Efficient fine mapping of the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>) by HEGS (High Efficiency Genome Scanning)/AFLP in barley

The hulled or naked caryopsis character of barley (Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F₂ population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1–7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.Communicated by G. Wenzel     

Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley

 The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach. The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2), 5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with regions carrying resistance to CCN and corn leaf aphid. Received: 6 January 1998 / Accepted: 1 April 1998     

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

 The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F₂ individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus. Received: 17 July 1998 / Accepted: 9 August 1998     

Variation in oil palm (Elaeis guineensis Jacq.) tissue culture-derived regenerants revealed by AFLPs with methylation-sensitive enzymes

Tissue culture-derived plants of oil palm (Elaeis guineensis Jacq.) can develop abnormal flowers in which stamen primordia are converted into carpel-like tissues (mantled fruit). This abnormality can be heritable; individual palms may show variation in mantling and reversion to the normal phenotype over time has been observed. Four sets of ortets (mother plant used as tissue source) and ramets (regenerated plants) were compared using standard amplified fragment length polymorphism (AFLP) analysis and AFLPs using methylation-sensitive enzymes. No polymorphisms were found when standard AFLPs were produced with ten different primer combinations. In contrast, when methylation-sensitive AFLPs were used, polymorphisms were detectable. Polymorphisms appeared as new bands in the ramets, suggesting that a reduction in methylation had occurred during tissue culture. The highest number of polymorphic bands (0.3%) was obtained when HpaII was used as the restriction endonuclease, indicating that the loss of methylation had occurred most frequently at the internal C within the HpaII recognition sequence 5’-CCGG-3’. Conversion of nine of the polymorphic bands into probes for Southern analysis confirmed that these were not due to partial digestion of the AFLP templates and showed that the majority were single-copy sequences. The exceptions were fragments showing homology to 25S ribosomal RNA genes and the chalcone synthase gene family. Examination of the Southern blots suggested that most of the single-copy sequences were partially de-methylated, and one example was found in which de-methylation affected only one allele. No polymorphism was consistently different between normal and abnormal clones in all the sets. This suggests that, whilst this method is an effective way of detecting variation in tissue culture-derived plants, different approaches will be required to identify the causal basis of the mantled fruit abnormality. Received: 25 May 2000 / Accepted: 28 August 2000     

High-resolution genetic mapping of the pepper (Capsicum annuum L.) resistance loci Me3 and Me4 conferring heat-stable resistance to root-knot nematodes (Meloidogyne spp.)

The PM687 line of Capsicum annuum L. has a single dominant gene, Me ₃ , that confers heat-stable resistance to root-knot nematodes (RKN). Me ₃ was mapped using doubled-haploid (DH) lines and F₂ progeny from a cross between the susceptible cultivar ’Yolo Wonder’ (’YW’) and the highly resistant line ’PM687’. Bulked-segregant analysis with DNA pools, from susceptible or resistant DH lines, was performed to identify RAPD and AFLP markers linked to Me ₃ . There was no polymorphism between bulks of ten DH lines using over 800 RADP primers (4,000 amplified fragments analysed). Using 512 AFLP primers (74,000 amplified fragments analysed), and bulked DNA templates from 20 resistant and 20 susceptible plants, we identified eight repulsion-phase and four coupling-phase markers linked to Me _3. Analysed in 103 DH progeny, they defined a 56.1-cM interval containing the target gene. The nearest were located 0.5, 1.0, 1.5 and 3.0 centimorgans (cM) on both sides of the gene. Analysis of the F₂ progeny (162 plants) with the nearest coupling-phase marker confirmed its close position. Another resistance gene to RKN, present in ’PM687’ (Me ₄ ), was shown to be linked to Me ₃ , 10 cM from it. In order to localize Me ₃ and Me ₄ on our reference intraspecific pepper linkage map, two AFLP markers were mapped. The Me ₃ nearest marker was 10.1cM from a RAPD marker named Q04_0.3 and 2.7cM from a RFLP marker named CT135. We investigated map-position orthologies between Me ₃ and two other nematode resistance genes, the tomato Mi-3 and the potato Gpa ₂ genes, which mapped in the telomeric region of the short arm of the tomato and potato chromosome 12 (or XII for potato). Received: 23 March 2000 / Accepted: 2 January 2001     

Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy

 The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5 cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26 000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy. Received: 25 April 1996 / Accepted: 26 June 1996     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998     

Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates

A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening. One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction and molecular isolation of disease resistance genes. Received: 22 May 2000 / Accepted: 25 September 2000     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

Molecular mapping of the photoperiod response gene ea7 in barley

 The gene ea ₇ determining photoperiod insensitivity under short day length was mapped on the short arm of chromosome 6H near the centromere. The gene was linked to the two flanking markers Xmwg2264 and Xmwg916 by 6.7 and 13.0 cM, respectively. Compared to Ppd-H1 (chromosome 2H) and Ppd-H2 (chromosome 1H), ea ₇ determines the strongest effect on flowering time with 55 and 18 days difference compared to photoperiod sensitive genotypes grown under short and long photoperiods, respectively. Allelic and homoeologous relationships to major genes and quantitative trait loci controlling flowering time in barley and wheat are discussed. Received: 10 March 1998 / Accepted: 7 April 1998     

Development and characterization of a codominant marker linked to root-knot nematode resistance, and its application to peach rootstock breeding

 The presence of a codominant AFLP marker, EAA/MCAT10, correlates with the primary source of resistance to root-knot nematodes (Meloidogyne incognita and M. javanica) in rootstock cultivars of peach [Prunus persica (L.) Batsch]. Two allelic DNA fragments of this AFLP marker were cloned, sequenced and converted to sequence tagged sites (STS). Four nucleotide differences (i.e. one addition and three substitutions) were observed between the two clones. Furthermore, there was a diagnostic Sau3 AI cleavage site (GATC) in the large fragment that was absent from the small fragment (GTTC at this site). The applicability of this STS marker system to peach germplasm improvement was evaluated: genomic DNAs of cross parents (i.e. ‘Lovell’ and ‘Nemared’), four F₁ hybrids (K62-67, K62-68, P101-40 and P101-41) and two F₂ populations (from K62-68 and P101-41), as well as DNA from a test panel of 18 rootstock cultivars or selections, were PCR-amplified with the Mij3F/Mij1R primer pair and then digested with Sau3 AI. The banding patterns showed that the EAA/MCAT10 STS markers can clearly distinguish the three genotypes – homozygous resistant, heterozygous resistant and homozygous susceptible – in the ‘Lovell’בNemared’ cross. In addition, results from the rootstock survey were consistent with each rootstock’s phenotypic response to nematode infection, except for ‘Okinawa’, ‘Flordaguard’ and ‘Yunnan’ where root-knot resistance may have arisen independently. Therefore, the EAA/MCAT10 STS markers will be a useful tool to initiate marker assisted selection studies in peach rootstock breeding for root-knot nematode resistance. Received: 4 January 1999 / Accepted: 4 January 1999     

Linkage mapping of two mutations that reduce phytic acid content of barley grain

 This study describes the inheritance and linkage map positions of two low phytic acid barley (Hordeum vulgare) mutations, lpa1-1 and lpa2-1, that dramatically reduce grain phytic acid content and increase inorganic seed phosphorus (P). Wide-cross, F₂ mapping populations were constructed by mating six-rowed varieties, ‘Steptoe’ and/or ‘Morex’, with two-rowed ‘Harrington’lpa donor lines homozygous for either lpa1-1 or lpa2-1. The barley lpa1-1 mutation showed normal inheritance patterns, whereas a deficiency of homozygous lpa2-1/lpa2-1 F₂ plants was observed. We identified a codominant, STS-PCR marker (aMSU21) that cosegregated with lpa1-1 in a population of 41 F₂ plants. The aMSU21 marker was then mapped to a locus on barley chromosome 2H, using a North American Barley Genome Mapping Project (NABGMP) doubled haploid population (‘Harrington’בMorex’). We determined that lpa2-1 is located within a recombination interval of approximately 30 cM between two AFLP markers that were subsequently mapped to barley chromosome 7H by integration with the same NABGMP population. Recent comparative mapping studies indicate conserved genetic map orders of several homologous molecular marker loci in maize and the Triticeae species that also show corresponding linkage to the biochemically similar lpa2 mutations of maize and barley. This observation suggests that barley and maize lpa2 mutations may affect orthologous genes. No such evidence for correspondence of the phenotypically similar lpa1 mutations of barley and maize has been revealed. Received: 22 September 1997 / Accepted: 2 December 1997     

Molecular mapping of the blast resistance gene, Pi44(t), in a line derived from a durably resistant rice cultivar

 A recombinant inbred line derived from a cross between CO39 and ‘Moroberekan’, RIL276, was found to be resistant to lineage 44 isolates of Pyricularia grisea in the Philippines. One hundred F₂ individuals were obtained from a backcross of RIL276 and CO39. Phenotypic analysis showed that RIL276 carries a single locus, tentatively named Pi44(t), conferring complete resistance to lineage 44 isolates of P. grisea. RFLP probes, STS primers and AFLP markers were applied to identify DNA markers linked to Pi44(t). Neither RFLP nor STS-PCR analysis gave rise to DNA markers linked to the locus. Using bulk segregant AFLP analysis, however, two dominant AFLP markers (AF₃₄₈ and AF₃₄₉) linked to Pi44(t) were identified. AF₃₄₉ and AF₃₄₈ were located at 3.3±1.5 cM and 11±3.5 cM from Pi44(t), respectively. These markers were mapped on chromosome 11 using an F₂ population derived from a cross between ‘Labelle’ and ‘Black Gora’. The location of AF₃₄₈ on chromosome 11 was confirmed using another F₂ mapping population derived from IR40931-26-3-3-5/ PI543851. DNA products at the loci linked to Pi44(t) were amplified from RIL276, ‘Labelle’ and PI543851 using the same primer pairs used to amplify AF₃₄₉ and AF₃₄₈. Sequence analysis of these bands showed 100% identity between lines. This result indicates that these AFLP markers could be used for the comparison of maps or assignment of linkage groups to chromosomes. Received: 12 May 1998 / Accepted: 13 November 1998     

Effect of amino acids,growth regulators and genotype on androgenesis in barley

The effects of an amino acid mixture and of plant growth regulators added to the FHG barley anther culture medium were examined using three barley cultivars (Cadette, Léger, and Igri) grown in two environments (growth cabinet and glasshouse). ‘Léger’ and ‘Igri’ were known as responsive, and ‘Cadette’ as recalcitrant to androgenesis. Our first experiment showed that the amino acid-supplemented medium was best for embryogenesis and regeneration of ‘Cadette’ and ‘Igri’ in both environments, and if ‘Léger’ in the growth cabinet. The addition of ABA and TDZ did not improve embryogenesis and plant regeneration, and PAA decreased them in the growth cabinet. The addition of the amino acid mixture in the FHG medium also reduced the percentage of albino plants in the growth cabinet, but growth regulators did not improve the percentage of albino plants, and in some cases increased it. In the growth cabinet, disregarding media, ‘Léger’ produced more embryos than ‘Cadette’ and ‘Igri’, and Léger' and ‘Igri’ produced more green plants than ‘Cadette‘. Percentages of albino plants were higher or ‘Cadette’ than for ‘Igri’ or ‘Léger’. In a second experiment, we compared seven hybrids with their parents for androgenic responsiveness. Hybrids had a higher ability to generate green plants than expected based upon the weighted average reflecting the contribution of each parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

A high-resolution linkage map of the vicinity of the rice submergence tolerance locus Sub1

Resistance to submergence stress is an important breeding objective in areas where rice cultivars are subjected to complete inundation for a week or more. The present study was conducted to develop a high-resolution map of the region surrounding the submergence tolerance gene Sub1 in rice, which derives from the Indian cultivar FR13A. Submergence screening of 8-day-old plants of F₃ families kept for 14 days submerged in 60 cm of water allowed an accurate classification of Sub1 phenotypes. Bulked segregant analysis was used to identify AFLP markers linked to Sub1. A population of 2950 F₂ plants segregating for Sub1 was screened with two RFLP markers flanking the Sub1 locus, 2.4 and 4.9 cM away. Submergence tolerance was measured in the recombinant plants, and AFLP markers closely linked to Sub1 were mapped. Two AFLP markers cosegregated with Sub1 in this large population, and other markers were localized within 0.2 cM of Sub1. The high-resolution map should serve as the basis for map-based cloning of this important locus, as it will permit the identification of BAC clones spanning the region. Received: 15 December 1999 / Accepted: 18 February 2000     

Comparative mapping of a gibberellic acid-insensitive dwarfing gene (Dwf2) on chromosome 4HS in barley

 We report the genetic mapping of Dwf2, a dominant gibberellic acid (GA₃)-insensitive dwarfing gene which has been previously described to cause a very short growth habit in barley (Hordeum vulgare) mutant ‘93/B694’. Using RFLP and microsatellite markers we performed segregation analysis in an F₂ population comprising 86 individuals developed from a cross of ‘93/B694’ (Dwf2) with ‘Bonus M2’ (dwf2). Dwf2 was mapped on the short arm of barley chromosome 4H proximal to microsatellite marker XhvOle (5.7 cM) and distal to RFLP marker Xmwg2299 (18.3 cM). The genetic localization of the Dwf2 gene at a homoeologous position to the multiallelic Rht-B1 and Rht-D1 loci in wheat suggests synteny of GA-insensitive dwarfing genes within the Triticeae. Moreover, the extremely prostrate growth habit exhibited in barley ‘93/B694’ (Dwf2) resembles that of wheat plants carrying the genes Rht-B1c (Rht3) or Rht-D1c (Rht10). Received: 1 July 1998 / Accepted: 17 September 1998     

Exogenous ethylene influences flower opening of cut roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis>) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh-ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-induced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhibited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.     

A <Emphasis Type="Italic">Pseudo-Response Regulator</Emphasis> is misexpressed in the photoperiod insensitive <Emphasis Type="Italic">Ppd-D1a</Emphasis> mutant of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Ppd-D1 on chromosome 2D is the major photoperiod response locus in hexaploid wheat (Triticum aestivum). A semi-dominant mutation widely used in the “green revolution” converts wheat from a long day (LD) to a photoperiod insensitive (day neutral) plant, providing adaptation to a broad range of environments. Comparative mapping shows Ppd-D1 to be colinear with the Ppd-H1 gene of barley (Hordeum vulgare) which is a member of the pseudo-response regulator (PRR) gene family. To investigate the relationship between wheat and barley photoperiod genes we isolated homologues of Ppd-H1 from a ‘Chinese Spring’ wheat BAC library and compared them to sequences from other wheat varieties with known Ppd alleles. Varieties with the photoperiod insensitive Ppd-D1a allele which causes early flowering in short (SD) or LDs had a 2 kb deletion upstream of the coding region. This was associated with misexpression of the 2D PRR gene and expression of the key floral regulator FT in SDs, showing that photoperiod insensitivity is due to activation of a known photoperiod pathway irrespective of day length. Five Ppd-D1 alleles were found but only the 2 kb deletion was associated with photoperiod insensitivity. Photoperiod insensitivity can also be conferred by mutation at a homoeologous locus on chromosome 2B (Ppd-B1). No candidate mutation was found in the 2B PRR gene but polymorphism within the 2B PRR gene cosegregated with the Ppd-B1 locus in a doubled haploid population, suggesting that insensitivity on 2B is due to a mutation outside the sequenced region or to a closely linked gene. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Beales and A. Turner contributed equally to the work.     

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.