Development and Validation of High-Resolution Melting Markers Derived from Rysto STS Markers for High-Throughput Marker-Assisted Selection of Potato Carrying Rysto.

Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Rysto, a gene responsible for extreme resistance to Potato virus Y (PVY) in potato, was performed in tetraploid potato 'Barbara' (Rrrr) and 'AC Chaleur' (rrrr) as well as their progeny selections. Three and two sequence variants were identified in Barbara resistant (R) selections and AC Chaleur susceptible (S) selections, respectively. Further analysis indicates that the variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harboring the STS markers. Two primer pairs, one targeting the region containing a 20-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in Barbara-R bulk and one visible fragment in AC Chaleur-S bulk; pair two produced one visible fragment in all samples. When subjected to high-resolution melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progeny of Barbara × AC Chaleur revealed 72 and 75 progeny with R and S melting profiles, respectively, which was consistent with YES3-3A and YES3-3B assays and phenotyping analysis, thus demonstrating the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for high-throughput marker-assisted selection for Rysto-conferred resistance to PVY was validated further with three populations involving Barbara as the R parent.

Molecular mapping of restorer-of-fertility 2 gene identified from a sugar beet (Beta vulgaris L. ssp. vulgaris) homozygous for the non-restoring restorer-of-fertility 1 allele.

KEY MESSAGE: By genetically eliminating the major restorer - of - fertility gene ( Rf ), a weak Rf gene was unveiled. It is an allele of Z , long known as an elusive Rf gene in sugar beet. In the hybrid breeding of sugar beet, maintainer-genotype selection is a laborious process because of the dependence on test crossing, despite the very low occurrence of this genotype. Marker-assisted selection (MAS) of the maintainer genotype is highly desired by sugar beet breeders. The major restorer-of-fertility gene (Rf) was identified as Rf1, and its non-restoring allele (rf1) was discriminated at the DNA level; however, some of the rf1rf1 selections retained an as yet unidentified Rf, another target locus for MAS. The objective of this study was to identify this Rf. An rfrf1 plant was crossed to a cytoplasmic male-sterile sugar beet and then backcrossed to obtain progeny segregating the unidentified Rf. The progeny exhibited partial male-fertility restoration that was unstable in single plants. The segregation ratio of restored vs. non-restored plants suggested the involvement of a single Rf in this male-fertility restoration, designated as Rf2. We confirmed the feasibility of molecular tagging of Rf2 by identifying four shared amplified fragment length polymorphism (AFLP) fragments specific to 17 restored plants. Bulked segregant analysis also was performed to screen the Rf2-linked AFLP markers, which were subsequently converted into 17 sequence-tagged site markers. All the markers, as well two additional chromosome-IV-assigned markers, were linked to each other to form a single linkage map, on which Rf2 was located. Our data suggested that Rf2 is likely an allele of Z, long known as an elusive Rf gene in sugar beet. We also discuss the importance of Rf2 for sugar beet breeding.

Expressed sequence tag analysis of khat (Catha edulis) provides a putative molecular biochemical basis for the biosynthesis of phenylpropylamino alkaloids

Khat (Catha edulis Forsk.) is a flowering perennial shrub cultivated for its neurostimulant properties resulting mainly from the occurrence of (S)-cathinone in young leaves. The biosynthesis of (S)-cathinone and the related phenylpropylamino alkaloids (1S,2S)-cathine and (1R,2S)-norephedrine is not well characterized in plants. We prepared a cDNA library from young khat leaves and sequenced 4,896 random clones, generating an expressed sequence tag (EST) library of 3,293 unigenes. Putative functions were assigned to > 98 percent of the ESTs, providing a key resource for gene discovery. Candidates potentially involved at various stages of phenylpropylamino alkaloid biosynthesis from L-phenylalanine to (1S,2S)-cathine were identified.

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.

Genetic mapping of common buckwheat using DNA, protein and morphological markers.

Genetic mapping of F(2) progeny (n = 225) of hybrids between the Sobano variety of common buckwheat (Fagopyrum esculentum Moench) and the Homo wild accession (F. esculentum var. homotropicum) was carried out using randomly amplified polymorphic DNA (RAPD), sequence-tagged site (STS) and seed protein subunit markers, and three morphological traits. Ten linkage groups were identified, involving 87 RAPD markers, 12 STS markers, four seed protein subunit (PS(62)/PS(59), PS(49.8)/PS(51.4), PS(44)/PS(42.9), and PS(39.9)/PS(37.8)) markers, and three morphological alleles controlling homo/long style (H/s), shattering habit (Sht/sht), and acute/obtuse achene ridge (Ac/ac), covering a total of 655.2 cM.

Functional genomics of rice pollen and seed development by genome-wide transcript profiling and Ds insertion mutagenesis.

Rice pollen and seed development are directly related to grain yield. To further improve rice yield, it is important for us to functionally annotate the genes controlling pollen/seed development and to use them for rice breeding. Here we first carried out a genome-wide expression analysis with an emphasis on genes being involved in rice pollen and seed development. Based on the transcript profiling, we have identified and functionally classified 82 highly expressed pollen-specific, 12 developing seed-specific and 19 germinating seed-specific genes. We then presented the utilization of the maize transposon Dissociation (Ds) insertion lines for functional genomics of rice pollen and seed development and as alternative germplasm resources for rice breeding. We have established a two-element Activator/Dissociation (Ac/Ds) gene trap tagging system and generated around 20,000 Ds insertion lines. We have subjected these lines for screens to obtain high and low yield Ds insertion lines. Some interesting lines have been obtained with higher yield or male sterility. Flanking Sequence Tags (FSTs) analyses showed that these Ds-tagged genes encoded various proteins including transcription factors, transport proteins, unknown functional proteins and so on. They exhibited diversified expression patterns. Our results suggested that rice could be improved not only by introducing foreign genes but also by knocking out its endogenous genes. This finding might provide a new way for rice breeder to further improve rice varieties.

A novel Rf gene controlling fertility restoration of Ogura male sterility by RNA processing of orf138 found in Japanese wild radish and its STS markers.

To reveal the molecular and genetic mechanism of fertility restoration in Ogura male sterility in Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides), we investigated fertility restoration of a plant that lacks the dominant type of orf687, a previously identified fertility restorer gene. A total of 100 F2 plants were made from the cross between a male-sterile strain with the Ogura cytoplasm, 'MS-Gensuke', and a Japanese wild radish plant. Segregation of pollen fertility in the F2 plants led us to assume that 2 dominant complementary genes controlled the fertility restoration of the plants. However, the fertility of 27 of 59 male-fertile plants was not completely restored, resulting in a group of plants with partial male fertility. Northern blot analysis of the CMS-associated gene orf138 indicated that one restorer allele (termed Rft) was involved in the processing of orf138 RNA. Rapid amplification of cDNA ends (RACE) and subsequent Northern blot analysis confirmed that the orf138 transcript lost a 5' part of the coding region of the orf138 gene in the restored plants. The accumulation of ORF138 protein was significantly reduced by Rft, but trace amounts of the protein were recognized in both partially male-fertile and male-sterile plants with Rft. The relationship of pollen fertility and segregation of co-dominant sequence tagged site (STS) markers in the F2 generation suggested that the penetrance of Rft was so low that Rft needs suitable conditions to function sufficiently for the complete restoration of fertility.

ACC synthase genes are polymorphic in watermelon (Citrullus spp.) and differentially expressed in flowers and in response to auxin and gibberellin.

The flowering pattern of watermelon species (Citrullus spp.) is either monoecious or andromonoecious. Ethylene is known to play a critical role in floral sex determination of cucurbit species. In contrast to its feminizing effect in cucumber and melon, in watermelon ethylene promotes male flower development. In cucumber, the rate-limiting enzyme of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), regulates unisexual flower development. To investigate the role of ethylene in flower development, we isolated four genomic sequences of ACS from watermelon (CitACS1-4). Both CitACS1 and CitACS3 are expressed in floral tissue. CitACS1 is also expressed in vegetative tissue and it may be involved in cell growth processes. Expression of CitACS1 is up-regulated by exogenous treatment with auxin, gibberellin or ACC, the immediate precursor of ethylene. No discernible differential floral sex-dependent expression pattern was observed for this gene. The CitACS3 gene is expressed in open flowers and in young staminate floral buds (male or hermaphrodite), but not in female flowers. CitACS3 is also up-regulated by ACC, and is likely to be involved in ethylene-regulated anther development. The expression of CitACS2 was not detected in vegetative or reproductive organs but was up-regulated by auxin. CitACS4 transcript was not detected under our experimental conditions. Restriction fragment length polymorphism (RFLP) and sequence tagged site (STS) marker analyses of the CitACS genes showed polymorphism among and within the different Citrullus groups, including watermelon cultivars, Citrullus lanatus var. lanatus, the central subspecies Citrullus lanatus var. citroides, and the desert species Citrullus colocynthis (L).

[A new method to obtain molecular marker of sequence-tagged site (STS) of Panax ginseng and P. quinquefolius].

OBJECTIVE: Searching a new molecular method to authenticate Panax ginseng and P. quenquefolium. METHOD: Single primers based on rDNA sequences of Panax species were designed to obtain polymorphic bands of P. ginseng and P. quinquefolius and then sequenced. Four PCR primers (two forword and two reverse primers) specific to P. ginseng and P. quinquefolius were designed. RESULT: Primer Pg-6F, Pg-479R only amplified 474 bp band for P. ginseng and primer Pq-442F, Pq-658R only amplified 217 bp band for P. quinquefolius. It is indicated that the four primers could serve as specific STS primers for Panax species. CONCLUSION: A new way to obtain STS primers of Panax species was established. This method is more quick and efficient than SCAR-PCR method and can serve as a model to obtain molecular markers for other Chinese material medica.

A new method to obtain molecular marker of sequence-tagged site (STS) of Panax ginseng and P. quinquefolius / 中国中药杂志

<p><b>OBJECTIVE</b>Searching a new molecular method to authenticate Panax ginseng and P. quenquefolium.</p><p><b>METHOD</b>Single primers based on rDNA sequences of Panax species were designed to obtain polymorphic bands of P. ginseng and P. quinquefolius and then sequenced. Four PCR primers (two forword and two reverse primers) specific to P. ginseng and P. quinquefolius were designed.</p><p><b>RESULT</b>Primer Pg-6F, Pg-479R only amplified 474 bp band for P. ginseng and primer Pq-442F, Pq-658R only amplified 217 bp band for P. quinquefolius. It is indicated that the four primers could serve as specific STS primers for Panax species.</p><p><b>CONCLUSION</b>A new way to obtain STS primers of Panax species was established. This method is more quick and efficient than SCAR-PCR method and can serve as a model to obtain molecular markers for other Chinese material medica.</p>

DeepSAGE--digital transcriptomics with high sensitivity, simple experimental protocol and multiplexing of samples.

Digital transcriptomics with pyrophosphatase based ultra-high throughput DNA sequencing of di-tags provides high sensitivity and cost-effective gene expression profiling. Sample preparation and handling are greatly simplified compared to Serial Analysis of Gene Expression (SAGE). We compare DeepSAGE and LongSAGE data and demonstrate greater power of detection and multiplexing of samples derived from potato. The transcript analysis revealed a great abundance of up-regulated potato transcripts associated with stress in dormant potatoes compared to harvest. Importantly, many transcripts were detected that cannot be matched to known genes, but is likely to be part of the abiotic stress-response in potato.

STS markers linked to the Rf1 fertility restorer gene of cotton.

Marker-assisted selection (MAS) can accelerate the process of plant breeding, and sequence-tagged site (STS) markers are highly specific for regions of DNA being used for MAS. The objective of this research was to develop STS markers tightly linked with Rf1, the fertility restoring gene for cytoplasmic male sterility (CMS) in cotton (Gossypium hirsutum L.). Bulked segregant analysis was employed to screen for Rf1-linked RAPD markers in a backcross population. Four RAPD markers were identified, three of which co-segregated with Rf1 (UBC147(1400), UBC607(500), and UBC679(700)). Another fragment, UBC169(800), co-segregated with the previously reported UBC169(700) in repulsion phase at a distance of 4.5 cM from Rf1. A marker published by others (UBC659(1500)) mapped to 2.7 cM from Rf1 and 1.8 cM from UBC169(800). Four sets of STS primer pairs were designed based on the RAPD fragment sequences. The primer pairs from the UBC147(1400) and UBC607(500) fragments both amplified a single fragment specific to fertile plants. The UBC679(700) and UBC659(1500) STS primer pairs each amplified one fragment specific to fertile plants and a monomorphic fragment. These four Rf1-linked STS markers were also present in the Rf1 donor species G. harknessii (D2-2). The three primer pairs that produced co-segregating STS markers also amplified fragments from G. trilobum (D8). However, the D8 fragment amplified by the UBC147(1400) STS primers was larger than that from D2-2, and G. trilobum does not restore fertility to CMS-D2-2 lines. These STS markers will be useful in the development of restorer parental lines in cotton CMS breeding efforts.

Identification of AFLP makers linked to non-seed shattering locus (sht1) in buckwheat and conversion to STS markers for marker-assisted selection.

Shattering habit in buckwheat has two forms: brittle pedicel and weak pedicel. Brittle pedicel is observed in wild buckwheat, but not in cultivated buckwheat. Brittle pedicel in buckwheat is produced by two complementary, dominant genes, Sht1 and Sht2. The sht1 locus is linked to the S locus; almost all common buckwheat cultivars possess the allele sht1. To detect molecular makers linked to the sht1 locus, we used amplified fragment-length polymorphism (AFLP) analysis in combination with bulked segregant analysis of segregating progeny of a cross between a non-brittle common buckwheat and a brittle self-compatible buckwheat line. We screened 312 primer combinations and constructed a linkage map around the sht1 locus by using 102 F2 plants. Five AFLP markers were linked to the sht1 locus. Two of these, e54m58/610 and e55m46/320, cosegregated with the sht1 locus without recombination. The two AFLP markers were converted to STS markers according to the sequence of the AFLPs. The STS markers are useful for marker-assisted selection of non-brittle pedicel plants and provides a stepping-stone for map-based cloning and characterization of the gene encoding non-brittle pedicel.

Use of serial analysis of gene expression technology to reveal changes in gene expression in Arabidopsis pollen undergoing cold stress.

We have characterized the global gene expression patterns of Arabidopsis pollen using Serial Analysis of Gene Expression (SAGE). A total of 21,237 SAGE tags were sequenced and 4,211 unique tags were identified. Interestingly, the number of unique tags in pollen was low compared with the SAGE library of the leaf constructed on a similar scale. The transcript profiles of pollen reflect accurately the characteristics of pollen as a reproductive organ. Functional classification of the expressed genes reveals that those involved in cellular biogenesis such as polygalacturonase, pectate lyase, and pectin methylesterase make up more than 40% of the total transcripts. However, genes involved in energy and protein synthesis, which are prevalent in leaves, were expressed at a relatively low level. The expression level of the great majority of transcripts was unaffected by cold treatment at 0 degrees C for 72 h, whereas pollen tube growth and seed production were substantially reduced. Interestingly, many genes thought to be responsible for cold acclimation such as COR, lipid transfer protein, and beta-amylase, that are highly induced in Arabidopsis leaves, were only expressed at their normal level or weakly induced in the pollen. The expression patterns of the cold-responsive transcripts identified by SAGE were confirmed by microarray analysis. Our results strongly suggest that poor accumulation of proteins that play a role in stress tolerance may be why Arabidopsis pollen is cold sensitive.

Fine mapping of the rice thermo-sensitive genic male-sterile gene tms5.

AnnongS-1, a thermo-sensitive genic male-sterile (TGMS) rice line, has a new TGMS gene. Genetic analysis indicated that the sterility of AnnongS-1 was controlled by a single resessive gene named tms5. In our previous studies based on an F(2) population from the cross between AnnongS-1 and Nanjing11, tms5 was mapped on chromosome 2. Recently, a RIL (recombinant inbred line) population from the same cross was developed and used for the fine mapping of the tms5 gene. Molecular marker techniques combined with BSA (bulked segregant analysis) were used. As a result, two AFLP markers (AF10, AF8), one RAPD marker (RA4), one STS marker (C365-1), one CAPs marker (G227-1) and four SSR markers (RM279, RM492, RM327, RM324) were found to be closely linked to tms5 gene. The DNA sequences of the RFLP marker of C365 and G227 were found in GenBank, and on the basis of these sequences, many primers were designed to amplify the two parents and their RIL population plants. Finally, the tms5 gene was mapped between STS marker C365-1 and CAPs marker G227-1 at a distance of 1.04 cM from C365-1 and 2.08 cM from G227-1.

Direct amplification of length polymorphism analysis differentiates Panax ginseng from P. quinquefolius.

The method of direct amplification of length polymorphism (DALP) was applied to authenticate Panax ginseng and P. quinquefolius. A 636-bp DALP fragment was present in all P. ginseng but absent in all the P. quinquefolius cultivars examined. We have shown that the use of DALP and conversion of specific polymorphic band to sequence-tagged site (STS) for quick authentication may be applied to authenticate related medicinal materials.

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

Y-chromosome microdeletion and its effect on reproductive decisions in taiwanese patients presenting with nonobstructive azoospermia.

OBJECTIVES: To investigate the position, extent, and frequency of Y chromosome microdeletions in Taiwanese patients presenting with nonobstructive azoospermia, and to investigate the effect of microdeletions on reproductive decisions. METHODS: We studied 176 consecutive men with azoospermia in our urology clinic. Polymerase chain reaction tests were performed in 94 patients with nonobstructive azoospermia, and a series of 27 sequence-tagged sites (STSs) mapped within intervals 5 and 6 of Yq11 was selected for analysis. Clinical genetics counseling was provided to couples with microdeletions, and these couples made their own choices about further treatment modalities. RESULTS: Among 94 patients screened for microdeletion, 11 (11.7%) showed microdeletions of one or more STSs. One had a deletion confined to the azoospermia factor b (AZFb) region (encompassing the RBM gene). Two were found to have deletions of both the AZFb and AZFc regions. Eight patients had deletions in the AZFc region (encompassing the DAZ gene). Five had deletions distal to the DAZ gene family. One had multiple, noncontiguous deletions. In 8 patients with testicular histology available, a lack of genotype/phenotype correlation was noted. Of the 11 couples with deletions, 3 thought microdeletion was a serious defect and opted for an artificial insemination of donor or adoption, 5 chose intracytoplasmic sperm injection, and the other 3 decided to undergo treatment with Chinese medicinal herbs. CONCLUSIONS: The most commonly deleted region in the Taiwanese population is AZFc. The genes implicated in Taiwanese spermatogenesis defects are the DAZ and RBM gene families. Twenty-seven percent of couples with microdeletions deferred assisted reproductive technologies because of concern about their underlying genetic defects.

Analysis of libraries encoded with GC tags: compound elution, tag decode analysis, and statistical sampling analysis.

Libraries encoded with electrophoric tags present a unique challenge with respect to library quality control and characterization. Libraries are prepared on Tentagel resin in 200-fold redundancy wherein each resin particle contains one compound per one tag set. The amount of compound present on the bead is ca. 200-500 pmole while tag levels are estimated at 0.5-1 pmol/bead. Several quality control protocols have been developed in order to accurately estimate bead yield and purity for the entire library, ensure high tag fidelity, and to determine the overall performance of individual synthons. This review provides a unique, collective portrait of Pharmacopeia's approach in assessing the quality of libraries prepared using its molecular encoding technology.

Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome.

A size-selected genomic library comprising 280,000 colonies and representing approximately 18% of the chickpea genome, was screened for (GA)n, (GAA)n and (TAA)n microsatellite-containing clones, of which 389 were sequenced. The majority (approximately 75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%-9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613 cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P > or = 0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.