High-resolution mapping of the barley leaf rust resistance gene Rph5 using barley expressed sequence tags (ESTs) and synteny with rice

The rapidly growing expressed sequence tag (EST) resources of species representing the Poacea family and availability of comprehensive sequence information for the rice (Oryza sativa) genome create an excellent opportunity for comparative genome analysis. Extensive synteny between rice chromosome 1 and barley (Hordeum vulgare L.) chromosome 3 has proven extremely useful for saturation mapping of chromosomal regions containing target genes of large-genome barley with conserved orthologous genes from the syntenic regions of the rice genome. Rph5 is a gene conferring resistance to the barley leaf rust pathogen Puccinia hordei. It was mapped to chromosome 3HS, which is syntenic with rice chromosome 1S. The objective of this study was to increase marker density within the sub-centimorgan region around Rph5, using sequence-tagged site (STS) markers that were developed based on barley ESTs syntenic to the phage (P1)-derived artificial chromosome (PAC) clones comprising the distal region of rice chromosome 1S. Five rice PAC clones were used as queries in a blastn search to screen 375,187 barley ESTs. Ninety-four non-redundant EST sequences were identified from the EST database and used as templates to design 174 pairs of primer combinations. As a result, 9 barley EST-based STS markers were incorporated into the ‘Bowman’ × ‘Magnif 102’ high-resolution map of the Rph5 region. More importantly, six markers, including five EST-derived STS sequences, were found to co-segregate with Rph5. The results of this study demonstrate the usefulness of rice genomic resources for efficient deployment of barley ESTs for marker saturation of targeted barley genomic regions.     

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.     

Comparative mapping of the barley Ppd-H1 photoperiod response gene region,which lies close to a junction between two rice linkage segments

Comparative mapping of cereals has shown that chromosomes of barley, wheat, and maize can be described in terms of rice "linkage segments." However, little is known about marker order in the junctions between linkage blocks or whether this will impair comparative analysis of major genes that lie in such regions. We used genetic and physical mapping to investigate the relationship between the distal part of rice chromosome 7L, which contains the Hd2 heading date gene, and the region of barley chromosome 2HS containing the Ppd-H1 photoperiod response gene, which lies near the junction between rice 7 and rice 4 linkage segments. RFLP markers were mapped in maize to identify regions that might contain Hd2 or Ppd-H1 orthologs. Rice provided useful markers for the Ppd-H1 region but comparative mapping was complicated by loss of colinearity and sequence duplications that predated the divergence of rice, maize, and barley. The sequences of cDNA markers were used to search for homologs in the Arabidopsis genome. Homologous sequences were found for 13 out of 16 markers but they were dispersed in Arabidopsis and did not identify any candidate equivalent region. The implications of the results for comparative trait mapping in junction regions are discussed.     

The gibberellic-acid insensitive dwarfing gene<Emphasis Type="Italic"> sdw3</Emphasis> 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.Communicated by R. Hagemann     

Fine mapping and comparative genomics integration of two quantitative trait loci controlling resistance to powdery mildew in a Spanish barley landrace

The intervals containing two major quantitative trait loci (QTL) from a Spanish barley landrace conferring broad spectrum resistance to Blumeria graminis were subjected to marker saturation. First, all the available information on recently developed marker resources for barley was exploited. Then, a comparative genomic analysis of the QTL regions with other sequenced grass model species was performed. As a result of the first step, 32 new markers were added to the previous map and new flanking markers closer to both QTL were identified. Next, syntenic integration revealed that the barley target regions showed homology with regions on chromosome 6 of rice (Oryza sativa), chromosome 10 of Sorghum bicolor and chromosome 1 of Brachypodium distachyon. A nested insertion of ancestral syntenic blocks on Brachypodium chromosome 1 was confirmed. Based on sequence information of the most likely candidate orthologous genes, 23 new barley unigene-derived markers were developed and mapped within the barley target regions. The assessment of colinearity revealed an inversion on chromosome 7HL of barley compared to the other three grass species, and nearly perfect colinearity on chromosome 7HS. This two-step marker enrichment allowed for the refinement of the two QTL into much smaller intervals. Inspection of all predicted proteins for the barley unigenes identified within the QTL intervals did not reveal the presence of resistance gene candidates. This study demonstrates the usefulness of sequenced genomes for fine mapping and paves the way for the use of these two loci in barley breeding programs.     

Candidate genes for barley mutants involved in plant architecture: an in silico approach

To individuate candidate genes (CGs) for a set of barley developmental mutants, a synteny approach comparing the genomes of barley and rice has been introduced. Based on map positions of mutants, sequenced RFLP markers linked to the target loci were selected. The markers were mapped in silico by BLAST searches against the rice genome sequence and chromosomal regions syntenous to barley target intervals were identified. Rice syntenous regions were defined for 15 barley chromosomal intervals hosting 23 mutant loci affecting plant height (brh1; brh2; sld4), shoot and inflorescence branching (als; brc1; cul-2, -3, -5, -15, -16; dub1; mnd6; vrs1), development of leaves (lig) and leaf-like organs (cal-b19, -C15, -d4; lks5; suKD-25; suKE-74; suKF-76; trd; trp). Annotation of 110 Mb of rice genomic sequence made it possible to screen for putative CGs which are listed together with the reasons supporting mutant–gene associations. For two loci, CGs were identified with a clear probability to represent the locus considered. These include FRIZZY PANICLE, a candidate for the brc1 barley mutant, and the rice ortholog of maize Liguleless1 (Lg1), a candidate for the barley lig locus on chromosome 2H. For this locus, the validity of the approach was supported by the PCR-amplification of a genomic fragment of the orthologous barley sequence. SNP mapping located this fragment on chromosome 2H in the region hosting the lig genetic locus. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Identification of perennial ryegrass (Lolium perenne (L.)) and meadow fescue (Festuca pratensis (Huds.)) candidate orthologous sequences to the rice Hd1(Se1) and barley HvCO1 CONSTANS-like genes through comparative mapping and microsynteny

Microsynteny with rice and comparative genetic mapping were used to identify candidate orthologous sequences to the rice Hd1(Se1) gene in Lolium perenne and Festuca pratensis. A F. pratensis bacterial artificial chromosome (BAC) library was screened with a marker (S2539) physically close to Hd1 in rice to identify the equivalent genomic region in F. pratensis. The BAC sequence was used to identify and map the same region in L. perenne. Predicted protein sequences for L. perenne and F. pratensis Hd1 candidates (LpHd1 and FpHd1) indicated they were CONSTANS-like zinc finger proteins with 61-62% sequence identity with rice Hd1 and 72% identity with barley HvCO1. LpHd1 and FpHd1 were physically linked in their respective genomes (< 4 kb) to marker S2539, which was mapped to L. perenne chromosome 7. The identified candidate orthologues of rice Hd1 and barley HvCO1 in L. perenne and F. pratensis map to chromosome 7, a region of the L. perenne genome which has a degree of conserved genetic synteny both with rice chromosome 6, which contains Hd1, and barley chromosome 7H, which contains HvCO1.     

Collinearity-based marker mining for the fine mapping of <Emphasis Type="Italic">Pm6</Emphasis>, a powdery mildew resistance gene in wheat

The genome sequences of rice (Oryza sativa L.) and Brachypodium distachyon and the comprehensive Triticeae EST (Expressed Sequence Tag) resources provide invaluable information for comparative genomics analysis. The powdery mildew resistance gene, Pm6, which was introgressed into common wheat from Triticum timopheevii, was previously mapped to the wheat chromosome bin of 2BL [fraction length (FL) 0.50–1.00] with limited DNA markers. In this study, we saturated the Pm6 locus in wheat using the collinearity-based markers by extensively exploiting these genomic resources. All wheat ESTs located in the bin 2BL FL 0.50–1.00 and their corresponding orthologous genes on rice chromosome 4 were firstly used to develop STS (Sequence Tagged Site) markers. Those identified markers that flanked the Pm6 locus were then used to identify the collinear regions in the genomes of rice and Brachypodium. Triticeae ESTs with orthologous genes in these collinear regions were further used to develop new conserved markers for the fine mapping of Pm6. Using two F₂ populations derived from crosses of IGVI-465 × Prins and IGVI-466 × Prins, we mapped a total of 29 markers to the Pm6 locus. Among them, 14 markers were co-segregated with Pm6 in the IGVI-466/Prins population. Comparative genome analysis showed that the collinear region of the 29 linked markers covers a ~5.6-Mb region in chromosome 5L of Brachypodium and a ~6.0-Mb region in chromosome 4L of rice. The marker order is conserved between rice and Brachypodium, but re-arrangements are present in wheat. Comparative mapping in the two populations showed that two conserved markers (CINAU123 and CINAU127) flanked the Pm6 locus, and an LRR-receptor-like protein kinase cluster was identified in the collinear regions of Brachypodium and rice. This putative resistance gene cluster provides a potential target site for further fine mapping and cloning of Pm6. Moreover, the newly developed conserved markers closely linked to Pm6 can be used for the marker-assisted selection (MAS) of Pm6 in wheat breeding programs.     

Fine mapping and syntenic integration of the semi-dwarfing gene sdw3 of barley

The barley mutant allele sdw3 confers a gibberellin-insensitive, semi-dwarf phenotype with potential for breeding of new semi-dwarfed barley cultivars. Towards map-based cloning, sdw3 was delimited by high-resolution genetic mapping to a 0.04 cM interval in a “cold spot” of recombination of the proximal region of the short arm of barley chromosome 2H. Extensive synteny between the barley Sdw3 locus (Hvu_sdw3) and the orthologous regions (Osa_sdw3, Sbi_sdw3, Bsy_sdw3) of three other grass species (Oryza sativa, Sorghum bicolor, Brachypodium sylvaticum) allowed for efficient synteny-based marker saturation in the target interval. Comparative sequence analysis revealed colinearity for 23 out of the 38, 35, and 29 genes identified in Brachypodium, rice, and Sorghum, respectively. Markers co-segregating with Hvu_sdw3 were generated from two of these genes. Initial attempts at chromosome walking in barley were performed with seven orthologous gene probes which were delimiting physical distances of 223, 123, and 127 kb in Brachypodium, rice, and Sorghum, respectively. Six non-overlapping small bacterial artificial chromosome (BAC) clone contigs (cumulative length of 670 kb) were obtained, which indicated a considerably larger physical size of Hvu_sdw3. Low-pass sequencing of selected BAC clones from these barley contigs exhibited a substantially lower gene frequency per physical distance and the presence of additional non-colinear genes. Four candidate genes for sdw3 were identified within barley BAC sequences that either co-segregated with the gene sdw3 or were located adjacent to these co-segregating genes. Identification of genic sequences in the sdw3 context provides tools for marker-assisted selection. Eventual identification of the actual gene will contribute new information for a basic understanding of the mechanisms underlying growth regulation in barley.     

Physical mapping of the barley stem rust resistance gene rpg4

The barley stem rust resistance gene rpg4 was physically and genetically localized on two overlapping BAC clones covering an estimated 300-kb region of the long arm of barley chromosome 7(5H). Initially, our target was mapped within a 6.0-cM region between the previously described flanking markers MWG740 and ABG391. This region was then saturated by integrating new markers from several existing barley and rice maps and by using BAC libraries of barley cv. Morex and rice cv. Nipponbare. Physical/genetic distances in the vicinity of rpg4 were found to be 1.0 Mb/cM, which is lower than the average for barley (4 Mb/cM) and lower than that determined by translocation breakpoint mapping (1.8 Mb/cM). Synteny at high resolution levels has been established between the region of barley chromosome 7(5H) containing the rpg4 locus and the subtelomeric region of rice chromosome 3 between markers S16474 and E10757. This 1.7-cM segment of the rice genome was covered by two overlapping BAC clones, about 250 kb of total length. In barley the markers S16474 and E10757 genetically delimit rpg4, lying 0.6 cM distal and 0.4 cM proximal to the locus, respectively.     

Analysis of the barley chromosome 2 region containing the six-rowed spike gene <Emphasis Type="Italic">vrs1</Emphasis> reveals a breakdown of rice–barley micro collinearity by a transposition

In cultivated barley (Hordeum vulgare ssp. vulgare), six-rowed spikes produce three times as many seeds per spike as do two-rowed spikes. The determinant of this trait is the Mendelian gene vrs1, located on chromosome 2H, which is syntenous with rice (Oryza sativa) chromosomes 4 and 7. We exploited barley–rice micro-synteny to increase marker density in the vrs1 region as a prelude to its map-based cloning. The rice genomic sequence, covering a 980 kb contig, identified barley ESTs linked to vrs1. A high level of conservation of gene sequence was obtained between barley chromosome 2H and rice chromosome 4. A total of 22 EST-based STS markers were placed within the target region, and the linear order of these markers in barley and rice was identical. The genetic window containing vrs1 was narrowed from 0.5 to 0.06 cM, which facilitated covering the vrs1 region by a 518 kb barley BAC contig. An analysis of the contig sequence revealed that a rice Vrs1 orthologue is present on chromosome 7, suggesting a transposition of the chromosomal segment containing Vrs1 within barley chromosome 2H. The breakdown of micro-collinearity illustrates the limitations of synteny cloning, and stresses the importance of implementing genomic studies directly in the target species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Towards map-based cloning of the barley stem rust resistance genes Rpg1 and rpg4 using rice as an intergenomic cloning vehicle

The barley stem rust resistance genes Rpg1 and rpg4 were mapped in barley on chromosomes 1P and 7M, respectively and the syntenous rice chromosomes identified as 6P and 3P by mapping common probes in barley and rice. Rice yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones were used to isolate probes mapping to the barley Rpg1 region. The rice BAC isolated with the pM13 probe was a particularly excellent source of probes. A high-resolution map of the Rpg1 region was established with 1400 gametes yielding a map density of 3.6 markers per 0.1 cM. A detailed physical map was established for the rice BAC fragment containing the Rpg1-flanking markers pM13 and B24. This fragment covers a barley genetic distance of 0.6 cM and a rice DNA physical distance of ca. 70 kb. The distribution of barley cross-overs in relation to the rice DNA physical distances was extremely uneven. The barley genetic distance between the pM13 marker and Rpg1 was 0.1 cM per ca. 55 kb, while on the proximal side it was 0.5 cm per ca. 15 kb. Three probes from the distal end of the pM13 BAC mapped 3.0 cm proximal of Rpg1 and out of synteny with rice. These experiments confirm the validity of using large insert rice clones as probe sources to saturate small barley (and other large genome cereals) genome regions with markers. They also establish a note of caution that even in regions of high microsynteny, there may be small DNA fragments that have transposed and are no longer in syntenous positions.     

High-density mapping and marker development for the powdery mildew resistance gene PmAS846 derived from wild emmer wheat (Triticum turgidum var. dicoccoides)

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important foliar disease of wheat worldwide. The dominant powdery mildew resistance gene PmAS846 was transferred to the hexaploid wheat lines N9134 and N9738 from wild emmer wheat (Triticum dicoccoides) in 1995, and it is still one of the most effective resistance genes in China. A high resolution genetic map for PmAS846 locus was constructed using two F₂ populations and corresponding F_2:3 families developed from the crosses of N9134/Shaanyou 225 and N9738/Huixianhong. Synteny between wheat and Brachypodium distachyon and rice was used to develop closely linked molecular markers to reduce the genetic interval around PmAS846. Twenty-six expressed sequence tag-derived markers were mapped to the PmAS846 locus. Five markers co-segregated with PmAS846 in the F₂ population of N9134/Shaanyou 225. PmAS846 was physically located to wheat chromosome 5BL bin 0.75–0.76 within a gene-rich region. The markers order is conserved between wheat and Brachypodium distachyon, but rearrangements are present in rice. Two markers, BJ261635 and CJ840011 flanked PmAS846 and narrowed PmAS846 to a region that is collinear with 197 and 112 kb genomic regions on Brachypodium chromosome 4 and rice chromosome 9, respectively. The genes located on the corresponding homologous regions in Brachypodium, rice and barley could be considered for further marker saturation and identification of potential candidate genes for PmAS846. The markers co-segregating with PmAS846 provide a potential target site for positional cloning of PmAS846, and can be used for marker-assisted selection of this gene.     

Quantitative Trait Locus Mapping and Candidate Gene Analysis for Plant Architecture Traits Using Whole Genome Re-Sequencing in Rice

Plant breeders have focused on improving plant architecture as an effective means to increase crop yield. Here, we identify the main-effect quantitative trait loci (QTLs) for plant shape-related traits in rice (Oryza sativa) and find candidate genes by applying whole genome re-sequencing of two parental cultivars using next-generation sequencing. To identify QTLs influencing plant shape, we analyzed six traits: plant height, tiller number, panicle diameter, panicle length, flag leaf length, and flag leaf width. We performed QTL analysis with 178 F₇ recombinant in-bred lines (RILs) from a cross of japonica rice line ‘SNUSG1’ and indica rice line ‘Milyang23’. Using 131 molecular markers, including 28 insertion/deletion markers, we identified 11 main- and 16 minor-effect QTLs for the six traits with a threshold LOD value > 2.8. Our sequence analysis identified fifty-four candidate genes for the main-effect QTLs. By further comparison of coding sequences and meta-expression profiles between japonica and indica rice varieties, we finally chose 15 strong candidate genes for the 11 main-effect QTLs. Our study shows that the whole-genome sequence data substantially enhanced the efficiency of polymorphic marker development for QTL fine-mapping and the identification of possible candidate genes. This yields useful genetic resources for breeding high-yielding rice cultivars with improved plant architecture.     

Towards Positional Isolation of Three Quantitative Trait Loci Conferring Resistance to Powdery Mildew in Two Spanish Barley Landraces

Three quantitative trait loci (QTL) conferring broad spectrum resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei, were previously identified on chromosomes 7HS, 7HL and 6HL in the Spanish barley landrace-derived lines SBCC097 and SBCC145. In the present work, a genome-wide putative linear gene index of barley (Genome Zipper) and the first draft of the physical, genetic and functional sequence of the barley genome were used to go one step further in the shortening and explicit demarcation on the barley genome of these regions conferring resistance to powdery mildew as well as in the identification of candidate genes. First, a comparative analysis of the target regions to the barley Genome Zippers of chromosomes 7H and 6H allowed the development of 25 new gene-based molecular markers, which slightly better delimit the QTL intervals. These new markers provided the framework for anchoring of genetic and physical maps, figuring out the outline of the barley genome at the target regions in SBCC097 and SBCC145. The outermost flanking markers of QTLs on 7HS, 7HL and 6HL defined a physical area of 4 Mb, 3.7 Mb and 3.2 Mb, respectively. In total, 21, 10 and 16 genes on 7HS, 7HL and 6HL, respectively, could be interpreted as potential candidates to explain the resistance to powdery mildew, as they encode proteins of related functions with respect to the known pathogen defense-related processes. The majority of these were annotated as belonging to the NBS-LRR class or protein kinase family.     

半矮秆基因brh1在大麦中的精细定位

选用从大麦、小麦和水稻中分离的RFLP标记 ,构建了大麦半矮秆基因brh1精细图谱。以快中子处理六棱大麦品种Steptoe的种子 ,从M2 代中选择出brh1突变体FN5 3。brh1是一个极易鉴别的形态学标记 ,通过对FN5 3×Morex的F2 代群体进行鉴定表明 ,brh1基因为隐性 ,前人通过BSA法将其初步定位在大麦第 1染色体 (7H)短臂上 ,靠近端粒区。这一区间还有一个控制秆锈病抗性的显性基因Rpg1。所以 ,brh1的精细定位不仅对研究其本身具有重要意义 ,同时 ,也为Rpg1的图位克隆和功能研究提供了更大的重组配子群体。定位实验全部以F2 中具有brh1特征的个体为对象完成 ,鉴定工作在苗期进行。在该精细图上 ,brh1区间长15 .2cM ,各标记间的平均距离为 0 .8cM。其中 ,大麦的cDNA克隆MWG2 0 74B与brh1共分离。 2 0 74A在靠近着丝点一侧 ,与brh1相距 0 .8cM。BCD12 9和R3139在定位群体内呈现与MWG2 0 74A共分离。CDO5 4 5位于端粒一侧 ,距离brh1为 0 .8cM。根据禾谷类作物基因组的共线性原理 ,CDO5 4 5成功定位在水稻的同源染色体即第 6染色体短臂brh1区间内。然而 ,由于在定位亲本间缺乏多态性 ,BCD12 9和MWG2 0 74的 2条主带A和B均未能定位在水稻的共线性区段内。推测MWG2 0 74的其他各带可能被定位在水稻的目标区间内 ,从而有