[Axillary recurrences after sentinel lymph node biopsy in initial breast cancer]. / Análisis de recurrencias axilares tras biopsia selectiva del ganglio centinela en cáncer de mama inicial.

OBJECTIVE: The aim of our study was to analyze the application of the Selective Sentinel Lymph Node Biopsy (SLNB) in early Breast Cancer of our population, through the analysis of axillary recurrences in patients with false negative sentinel node procedures without complete axillary lymphadenectomy, after a subsequent clinical follow-up. MATERIAL AND METHODS: A total of 218 early Breast Cancer patients who underwent SLNB after being diagnosed of early breast cancer (T1-2N0) with complete axillary dissection only when the SLNB was positive in the histopathological analysis. In every case, a 2-day protocol was used to localize the sentinel node after injection of (99m)Tc-Nanocolloid. RESULTS: The mean subsequent clinical follow-up was 27 months. A total of 413 sentinel nodes were removed with a median of 1.89/p (range 1-5). Infiltration was detected in 33.9% of patients (59.45% macrometastasis, 22.97% micrometastasis and 17.5% Isolated Tumor Cells (ITC)) and negative for the other nodes excised after conventional lymphadenectomy in 60% of cases. In our population, there was only one case of false negative (FN) SLN due to massive lymphatic blockage, and an abnormal lymph node without uptake adjacent to the SLN was identified intraoperatively. No case of axillary recurrence was detected during an average follow-up of 27 months. CONCLUSION: The absence of axillary recurrences in our population with negative SLNB without complete axillary dissection demonstrates the appropriate local control offered by this procedure in early Breast Cancer.

Strong correlation of wild barley (Hordeum spontaneum) population structure with temperature and precipitation variation.

In this study, we present the genetic analysis of a new collection of wild barley (Hordeum spontaneum) using 42 simple sequence repeat (SSR) markers that represent the seven chromosomes. The Barley1K (B1K) infrastructure consists of 1020 accessions collected in a hierarchical sampling mode (HSM) from 51 sites across Israel and represents the wide adaptive niche of the modern barley's ancestor. According to the genetic structure analysis, the sampled sites can be divided into seven groups, and sampled microsites located on opposing slopes or in different soil types did not show significant genetic differentiation. Although the genetic analysis indicates a simple isolation-by-distance model among the populations, examination of the genetic populations' structure with abiotic parameters in an ordination analysis revealed that the combination of elevation, mid-day temperature and rainfall explains a high proportion of the variance in the principal components analysis. Our findings demonstrate that the current populations have therefore been shaped and distinguished by non-selective forces such as migration; however, we suggest that aridity and temperature gradients played major roles as selective forces in the adaptation of wild barley in this part of the Fertile Crescent. This unique collection is a prelude for the investigation of the molecular basis underlying plant adaptation and responsiveness to harsh environments.

EST-derived single nucleotide polymorphism markers for assembling genetic and physical maps of the barley genome.

In a panel of seven genotypes, 437 expressed sequence tag (EST)-derived DNA fragments were sequenced. Single nucleotide polymorphisms (SNPs) that were polymorphic between the parents of three mapping populations were mapped by heteroduplex analysis and a genome-wide consensus map comprising 216 EST-derived SNPs and 4 InDel (insertion/deletion) markers was constructed. The average frequency of SNPs amounted to 1/130 bp and 1/107.8 bp for a set of randomly selected and a set of mapped ESTs, respectively. The calculated nucleotide diversities (pi) ranged from 0 to 40.0 x 10(-3) (average 3.1 x 10(-3)) and 0.52 x 10(-3) to 39.51 x 10(-3) (average 4.37 x 10(-3)) for random and mapped ESTs, respectively. The polymorphism information content value for mapped SNPs ranged from 0.24 to 0.50 with an average of 0.34. As expected, combination of SNPs present in an amplicon (haplotype) exhibited a higher information content ranging from 0.24 to 0.85 with an average of 0.50. Cleaved amplified polymorphic sequence assays (including InDels) were designed for a total of 87 (39.5%) SNP markers. The high abundance of SNPs in the barley genome provides avenues for the systematic development of saturated genetic maps and their integration with physical maps.

Single nucleotide polymorphisms in rye (Secale cereale L.): discovery, frequency, and applications for genome mapping and diversity studies.

To elucidate the potential of single nucleotide polymorphism (SNP) markers in rye, a set of 48 barley EST (expressed sequence tag) primer pairs was employed to amplify from DNA prepared from five rye inbred lines. A total of 96 SNPs and 26 indels (insertion-deletions) were defined from the sequences of 14 of the resulting amplicons, giving an estimated frequency of 1 SNP per 58 bp and 1 indel per 214 bp in the rye transcriptome. A mean of 3.4 haplotypes per marker with a mean expected heterozygosity of 0.66 were observed. The nucleotide diversity index (pi) was estimated to be in the range 0.0059-0.0530. To improve assay cost-effectiveness, 12 of the 14 SNPs were converted to a cleaved amplified polymorphic sequence (CAPS) format. The resulting 12 SNP loci mapped to chromosomes 1R, 3R, 4R, 5R, 6R, and 7R, at locations consistent with their known map positions in barley. SNP genotypic data were compared with genomic simple sequence repeat (SSR) and EST-derived SSR genotypic data collected from the same templates. This showed a broad equivalence with respect to genetic diversity between these different data types.

A high density barley microsatellite consensus map with 775 SSR loci.

A microsatellite or simple sequence repeat (SSR) consensus map of barley was constructed by joining six independent genetic maps based on the mapping populations 'Igri x Franka', 'Steptoe x Morex', 'OWB(Rec) x OWB(Dom)', 'Lina x Canada Park', 'L94 x Vada' and 'SusPtrit x Vada'. Segregation data for microsatellite markers from different research groups including SCRI (Bmac, Bmag, EBmac, EBmag, HVGeneName, scsssr), IPK (GBM, GBMS), WUR (GBM), Virginia Polytechnic Institute (HVM), and MPI for Plant Breeding (HVGeneName), generated in above mapping populations, were used in the computer program RECORD to order the markers of the individual linkage data sets. Subsequently, a framework map was constructed for each chromosome by integrating the 496 "bridge markers" common to two or more individual maps with the help of the computer programme JoinMap 3.0. The final map was calculated by following a "neighbours" map approach. The integrated map contained 775 unique microsatellite loci, from 688 primer pairs, ranging from 93 (6H) to 132 (2H) and with an average of 111 markers per linkage group. The genomic DNA-derived SSR marker loci had a higher polymorphism information content value (average 0.61) as compared to the EST/gene-derived SSR loci (average 0.48). The consensus map spans 1,068 cM providing an average density of one SSR marker every 1.38 cM. Such a high-density consensus SSR map provides barley molecular breeding programmes with a better choice regarding the quality of markers and a higher probability of polymorphic markers in an important chromosomal interval. This map also offers the possibilities of thorough alignment for the (future) physical map and implementation in haplotype diversity studies of barley.

A high-density consensus map of barley to compare the distribution of QTLs for partial resistance to Puccinia hordei and of defence gene homologues.

A consensus map of barley was constructed based on three reference doubled haploid (DH) populations and three recombinant inbred line (RIL) populations. Several sets of microsatellites were used as bridge markers in the integration of those populations previously genotyped with RFLP or with AFLP markers. Another set of 61 genic microsatellites was mapped for the first time using a newly developed fluorescent labelling strategy, referred to as A/T labelling. The final map contains 3,258 markers spanning 1,081 centiMorgans (cM) with an average distance between two adjacent loci of 0.33 cM. This is the highest density of markers reported for a barley genetic map to date. The consensus map was divided into 210 BINs of about 5 cM each in which were placed 19 quantitative trait loci (QTL) contributing to the partial resistance to barley leaf rust (Puccinia hordei Otth) in five of the integrated populations. Each parental barley combination segregated for different sets of QTLs, with only few QTLs shared by any pair of cultivars. Defence gene homologues (DGH) were identified by tBlastx homology to known genes involved in the defence of plants against microbial pathogens. Sixty-three DGHs were located into the 210 BINs in order to identify candidate genes responsible for the QTL effects. Eight BINs were co-occupied by a QTL and DGH(s). The positional candidates identified are receptor-like kinase, WIR1 homologues and several defence response genes like peroxidases, superoxide dismutase and thaumatin.

Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome.

A set of 111,090 barley expressed sequence tags (ESTs) was searched for the presence of microsatellite motifs [simple sequence repeat (SSRs)] and yielded 2,823 non-redundant SSR-containing ESTs (SSR-ESTs). From this, a set of 754 primer pairs was designed of which 525 primer pairs yielded an amplicon and as a result, 185 EST-derived microsatellite loci (EST-SSRs) were placed onto a genetic map of barley. The markers show a uniform distribution along all seven linkage groups ranging from 21 (7H) to 35 (3H) markers. Polymorphism information content values ranged from of 0.24 to 0.78 (average 0.48). To further investigate the physical distribution of the EST-SSRs in the barley genome, a bacterial artificial chromosomes (BAC) library was screened. Out of 129 markers tested, BAC addresses were obtained for 127 EST-SSR markers. Twenty-seven BACs, forming eight contigs, were hit by two or three EST-SSRs each. This unexpectedly high incidence of EST-SSRs physically linked at the sub-megabase level provides additional evidence of an uneven distribution of genes and the segmentation of the barley genome in gene-rich and gene-poor regions.

Expression genetics and haplotype analysis reveal cis regulation of serine carboxypeptidase I (Cxp1), a candidate gene for malting quality in barley (Hordeum vulgare L.).

Using a cDNA array-based functional genomics approach in barley, several candidate genes for malting quality including serine carboxypeptidase I (Cxp1) were previously identified (Potokina et al. in Mol Breed 14:153, 2004). The gene was mapped as a single nucleotide polymorphism (SNP) marker on chromosome 3H using the Steptoe (feeding grade)xMorex (malting grade) mapping population. Subsequently, the relative level of Cxp1 expression was determined by real-time RT-PCR for each of the 134 progeny lines and mapped as a quantitative trait. Only one quantitative trait locus (QTL) could be identified that significantly influenced the level of the Cxp1 expression. The expressed QTL maps to the same region on chromosome 3H as does the structural gene and corresponds to a QTL for "diastatic power," one among several traits measured to assess malting quality. An analysis of 90 barley cultivars sampled from a worldwide collection revealed six SNPs at the Cxp1 locus, three of which display complete linkage disequilibrium and define two haplotypes. The Cxp1 expression level in a set of barley accessions showing haplotype I was significantly higher than that of accessions displaying haplotype II. The data provide evidence that (1) the expression of Cxp1 is regulated in cis and that (2) the level of diastatic power in the barley seed is influenced by the level of Cxp1 expression.

The gibberellic-acid insensitive dwarfing gene sdw3 of barley is located on chromosome 2HS in a region that shows high colinearity with rice chromosome 7L.

In this study, comparative high resolution genetic mapping of the GA-insensitive dwarfing gene sdw3 of barley revealed highly conserved macrosynteny of the target region on barley chromosome 2HS with rice chromosome 7L. A rice contig covering the sdw3-orthologous region was identified and subsequently exploited for marker saturation of the target interval in barley. This was achieved by (1) mapping of rice markers from the orthologous region of the rice genetic map, (2) mapping of rice ESTs that had been physically localized on the rice contig, or (3) mapping of barley ESTs that show strong sequence similarity to coding sequences present in the rice contig. Finally, the sdw3 gene was mapped to an interval of 0.55 cM in barley, corresponding to a physical distance of about 252 kb in rice, after employing orthologous EST-derived rice markers. Three putative ORFs were identified in this interval in rice, which exhibited significant sequence similarity to known signal regulator genes from different species. These ORFs can serve as starting points for the map-based isolation of the sdw3 gene from barley.

Snipping polymorphisms from large EST collections in barley (Hordeum vulgare L.).

The public EST (expressed sequence tag) databases represent an enormous but heterogeneous repository of sequences, including many from a broad selection of plant species and a wide range of distinct varieties. The significant redundancy within large EST collections makes them an attractive resource for rapid pre-selection of candidate sequence polymorphisms. Here we present a strategy that allows rapid identification of candidate SNPs in barley (Hordeum vulgare L.) using publicly available EST databases. Analysis of 271,630 EST sequences from different cDNA libraries, representing 23 different barley varieties, resulted in the generation of 56,302 tentative consensus sequences. In all, 8171 of these unigene sequences are members of clusters with six or more ESTs. By applying a novel SNP detection algorithm (SNiPpER) to these sequences, we identified 3069 candidate inter-varietal SNPs. In order to verify these candidate SNPs, we selected a small subset of 63 present in 36 ESTs. Of the 63 SNPs selected, we were able to validate 54 (86%) using a direct sequencing approach. For further verification, 28 ESTs were mapped to distinct loci within the barley genome. The polymorphism information content (PIC) and nucleotide diversity (pi) values of the SNPs identified by the SNiPpER algorithm are significantly higher than those that were obtained by random sequencing. This demonstrates the efficiency of our strategy for SNP identification and the cost-efficient development of EST-based SNP-markers.

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.).

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.

Differential gene expression during seed germination in barley (Hordeum vulgare L.).

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process.

EST analysis in barley defines a unigene set comprising 4,000 genes.

We report the generation of 13,109 EST (Expressed Sequence Tag) sequences from barley as a first step towards the generation of a unigene set for this organism. Sequences were generated from three libraries encompassing 7,568 cDNA clones. Comparisons to nucleic acid and protein sequence databases enabled the assignment of putative functions to the mRNAs. The results of the searches against protein databases were parsed and built into a regularly updated database, available over the World Wide Web. The Stack_Pack clustering system has been applied to survey the level of redundancy, which was calculated to amount to 69%, thus we identified 4,000 different barley genes. To prove the usability of the results of the clustering process for further experiments, we subjected alignments with sequences similar to elongation factor 1 alpha to additional analysis. These sequences represented the largest group with identical putative functions (228 members) and clustering based on the analysis of 3; sequences subdivided the group into five different assemblies. Alignments of the consensus sequences facilitated the development of PCR assays suitable for genetic mapping of four of the different gene-family members, which reside on chromosomes 2H, 4H and 5H, thus demonstrating the suitability of the cluster-results as a basis for in-depth analyses of barley gene families.

Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.).

Recent advances in DNA sequence analysis and the establishment of high-throughput assays have provided the framework for large-scale discovery and analysis of DNA sequence variation. In this context, single nucleotide polymorphisms (SNPs) are of particular interest. To initiate a systematic approach to develop an SNP map of barley (Hordeum vulgare L.), we have employed denaturing high-performance liquid chromatography (DHPLC) to analyse segregating SNP patterns in a doubled-haploid (DH) mapping population. To this end, SNPs between the parental genotypes were identified using a direct sequencing approach. Once a SNP was established between the parents, the optimal melting temperature of the PCR fragment containing the SNP was predicted for its analysis by DHPLC. Following the detection of the optimal temperature, the DH lines were analysed for the presence of either of the alleles. To test the utility of the analysis, data from previously mapped RFLP markers from which these SNPs were derived were compared. Results from these experiments indicate that DHPLC can be efficiently employed in analysing SNPs on a high-throughput scale.

New evidence for the synteny of rice chromosome 1 and barley chromosome 3H from rice expressed sequence tags.

To provide improved access to the wealth of resources and genomic information that is presently being developed for rice a set of 88 rice expressed sequence tags (ESTs) previously mapped on rice chromosome I in the cross 'Nipponbare' x 'Kasalath' was used for comparative mapping in a cross of the barley cultivars 'Igri' and 'Franka'. As expected. most (89%) of the clones gave distinct banding patterns in barley of which about one-third was polymorphic between 'Igri' and 'Franka'. These polymorphisms were mapped, and most of these (56%) confirmed that rice chromosome 1 and barley chromosome 3H are syntenous. All single-copy markers identified conserved collinear positions, while markers with multiple copies did so in a few cases only. The markers that were not fitting in the collinear order were distributed randomly across the barley genome. The comparative maps of barley chromosome 3H and rice chromosome 1 comprise in total 26 common markers covering more than 95% of the genetic length of both chromosomes. A 30-fold reduction of recombination is seen around the barley centromere, and synteny may be interrupted in this region. However, the good overall synteny on a mesoscale (1-10 cM) justifies the use of rice as a platform for map-based cloning in barley.

Generation and comparison of EST-derived SSRs and SNPs in barley (Hordeum vulgare L.).

The progress of genome sequencing projects of model plants like barley, combined with the recent advances of high throughput assays, has provided a wealth of sequence information. This information is being employed to develop a high density transcript map of barley (Hordeum vulgare L.). To achieve this goal, the available EST database is being used as a resource for the development of novel microsatellite (SSR) and single nucleotide polymorphism (SNP) markers. So far, a total of 692 microsatellites representing different di-, tri- and tetra-nucleotide repeats were identified from a set of 19,000 EST sequences. Non-redundant SSRs have been used for mapping and so far 76 microsatellite loci were mapped. In addition to the 180 SNP primer pairs, which were designed to target specific ESTs, 72 were polymorphic among the seven genotypes examined here. Of these, 60 SNPs have been mapped applying a denaturing HPLC approach. To examine the potential of the EST-derived markers for pedigree studies, EST-derived SSRs (75 loci) and SNPs (72 loci) were used to fingerprint a set of seven genotypes. The results show that although both marker types yielded similar groupings, a larger data set of both SSRs and SNPs is necessary to obtain stable clusters in unrelated germplasm.

Sequence analysis and gene identification in a set of mapped RFLP markers in barley (Hordeum vulgare).

The "Igri/Franka" (I/F) map ranks among the most comprehensive genetic linkage maps of barley (Hordeum vulgare), containing a large number of markers derived from cDNA and genomic PstI clones. Fourty-three cDNA clones and 259 genomic clones were at least partially sequenced and compared with the major data bases of protein and nucleic acid sequences. Of the cDNA clones, 53% show significant similarity to known sequences in protein data bases. A comparison of sequences from genomic clones to nucleic acid sequence data bases revealed similarities for 9% of the clones. For cDNA sequences analyzed the same way, significant similarities were observed for 35% of the clones. These results show that genomic PstI clones, although containing genes at a significant frequency, represent an inappropriate source for an efficient, systematic gene identification in barley. Sequence information obtained in the context of the present study provides a resource for the conversion of these markers into sequence-tagged site (STS) markers and their use in PCR assays.

Molecular characterization of two lipoxygenases from barley.

Two full-length lipoxygenase cDNA sequences (LoxB and LoxC) from barley (Hordeum distichum cv. L. Triumph) are described. The cDNAs share high homology with the barley LoxA cDNA. Southern blotting experiments indicate single copy numbers of the three lipoxygenase genes. RFLP mapping revealed the presence of single lipoxygenase loci. LoxA and LoxB map on chromosome 4 and LoxC on chromosome 7. Two isoenzymes, LOX1 and LOX2, have been purified previously from germinating barley and characterized. LOX1 is encoded by LoxA, while LOX2 is encoded by LoxC. The product related to the third cDNA (loxB) has not been identified so far, suggesting a low protein abundance for the corresponding isoform in barley. Transcripts corresponding with these LOX genes are predominantly observed in grain and in seedling, whereas transcripts corresponding to LoxB and LoxC are also observed in mature vegetative tissue. No lipoxygenase mRNA could be detected in aleurone layer of germinating grain. No significant differences in lipoxygenase mRNA levels were observed in developing grains grown under dormant or non-dormant conditions, suggesting that LOX is not directly involved in induction of grain dormancy.

Molecular mapping of two dwarfing genes differing in their GA response on chromosome 2H of barley.

The two recessive dwarfing mutants gai (GA-ins) and gal (GA-less), differing in their response to exogenously applied gibberellic acid (GA(3)), were mapped in the centromere region and on the long arm, respectively, of the barley chromosome 2H. The gene gai, which determines reduced plant height and GA insensitivity pleiotropically, was found to co-segregate with the two RFLP markers Xmwg2058 and Xmwg2287. Both markers are known to map close to the centromere. The GA-sensitive dwarfing gene gal was found to be linked to the three co-segregating RFLP markers Xmwg581, Xmwg882 and Xmwg2212 (proximal) and XksuG5 (distal) by 3.6 and 9.5. cM, respectively. The distance between the two mutant loci was estimated to be about 55 cM. Homoeologous relationships between the dwarfing genes within the Triticeae are discussed.