5, 1.5, and 2.6 cM between the resistance gene and the markers, respectively. The resistance gene was located between markers RM6293 and RM5926 at the end side of long arm and the other 4 markers at the centromere side. Finally, Xa32(t) was mapped within a length of 2.0 cM on the long arm of rice chromosome 11.

EST, COSII, and arbitrary gene markers give similar estimates of nucleotide diversity in cultivated tomato (Solanum lycopersicum L.)

Because cultivated tomato (Solanum lycopersicum L.) is low in genetic diversity, public, verified single nucleotide polymorphism (SNP) markers within the species are in demand. To promote marker development we resequenced approximately 23 kb in a diverse set of 31 tomato lines including TA496. Three classes of markers were sampled: (1) 26 expressed-sequence tag (EST), all of which were predicted to be polymorphic based on TA496, (2) 14 conserved ortholog set II (COSII) or unigene, and (3) ten published sequences, composed of nine fruit quality genes and one anonymous RFLP marker. The latter two types contained mostly noncoding DNA. In total, 154 SNPs and 34 indels were observed. The distributions of nucleotide diversity estimates among marker types were not significantly different from each other. Ascertainment bias of SNPs was evaluated for the EST markers. Despite the fact that the EST markers were developed using SNP prediction within a sample consisting of only one TA496 allele and one additional allele, the majority of polymorphisms in the 26 EST markers were represented among the other 30 tomato lines. Fifteen EST markers with published SNPs were more closely examined for bias. Mean SNP diversity observations were not significantly different between the original discovery sample of two lines (53 SNPs) and the 31 line diversity panel (56 SNPs). Furthermore, TA496 shared its haplotype with at least one other line at 11 of the 15 markers. These data demonstrate that public EST databases and noncoding regions are a valuable source of unbiased SNP markers in tomato.

Genetic Dissection of Seed Production Traits and Identification of a Major‐Effect Seed Retention QTL in Hybrid Leymus (Triticeae) Wildryes

ABSTRACTMany native grasses display seed shattering and other seed production problems. Basin wildrye [Leymus cinereus (Scribn. & Merr.) Á. Löve] and creeping wildrye [L. triticoides (Buckley) Pilg.] are perennial Triticeae grasses native to western North America. In this study, variation in the number of florets per inflorescence, percent seed set, seed mass, seed shattering, and seed dormancy was measured in clonally replicated multiyear field evaluations of 164 progeny derived from a backcross between a creeping × basin wildrye hybrid and a true creeping wildrye tester. Genomewide quantitative trait locus (QTL) scans detected independent gene effects for all five traits and one pleiotropic QTL, located on Triticeae homeologous chromosome group 6, associated with major seed shattering (43.1% R2, 26.9 log of odds likelihood ratio [LOD]) and seed dormancy (24.1% R2, 10.6 LOD) effects. We hypothesize that a dominant basin wildrye gene creates an abscission that promotes seed disarticulation but hinders seed development and seed ripening in the creeping wildrye backcross family. Alignments of Leymus chromosome 6 expressed sequence tag markers to the rice (Oryza) genome reveal well‐defined correspondence of Leymus wildrye and American wildrice (Zizania palustris L.) seed shatter QTLs.

DEVELOPMENT AND CHARACTERIZATION OF POLYMORPHIC EST MARKERS IN COMMON CARP

Expressed sequence tags(ESTs) are of high value in genome mapping and marker-assisted selection(MAS).This study aims to develop EST markers for genome mapping and other studies of common carp(Cyprinus carpio L).A total of 7381 EST sequences(400—3500bp) were downloaded from the EST database of the GenBank as the source data of the present analyses.Sixty-seven pairs of primers(designated as HB1-67) were designed in the 3′ untranslated region(UTR) of the common carp ESTs,47 of which can successfully amplify specific products from genomic DNA.Twelve polymorphic EST markers were identified in a backcross mapping family of common carp by single-strand conformation polymorphism(SSCP) analysis.Six of the 12 polymorphic EST markers were derived from common carp genes of known function,and the rest were from common carp ESTs homologous to putative genes in zebrafish(Danio rerio) or channel catfish(Ictalurus punctatus).Allele distribution of these polymorphic EST markers was found to follow the Mendelian segregation(1∶1 or 3∶1) in both the backcross family and gynogenetic haploids,and the number of SSCP bands in haploid population was half of that in diploid family.The gene diversity(H=0.437) of Dongting Lake population of common carp revealed by 4 polymorphic EST markers is much lower than that by previous microsatellite analysis(close to 1).The results of present study indicated that EST-SSCP markers,with codominant inheritance,can be used as a powerful tool in the genetics of not only diploid but also haploid common carp.As Type I marker derived from coding region with relatively lower genetic variability,EST-SSCP markers are still of high potentials in such studies as genome mapping(including genetic linkage maps and gene maps,as well as comparative mapping),genetic structure,and genetic adaptability of fish populations.

Genetic Diversity Analysis and Single-nucleotide Polymorphism Marker Development in Cultivated Bulb Onion Based on Expressed Sequence Tag–Simple Sequence Repeat Markers

Bulb onion (Allium cepa L.) is a globally significant crop, but the structure of genetic variation within and among populations is poorly understood. We broadly surveyed genetic variation in a cultivated onion germplasm using simple sequence repeat (SSR) markers and sequenced regions flanking expressed sequence tag (EST)-SSRs to develop single-nucleotide polymorphism (SNP) markers. Samples from 89 inbred and open-pollinated (OP) bulb onion populations of wide geographical adaptation and four related Allium L. accessions were genotyped with 56 EST-SSR and four genomic SSR markers. Multivariate analysis of genetic distances among populations resolved long-day, short-day, and Indian populations. EST-SSR markers frequently revealed two major alleles at high frequency in OP populations. The median proportion of single-locus polymorphic loci was 0.70 in OP and landrace populations compared with 0.43 in inbred lines. Resequencing of 24 marker amplicons revealed additional SNPs in 17 (68%) and five SNP assays were developed from these, suggesting that resequencing of EST markers can readily provide SNP markers for purity testing of inbreds and other applications in Allium genetics.

Development and annotation of perennial Triticeae ESTs and SSR markers

Triticeae contains hundreds of species of both annual and perennial types. Although substantial genomic tools are available for annual Triticeae cereals such as wheat and barley, the perennial Triticeae lack sufficient genomic resources for genetic mapping or diversity research. To increase the amount of sequence information available in the perennial Triticeae, three expressed sequence tag (EST) libraries were developed and annotated for Pseudoroegneria spicata, a mixture of both Elymus wawawaiensis and E. lanceolatus, and a Leymus cinereus x L. triticoides interspecific hybrid. The ESTs were combined into unigene sets of 8 780 unigenes for P. spicata, 11 281 unigenes for Leymus, and 7 212 unigenes for Elymus. Unigenes were annotated based on putative orthology to genes from rice, wheat, barley, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Simple sequence repeat (SSR) markers were developed, tested for amplification and polymorphism, and aligned to the rice genome. Leymus EST markers homologous to rice chromosome 2 genes were syntenous on Leymus homeologous groups 6a and 6b (previously 1b), demonstrating promise for in silico comparative mapping. All ESTs and SSR markers are available on an EST information management and annotation database (http://titan.biotec.uiuc.edu/triticeae/).

Linkage maps of the apple (Malus × domestica Borkh.) cvs ‘Ralls Janet’ and ‘Delicious’ include newly developed EST markers

Two apple genetic linkage maps were constructed using amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), random amplified polymorphic DNAs (RAPDs), and expressed sequence tag (EST)-derived markers in combination with a pseudo-testcross mapping strategy in which the cultivars ‘Ralls Janet’ and ‘Delicious’ were used as the respective seed parents. Mitsubakaido (Malus sieboldii) was used as the pollen parent for each of the segregating F1 populations. Expressed sequence tag data were obtained from the random sequencing of cDNA libraries constructed from in vitro cultured shoots and maturing fruits of cv ‘Fuji’, which is the offspring of a cross between ‘Ralls Janet’ and ‘Delicious’. In addition, a number of published gene sequences were used to develop markers for mapping. The ‘Ralls Janet’ map consisted of 346 markers (178 AFLPs, 95 RAPDs, 54 SSRs, 18 ESTs, and the S locus) in 17 linkage groups, with a total length of 1082 cM, while that of ‘Delicious’ comprised 300 markers (120 AFLPs, 81 RAPDs, 64 SSRs, 32 ESTs, and the S, Rf, and MdACS-1 loci) on 17 linkage groups spanning 1031 cM. These maps are amenable to comparisons with previously published maps of ‘Fiesta’ and ‘Discovery’ (Liebhard et al., Mol Breed 10:217–241, 2002; Liebhard et al., Theor Appl Genet 106:1497–1508, 2003a) because several of the SSRs (one to three markers per linkage group) were used in all of the maps. Distorted marker segregation was observed in three and two regions of the ‘Ralls Janet’ and ‘Delicious’ maps, respectively. These regions were localized in different parts of the genome from those in previously reported apple linkage maps. This marker distortion may be dependent on the combinations of cultivars used for map construction.

TriMEDB: A database to integrate transcribed markers and facilitate genetic studies of the tribe Triticeae

BackgroundThe recent rapid accumulation of sequence resources of various crop species ensures an improvement in the genetics approach, including quantitative trait loci (QTL) analysis as well as the holistic population analysis and association mapping of natural variations. Because the tribe Triticeae includes important cereals such as wheat and barley, integration of information on the genetic markers in these crops should effectively accelerate map-based genetic studies on Triticeae species and lead to the discovery of key loci involved in plant productivity, which can contribute to sustainable food production. Therefore, informatics applications and a semantic knowledgebase of genome-wide markers are required for the integration of information on and further development of genetic markers in wheat and barley in order to advance conventional marker-assisted genetic analyses and population genomics of Triticeae species.DescriptionThe Triticeae mapped expressed sequence tag (EST) database (TriMEDB) provides information, along with various annotations, regarding mapped cDNA markers that are related to barley and their homologues in wheat. The current version of TriMEDB provides map-location data for barley and wheat ESTs that were retrieved from 3 published barley linkage maps (the barley single nucleotide polymorphism database of the Scottish Crop Research Institute, the barley transcript map of Leibniz Institute of Plant Genetics and Crop Plant Research, and HarvEST barley ver. 1.63) and 1 diploid wheat map. These data were imported to CMap to allow the visualization of the map positions of the ESTs and interrelationships of these ESTs with public gene models and representative cDNA sequences. The retrieved cDNA sequences corresponding to each EST marker were assigned to the rice genome to predict an exon-intron structure. Furthermore, to generate a unique set of EST markers in Triticeae plants among the public domain, 3472 markers were assembled to form 2737 unique marker groups as contigs. These contigs were applied for pairwise comparison among linkage maps obtained from different EST map resources.ConclusionTriMEDB provides information regarding transcribed genetic markers and functions as a semantic knowledgebase offering an informatics facility for the acceleration of QTL analysis and for population genetics studies of Triticeae.

Genomic targeting and mapping of tiller inhibition gene (tin3) of wheat using ESTs and synteny with rice

Changes in plant architecture have been central to the domestication of wild species. Tillering or the degree of branching determines shoot architecture and is a key component of grain yield and/or biomass. Previously, a tiller inhibition mutant with monoculm phenotype was isolated and the mutant gene (tin3) was mapped in the distal region of chromosome arm 3AmL of Triticum monococcum. As a first step towards isolating a candidate gene for tin3, the gene was mapped in relation to physically mapped expressed sequence tags (ESTs) and sequence tag site (STS) markers developed based on synteny with rice. In addition, we investigated the relationship of the wheat region containing tin3 with the corresponding region in rice by comparative genomic analysis. Wheat ESTs that had been previously mapped to deletion bins provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 region. The tin3 gene was mapped to a 324-kb region spanned by two overlapping bacterial artificial chromosomes (BACs) of rice chromosome arm 1L. Wheat-rice synteny was exceptionally high at the tin3 region despite being located in the high-recombination, gene-rich region of wheat. Identification of tightly linked flanking EST and STS markers to the tin3 gene and its localization to highly syntenic rice BACs will assist in the future development of a high-resolution map and map-based cloning of the tin3 gene.

Comparative study of the discriminating capacity and effectiveness of AFLP, STMS and EST markers in assessing genetic relationships among evergreen azaleas

AbstractThe application of amplified fragment length polymorphism (AFLP), sequence tagged microsatellite site (STMS) and expressed sequence tag (EST) markers to study the genetic relationships in an evergreen azalea gene pool was investigated. STMS and EST markers revealed a higher genetic distance detection capacity than AFLPs, which, nevertheless, were the most efficient marker system due to their highest polymorphism detection capacity. Similarity matrices showed weak, yet significant, correlations when Mantel's test was applied. To assess the usefulness of the overall information provided by these marker data for establishing phylogenetic relationships and horticultural classification, cluster analysis was performed. The joint AFLP, STMS and EST data were demonstrated to be remarkably effective for group discrimination and phylogenetic studies. The use of these polymerase chain reaction marker systems is discussed in terms of the choice of appropriate marker techniques for different aspects of evergreen azalea germplasm evaluation.

Barley EST Markers Enhance Map Saturation and QTL Mapping in Diploid Wheat

A linkage map was constructed using PCR-based markers developed from barley expressed sequence tags (ESTs) for recombinant inbred (RI) lines (F10) derived from a cross between Triticum monococcum L. strain KT3-5 and T. boeoticum Boiss. strain KT1-1. Among the primer sets of 2,695 barley ESTs, 242 EST markers were integrated into the frame of a previously constructed diploid wheat linkage map with 96 RFLP markers. The total map length comprising barley EST, previously mapped RFLP and morphological markers was 1,038.1 cM, and the average distance between two adjacent loci was 3.1 cM. The linkage map showed saturation with the barley EST markers by covering extended telomeric regions and increasing the resolution compared with the previous RFLP map. Order of barley ESTs on the diploid wheat linkage map mostly showed a collinearity to that on the barley linkage map constructed previously, although the long arms of the homoeologous linkage groups 4 and 5 showed structural changes between diploid wheat and barley. Phenotypes of nine quantitative traits were scored for the diploid wheat RI lines and their parents. Composite interval mapping enabled to detect 24 quantitative trait loci for the traits. Thus, barley ESTs could become powerful tools to reveal homoeologous relationships between barley and wheat chromosomes, and valuable resources to develop genetic markers for wheat genomes.

Comparative linkage mapping of Oryza glumaepatula and Oryza sativa interspecific crosses based on microsatellite and expressed sequence tag markers

Molecular linkage maps representing the rice genome have been an important tool for breeding programs because they allow the elucidation of polygenic traits and are an efficient tool for monitoring wild introgressions in interspecific crosses. Common markers among rice genetic maps are important in defining the homology of chromosomes and the synteny between genomic target regions. We used 148 markers (expressed sequence tags, microsatellites and single nucleotide polymorphisms) to construct a molecular linkage map based on co-dominant markers for an interspecific backcross population using a wild rice (Oryza glumaepatula) from Brazil and performed a comparative analysis with other interspecific maps. The comparative analysis revealed a Spearman correlation index of 0.86 for marker order conservation to a previous map constructed for an interspecific cross using the same wild parent. Approximately 90% of markers common to other interspecific maps kept the same order. These results indicate that it will be possible to generate a unique genetic map using the wild donor and that it may be a helpful tool for breeding programs because plants derived from different interspecific populations can be rapidly scanned using markers associated with useful wild traits.

Application of comparative genomics in developing molecular markers tightly linked to the virus resistance geneRsv4 in soybean

The Rsv4 gene confers resistance to all the known strain groups of soybean mosaic virus in soybean (Glycine max (L.) Merr.). To construct a fine genetic map near Rsv4 in soybean, we employed a comparative genomics approach that used genome sequence information of the model legume Lotus japonicus. Sequences of the soybean expressed sequence tags (ESTs) AI856415 and BF070293 mapping to one side of the Rsv4 gene showed high similarity with gene sequences of the transformation-competent artificial chromosome (TAC) clone LjT32P24 of Lotus. LjT32P24 is tightly linked to another sequenced TAC clone, LjT26I01, in Lotus. A new marker, AW307114A, developed from soybean EST AW307114, which is homologous to a Lotus gene within LjT26I01, was mapped to the other side of the Rsv4 gene. The identification of the microsyntenic relationship facilitated the development of additional 2 EST markers between BF070293-S and AW307114A bracketing the Rsv4 gene. Several other markers developed in this study were mapped to putative homoeologous or duplicated chromosomal regions in soybean. Alignment between the soybean maps indicated that Rsv4 is located near a local chromosomal rearrangement. This targeted comparative mapping serves to provide a foundation for marker-assisted selection and cloning of the Rsv4 gene.

Mapping of the rice (Oryza sativa L.) thermo-sensitive genic male sterile gene tms5 with EST and SSR markers

With the cDNA suppression subtraction hybridization method, a spikelet-specific cDNA library was constructed that expressed at meiosis stage in rice. A total of 121 cDNA fragments were selected from the library and used as EST (expressed sequence tags) markers to detect the polymorphism between Annong N, a normal fertile Indica rice line and Annong S-1, its spontaneous mutant with thermo-sensitive genic male sterility, using the RFLP (restriction fragment length polymorphism) technique. HN57, one of the EST probes, could detect polymorphism between them. The results of segregation analysis with the F2 population developed from Annong S-1 and Annong N indicate that HN57 co-segregates with the thermo-sensitive genic male-sterility controlled by tms5, the recessive gene in Annong S-1. This marker is located on the 31.2-cM region of the chromosome 2 of RGP (rice genome research program) genetic map. To further determine the location of tms5, 80 SSR (simple sequence repeat) markers around this region were developed, and 12 of them were polymorphic. And finally, the tms5 was mapped within region of 181 kb by using these new markers.

Mapping of expressed sequence tag markers with a cDNA‐amplified fragment length polymorphism method in Japanese quail (Coturnix japonica)

ABSTRACTIn our previous study, a Kobe‐NIBS Japanese quail (KNQ) linkage map was constructed mainly using amplified fragment length polymorphism (AFLP) markers. In order to compare chicken and quail chromosomes, we developed expressed sequence tag (EST) markers derived from cDNA‐AFLP fragments and localized these markers on the linkage map. Using a total of 128 AFLP primer combinations, 24 polymorphic bands were obtained between a neurofilament‐deficient mutant quail line male and a muscular disorder quail line female, which were the parents of the KNQ resource family. Nine of the 24 markers were mapped by linkage analysis. These markers were mapped to seven linkage groups, namely 1, 2, 3, 6, 8, 15 and 42. A subsequent homology search using chicken genome sequences strongly suggests that these linkage groups correspond with chicken chromosomes 1, 2, 3, 5, 15, 23 and 26.

Molecular markers derived from EST: their development and applications in comparative genomics

Expressed sequence tags (EST), which have been increasing rapidly in number recently, provide important resources for the development of molecular markers. Compared with conventional markers derived from genomic DNA, the EST-derived markers are a novel type of molecular tool with remarkable advantages such as being easy to develop, more informative, and highly transferable. EST markers have been used in many research fields. In the present paper, five kinds of recently developed EST markers, including EST-PCR, EST-SSR, EST-SNP, EST-RFLP, and EST-AFLP, as well as their development strategies based on bioinformatics, are introduced. Moreover, the applications of these markers in comparative genomics studies, including comparative mapping, genetic diversity evaluation, and phylogenetics and so on, are also reviewed.

Uneven distribution of expressed sequence tag loci on maize pachytene chromosomes

Examining the relationships among DNA sequence, meiotic recombination, and chromosome structure at a genome-wide scale has been difficult because only a few markers connect genetic linkage maps with physical maps. Here, we have positioned 1195 genetically mapped expressed sequence tag (EST) markers onto the 10 pachytene chromosomes of maize by using a newly developed resource, the RN-cM map. The RN-cM map charts the distribution of crossing over in the form of recombination nodules (RNs) along synaptonemal complexes (SCs, pachytene chromosomes) and allows genetic cM distances to be converted into physical micrometer distances on chromosomes. When this conversion is made, most of the EST markers used in the study are located distally on the chromosomes in euchromatin. ESTs are significantly clustered on chromosomes, even when only euchromatic chromosomal segments are considered. Gene density and recombination rate (as measured by EST and RN frequencies, respectively) are strongly correlated. However, crossover frequencies for telomeric intervals are much higher than was expected from their EST frequencies. For pachytene chromosomes, EST density is about fourfold higher in euchromatin compared with heterochromatin, while DNA density is 1.4 times higher in heterochromatin than in euchromatin. Based on DNA density values and the fraction of pachytene chromosome length that is euchromatic, we estimate that approximately 1500 Mbp of the maize genome is in euchromatin. This overview of the organization of the maize genome will be useful in examining genome and chromosome evolution in plants.

Genomic differentiation of Hordeum chilense from H. vulgare as revealed by repetitive and EST sequences

Hordeum vulgare, cultivated barley, and its wild relative, H. chilense, have several important traits that might be useful for wheat improvement. Here, in situ hybridization and barley expressed sequence tag (EST) markers were used to characterize and compare the chromosomes of H. chilense with those of H. vulgare. FISH with four repetitive DNA sequences, AG, AAG, 5S rDNA and 45S rDNA, was applied to the mitotic chromosomes of H. vulgare, H. chilense and available wheat-H. chilense addition and substitution lines. FISH with the AAG repeat differentiated the individual chromosomes of H. chilense and H. vulgare. The patterns of FISH signals in the two species differed greatly. The 45S rDNA signals were observed on two pairs of chromosomes in both species, while the 5S rDNA signals were observed on four pairs of chromosomes in H. vulgare and on one pair in H. chilense. The AG repeat showed FISH signals at the centromeric regions of all chromosomes of H. vulgare but none of the chromosomes of H. chilense. These results indicate that the chromosomes of the two species are highly differentiated. To study the homoeology between the two species, 209 EST markers of H. vulgare were allocated to individual chromosomes of H. chilense. One hundred and forty of the EST markers were allocated to respective chromosomes of H. chilense using the wheat-H. chilense addition and substitution lines. Twenty-six EST markers on average were allocated to each chromosome except to the chromosome 2H(ch)S, to which only 10 markers were allocated. Ninety percent of the allocated EST markers in H. chilense were placed on H. vulgare chromosomes of the same homo-eologous group, indicating that the expressed sequences of the two species were highly conserved. These EST markers would be useful for detecting chromatin introgressed from these species into the wheat genome.

The medaka midblastula transition as revealed by the expression of the paternal genome

At midblastula transition (MBT), zygotic gene transcription is activated, cells become motile and cell division becomes asynchronous. The onset of the medaka ( Oryzias latipes) MBT was examined using expressed sequence tag (EST) markers. Among 187 randomly chosen medaka EST markers, 33 EST markers and two genes (eIF-4C and hsc70) showed polymorphisms in terms of insertion/deletions or restriction sites between the two parental inbred strains, one from the northern Japanese population and the other from the southern Japanese population. There was no evidence of zygotic expression of these EST markers before Stage 10 (early blastula stage), whereas expression of 12 genes was found from Stage 11 on. These results suggest that the medaka MBT in terms of first time transcription of paternal genes in the life of the embryo begins at Stage 11 (late blastula stage).