RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat

A population of single chromosome recombinant lines was developed from the cross between a frost-sensitive, vernalization-insensitive substitution line, ‘Chinese Spring’ (Triticum spelta 5A) and a frost-tolerant, vernalization-sensitive line, ‘Chinese Spring’ (‘Cheyenne’ 5A), and used to map the genes Vrn1 and Fr1 controlling vernalization requirement and frost tolerance, respectively, relative to RFLP markers located on this chromosome. The Vrn1 and Fr1 loci were located closely linked on the distal portion of the long arm of 5AL, but contrary to previous observations, recombination between them was found. Three RFLP markers, Xpsr426, Xcdo504 and Xwg644 were tightly linked to both. The location of Vrn1 suggests that it is homoeologous to other spring habit genes in related species, particularly the Sh2 locus on chromosome 7 (5H) of barley and the Sp1 locus on chromosome 5R of rye.     

Two loci on wheat chromosome 5A regulate the differential cold-dependent expression of the cor14b gene in frost-tolerant and frost-sensitive genotypes

Although cold acclimation in cereals involves the expression of many cold-regulated genes, genetic studies have shown that only very few chromosomal regions carry loci that play an important role in frost tolerance. To investigate the genetic relationship between frost tolerance and the expression of cold-regulated genes, the expression and regulation of the wheat homolog of the barley cold-regulated gene cor14b was studied at various temperatures in frost-sensitive and frost-tolerant wheat genotypes. At 18/15 °C (day/night temperatures) frost-tolerant plants accumulated cor14b mRNAs and expressed COR14b proteins, whereas the sensitive plants did not. This result indicates that the threshold temperature for induction of the wheat cor14b homolog is higher in frost-resistant plants, and allowed us to use this polymorphism in a mapping approach. Studies made with chromosome substitution lines showed that the polymorphism for the threshold induction temperature of the wheat cor14b homolog is controlled by a locus(i) located on chromosome 5A of wheat, while the cor14b gene was mapped in Triticum monococcum on the long arm of chromosome 2A^m. The analysis of single chromosome recombinant lines derived from a cross between Chinese Spring/Triticum spelta 5A and Chinese Spring/Cheyenne 5A identified two loci with additive effects that are involved in the genetic control of cor14b mRNA accumulation. The first locus was tightly linked to the marker psr911, while the second one was located between the marker Xpsr2021 and Frost resistance 1 (Fr1). Received: 20 July 1999 / Accepted: 15 November 1999     

Deletion-based physical mapping of barley chromosome 7H

Chromosomal mutations in barley (Hordeum vulgare, 2n=2x=14, HH) chromosome 7H added to the common wheat (Triticum aestivum, 2n=6x=42, AABBDD) cultivar Chinese Spring were induced genetically by the gametocidal activity of certain alien chromosomes derived from wild species of the genus Aegilops. The rearranged barley chromosomes were characterized by C-banding, FISH and GISH. Twenty two deletion or translocation chromosomes in a hemizygous condition were selected for deletion mapping of 17 AFLP and 28 STS markers that are specific to 7H. Of the 22 breakpoints in chromosome 7H, seven involved the short arm (7HS), 12 the long arm (7HL) and three were in the centromeric region. The seven 7HS breakpoints separated all four 7HS-specific AFLP markers and split the 21 STS markers into six groups. One breakpoint occurred between two STS markers formerly occupying the same position in the genetic map. All seven 7HS breakpoints were separated from each other by either the AFLP or STS markers. The 12 breakpoints in 7HL divided the 13 7HL-specific AFLP markers into seven groups, and the seven STS markers into three groups. On the other hand, the 12 breakpoints in 7HL were divided into six groups by the AFLP markers and into two groups by the STS markers. This deletion-based map was in accordance with previously published genetic and physical maps using the same STS markers. The breakpoints, AFLP markers and STS markers were arrayed in a consistent order. Received: 5 February 2001 / Accepted: 19 February 2001     

Varietal and chromosome 2H locus-specific frost tolerance in reproductive tissues of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) detected using a frost simulation chamber

Exposure of flowering cereal crops to frost can cause sterility and grain damage, resulting in significant losses. However, efforts to breed for improved low temperature tolerance in reproductive tissues (LTR tolerance) has been hampered by the variable nature of natural frost events and the confounding effects of heading time on frost-induced damage in these tissues. Here, we establish conditions for detection of LTR tolerance in barley under reproducible simulated frost conditions in a custom-built frost chamber. An ice nucleator spray was used to minimize potential effects arising from variation in naturally occurring extrinsic nucleation factors. Barley genotypes differing in their field tolerance could be distinguished. Additionally, an LTR tolerance quantitative trait locus (QTL) on the long arm of barley chromosome 2H could be detected in segregating families. In a recombinant family, the QTL was shown to be separable from the effects of the nearby flowering time locus Flt-2L. At a minimum temperature of −3.5°C for 2 h, detection of the LTR tolerance locus was dependent on the presence of the nucleator spray, suggesting that the tolerance relates to freezing rather than chilling, and that it is not the result of plant-encoded variation in ice-nucleating properties of the tiller surface. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

QTL analysis of Fusarium head blight resistance using a high-density linkage map in barley

Fusarium head blight (FHB) resistance was evaluated in a set of recombinant inbred (RI) lines from a cross between Russia 6 (resistant) and H.E.S. 4 (susceptible), which had one of the widest differences of FHB resistance reactions among ca. 5,000 barley germplasm accessions in Okayama University. Field-grown spikes were sampled and inoculated by the ‘cut-spike test’. Resistance reactions on the parents and RI lines were scored by eleven grades, from resistant (0) to susceptible (10). Quantitative trait loci (QTL) analysis detected three QTL: two located on the long arm of chromosome 2H, and another on the short arm of chromosome 5H. A QTL located on chromosome 2H was coincident with the vrs1 locus, which governs inflorescence row type. The other QTL on chromosome 2H was positioned in the vicinity of cleistogamy locus (cly1 or Cly2) that determines inflorescence opening/closing. Resistant gene analog (RGA) and expressed sequence tag (EST) markers with homology for disease resistance genes were integrated into the high-density linkage map. Most of these markers were not localized near the identified resistance QTL, except for one RGA marker (FXLRRfor_XLRRrev170) localized in the vicinity of the cly1/Cly2 locus. Five AFLP markers localized in the vicinity of the identified QTL were sequenced to convert them into sequence tagged site (STS) markers. Genotyping of each RI line using two AFLP–STS markers and the vrs1 locus indicated that the RI lines with three Russia 6 QTL alleles exhibited the same level of high FHB resistance reactions as Russia 6. In contrast, RI lines with three susceptible alleles showed reactions close to H.E.S. 4. Therefore, the markers closely linked to the QTL can be efficiently used for the selection of resistance.     

Genetic and molecular analyses of resistance to a variant of Puccinia striiformis in barley

Seedlings of 62 Australian barley cultivars and two exotic barley genotypes were assessed for resistance to a variant of Puccinia striiformis, referred to as “Barley Grass Stripe Rust” (BGYR), first detected in Australia in 1998, which is capable of infecting wild Hordeum species and some genotypes of cultivated barley. Fifty-three out of 62 cultivated barley cultivars tested were resistant to the pathogen. Genetic analyses of seedling resistance to BGYR in six Australian barley cultivars and one Algerian barley landrace indicated that they carried either one or two major resistance genes to the pathogen. A single recessive seedling resistance gene, rpsSa3771, identified in Sahara 3771, was located on the long arm of chromosome 1 (7 H), flanked by the restriction fragment length polymorphism (RFLP) markers Xwg420 and Xcdo347 at genetic distances of 12.8 and 21.9 cM, respectively. Mapping resistance to BGYR at adult plant growth stages using the doubled haploid (DH) population Clipper × Sahara 3771 identified two major quantitative trait loci (QTL), one on the long arm of chromosome 3 (3 H) and the second on the long arm of chromosome 1 (7 H), accounting for 26 % and 18 % of the total phenotypic variation, respectively. The QTL located on chromosome 7HL corresponded to seedling resistance gene rpsSa3771 and the second QTL was concluded to correspond to a single APR gene, designated rpsCl, contributed by cultivar Clipper.     

Structure–function analysis of the barley genome: the gene-rich region of chromosome 2HL

A major gene-rich region on the end of the long arm of Triticeae group 2 chromosomes exhibits high recombination frequencies, making it an attractive region for positional cloning. Traits known to be controlled by this region include chasmogamy/cleistogamy, frost tolerance at flowering, grain yield, head architecture, and resistance to Fusarium head blight and rusts. To assist these cloning efforts, we constructed detailed genetic maps of barley chromosome 2H, including 61 polymerase chain reaction markers. Colinearity with rice occurred in eight distinct blocks, including five blocks in the terminal gene-rich region. Alignment of rice sequences from the junctions of colinear chromosome segments provided no evidence for the involvement of long (>2.5 kb) inverted repeats in generating inversions. However, reuse of some junction sequences in two or three separate evolutionary breakage/fusion events was implicated, suggesting the presence of fragile sites. Sequencing across 91 gene fragments totaling 107 kb from four barley genotypes revealed the highest single nucleotide substitution and insertion–deletion polymorphism levels in the terminal regions of the chromosome arms. The maps will assist in the isolation of genes from the chromosome 2L gene-rich region in barley and wheat by providing markers and accelerating the identification of the corresponding points in the rice genome sequence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

QTL mapping of chromosomal regions conferring reproductive frost tolerance in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Spring radiation frost is a major abiotic stress in southern Australia, reducing yield potential and grain quality of barley by damaging sensitive reproductive organs in the latter stages of development. Field-based screening methods were developed, and genetic variation for reproductive frost tolerance was identified. Mapping populations that were segregating for reproductive frost tolerance were screened and significant QTL identified. QTL on chromosome 2HL were identified for frost-induced floret sterility in two different populations at the same genomic location. This QTL was not associated with previously reported developmental or stress-response loci. QTL on chromosome 5HL were identified for frost-induced floret sterility and frost-induced grain damage in all three of the populations studied. The locations of QTL were coincident with previously reported vegetative frost tolerance loci close to the vrn-H1 locus. This locus on chromosome 5HL has now been associated with response to cold stress at both vegetative and reproductive developmental stages in barley. This study will allow reproductive frost tolerance to be seriously pursued as a breeding objective by facilitating a change from difficult phenotypic selection to high-throughput genotypic selection.     

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     

Marker development and characterisation of Hordeum bulbosum introgression lines: a resource for barley improvement

A set of 110 diploid putative introgression lines (ILs) containing chromatin introgressed from the undomesticated species Hordeum bulbosum L. (bulbous barley grass) into cultivated barley (Hordeum vulgare L.) has been identified using a high-copy number retrotransposon-like PCR marker, pSc119.1, derived from rye (Secale cereale L.). To evaluate these lines, 92 EST-derived markers were developed by marker sequencing across four barley cultivars and four H. bulbosum genotypes. Single nucleotide polymorphisms and insertions/deletions conserved between the two species were then used to develop a set of fully informative cleaved amplified polymorphic sequence markers or size polymorphic insertion/deletion markers. Introgressed chromatin from H. bulbosum was confirmed and genetically located in 88 of these lines using 46 of the EST-derived PCR markers. A total of 96 individual introgressions were detected with most of them (94.8%) extending to the most distal marker for each respective chromosome arm. Introgressions were detected on all chromosome arms except chromosome 3HL. Interstitial or sub-distal introgressions also occurred, with two located on chromosome 2HL and one each on 3HS, 5HL and 6HS. Twenty-two putative ILs that were positive for H. bulbosum chromatin using pSc119.1 have not had introgressions detected with these single-locus markers. When all introgressions are combined, more than 36% of the barley genetic map has now been covered with introgressed chromatin from H. bulbosum. These ILs represent a significant germplasm resource for barley improvement that can be mined for diverse traits of interest to barley breeders and researchers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytology of Potato Callus Cells in Relation to their Frost Hardiness

Structure of callus cells of frost-sensitive and frost-tolerant Solanum species and a frost-tolerant cell line (D20-1), selected from S. tuberosum cv. Desirée callus, was studied. Like frost-tolerant species S. commersonii, cells of the frost-tolerant cell line contained starch grains in their plastids. The cells of this frost-tolerant line also possessed an increased number of microbodies containing protein crystals which suggests the involvement of proteins in frost tolerance but the mechanism may differ from that in frost-tolerant species.     

Comparative RFLP-based genetic maps of barley chromosome 5 (1H) and rye chromosome 1R

Summary A genetic map of barley chromosome 5 (1H) was constructed using DNA markers. Seventeen loci were mapped to 15 locations, and these included the known-function loci (in order from the most distal on the long arm) XAdh (alcohol dehydrogenase), XLec (homologous to wheat germ agglutinin), XHor3 (D-hordein), XPpdk (pyruvate orthophosphate dikinase), centromere, XIcal (chymotrypsin inhibitor), and 6 loci in the B- and C-hordein cluster towards the end of the short arm. The gene order on the barley map agreed closely with that of chromosome 1 of rye. Intervarietal comparisons showed that single-copy cDNA and genomic DNA probes revealed about twice the level of RFLPs found in wheat.     

Genetic study of glutathione accumulation during cold hardening in wheat

The effect of cold hardening on the accumulation of glutathione (GSH) and its precursors was studied in the shoots and roots of wheat (Triticum aestivum L.) cv. Cheyenne (Ch, frost-tolerant) and cv. Chinese Spring (CS, moderately frost-sensitive), in a T. spelta L. accession (Tsp, frost-sensitive) and in chro- mosome substitution lines CS (Ch 5A) and CS (Tsp 5A). The fast induction of total glutathione accumulation was detected during the first 3 d of hardening in the shoots, especially in the frost-tolerant Ch and CS (Ch 5A). This observation was corroborated by the study of de novo GSH synthesis using [³⁵S]sulfate. In Ch and CS (Ch 5A) the total cysteine, γ-glutamylcysteine (precursors of GSH), hydroxymethylglutathione and GSH contents were greater during the 51-d treatment than in the sensitive genotypes. After 35 d hardening, when the maximum frost tolerance was observed, greater ratios of reduced to oxidised hydroxymethylglutathione and glutathione were detected in Ch and CS (Ch 5A) compared to the sensitive genotypes. A correspondingly greater glutathione reductase (EC 1.6.4.2) activity was also found in Ch and CS (Ch 5A). It can be assumed that chromosome 5A of wheat has an influence on GSH accumulation and on the ratio of reduced to oxidised glutathione as part of a complex regulatory function during hardening. Consequently, GSH may contribute to the enhancement of frost tolerance in wheat. Received: 24 March 1999 / Accepted: 19 July 1999     

Introgression of an intermediate <Emphasis Type="Italic">VRNH1</Emphasis> allele in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) leads to reduced vernalization requirement without affecting freezing tolerance

The process of vernalization is mainly controlled by two genes in winter barley (Hordeum vulgare L.), VRNH1 and VRNH2. A recessive allele at VRNH1 and a dominant allele at VRNH2 must be present to induce a vernalization requirement. In addition, this process is usually associated with greater low-temperature tolerance. Spanish barleys originated in areas with mild winters and display a reduced vernalization requirement compared with standard winter cultivars. The objective of this study was to investigate the genetic origin of this reduced vernalization requirement and its effect on frost tolerance. We introgressed the regions of a typical Spanish barley line that carry VRNH1 and VRNH2 into a winter cultivar, Plaisant, using marker-assisted backcrossing. We present the results of a set of 12 lines introgressed with all four possible combinations of VRNH1 and VRNH2, which were evaluated for vernalization requirement and frost tolerance. The reduced vernalization requirement of the Spanish parent was confirmed, and was found to be due completely to the effect of the VRNH1 region. The backcross lines showed no decline in frost tolerance compared with that of the recurrent parent unless they carried an extra segment of chromosome 5H. This extra segment, a carryover of the backcross process, apparently contained the well-known frost tolerance quantitative trait locus Fr-H2. We demonstrate that it is possible to manipulate the vernalization requirement with only minor effects on frost tolerance. This finding opens the path to creating new types of barley cultivars that are better suited to specific environments, especially in a climate-change scenario.     

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.     

Molecular mapping of a fertility restoration locus (Rfm1) for cytoplasmic male sterility in barley (Hordeum vulgare L.)

The Rfm1a gene restores the fertility of msm1 cytoplasmic male-sterile lines in barley. We identified three RAPD markers linked to the Rfm1 locus (CMNB-07/800, OPI-18/900, and OPT-02/700) using isogenic lines and segregating BC₁F₁ and F₂ populations. Using a previously developed linkage map of barley, we located CMNB-07/800 and OPT-02/700 beside MWG2218 on chromosome 6HS. The linkage between MWG2218 and the Rfm1 locus was demonstrated using the segregating BC₁F₁ and F₂ populations. To confirm the chromosomal locations of these markers, we converted them to STSs and tested against two sets of wheat–barley chromosome addition lines. These STS markers, CMNB-07/800, OPT-02/700, and MWG2218, were amplified only in the addition lines possessing the chromosome 6H, thereby providing additional evidence the Rfm1 locus is located on chromosome 6H. Homoeologous relationships among fertility restoration genes in Triticeae are discussed. Received: 27 March 2000 / Accepted: 25 June 2000     

Mapping of powdery mildew resistance genes in a newly determined accession of Hordeum vulgare ssp. spontaneum

The accession PI466197 of wild barley (Hordeum vulgare ssp. spontaneum) with a newly identified resistance to powdery mildew caused by Blumeria graminis f.sp. hordei was studied with the aim to localise the genes determining resistance on a barley genetic map using DNA markers. Molecular analysis was performed in the F₂ population of the cross between the winter variety ‘Tiffany’ and the resistant accession PI466197, consisting of 113 plants. DNA markers, 17 simple sequence repeats (SSRs), four sequence-tagged sites (STSs) and one cleaved amplified polymorphic sequence (CAPS) marker developed from the Mla locus sequence were used for genetic mapping and a two-locus model of resistance was shown. One of the resistance genes originating from H. vulgare ssp. spontaneum PI466197 was localised between the markers RGH1aE1 and Bmac0213 on the short arm of chromosome 1H, which is the position consistent with the Mla locus. The other gene was proven to be highly significantly linked with GBMS247, Bmac0134 and MWG878 on the short arm of chromosome 2H. The flanking markers were Bmac0134 and MWG878, assigned 4 and 8 cM from the resistance gene, respectively. Until now, no gene conferring powdery mildew resistance originating from H. vulgare has been located on the short arm of barley chromosome 2H.