A Novel STS Marker for Polyphenol Oxidase Activity in Bread Wheat

The enzyme activity of polyphenol oxidase (PPO) in grain has been related to undersirable brown discoloration of bread wheat (Triticum aestivum L.) based end-products, particularly for Asian noodles. Breeding wheat cultivars with low PPO activity is the best approach to reduce the undesirable darkening. Molecular markers could greatly improve selection efficiency in breeding programs. Based on the sequences of PPO genes (GenBank Accession Numbers AY596268, AY596269 and AY596270) conditioning PPO activity during kernel development, 28 pairs of primers were designed using the software ‘DNAMAN’. One of the markers from AY596268, designated as PPO18, can amplify a 685-bp and an 876-bp fragment in the cultivars with high and low PPO activity, respectively. The difference of 191-bp size was detected in the intron region of the PPO gene. The STS marker PPO18 was mapped to chromosome 2AL using a DH population derived from a cross Zhongyou 9507× CA9632, a set of nulli-tetrasomic lines and ditelosomic line 2AS of Chinese Spring. QTL analysis indicated that the PPO gene co-segregated with the STS marker PPO18 and is closely linked to Xgwm312 and Xgwm294 on chromosome 2AL, explaining 28–43% of phenotypic variance for PPO activity across three environments. A total of 233 Chinese wheat cultivars and advanced lines were used to validate the correlation between the polymorphic fragments of PPO18 and grain PPO activity. The results showed that PPO18 is a co-dominant, efficient and reliable molecular marker for PPO activity and can be used in wheat breeding programs targeted for noodle quality improvement.     

A novel molecular marker for the polyphenol oxidase gene located on chromosome 2B in common wheat

Polyphenol oxidase (PPO) is a major cause of time-dependent darkening and discoloration in Asian noodles and other wheat-based products. One of the best ways to reduce this undesirable darkening is to breed new wheat cultivars with low PPO activity using efficient and reliable markers. Based on the sequence of a PPO gene SSPPO-B1 (GenBank accession no. AB254804) located on chromosome 2B of common wheat, 26 pairs of primers were designed to detect polymorphisms between wheat cultivars with low and high PPO activity. F-8, one of these primer pairs, amplified double fragments (band ??a?? of approximately 400?bp and band ??b?? of approximately 600?bp) in the cultivars with low PPO activity, and a single fragment (only band a) in the cultivars with high PPO activity. The differences between the fragments a and b include five indels and several single nucleotide polymorphisms, which occurred in intron II of the PPO gene. F-8 can be used as a sequence-tagged site marker to discriminate between two alleles Ppo-B1a (GQ303713) and Ppo-B1b (AB254804). The screening of 284 accessions of the core collection of Chinese wheat germplasms using the marker F-8 showed that the double fragments were present in 188 accessions, and the single fragments were present in the remaining 96 accessions. Statistical analysis revealed that the cultivars with the double fragments had significantly lower mean PPO activity than those with the single fragments. We also screened the 284 accessions using two additional markers, PPO18 for Ppo-A1 on chromosome 2A and STS01 for Ppo-D1 on chromosome 2D. Results showed that the combination of markers F-8, PPO18, and STS01 could reliably predict PPO activity. These markers can be used in wheat breeding programs for low PPO activity selection to improve the quality of wheat-based products.     

Gene markers for grain polyphenol oxidase activity in common wheat

Polyphenol oxidase (PPO) in grain is regarded as a major factor resulting in time-dependent darkening of wheat end products, particularly for Asian noodles and steamed bread. Breeding wheat cultivars with low PPO activity using efficient and reliable markers is one of the best ways to reduce the undesirable darkening. In the present study, we developed a gene-specific marker (PPO05) for low PPO activity from the sequence AY515506. This marker detected double PCR fragments (<750 and >750 bp) in the cultivars with low PPO activity and a single PCR fragment (<750 bp) in the cultivars with high PPO activity. Screening of this marker on 235 Chinese wheat micro-core collections showed that the double fragments were present in 113 genotypes and the single fragments in the remaining 122 genotypes. Statistic analysis revealed that the cultivars with the double fragments had significantly lower mean PPO activity than those with single fragments. Through sequence analysis and blast search in NCBI, we found that the cultivars with the double fragments contained the PPO-2Ab allele, while the cultivars with the single fragments contained the PPO-2Aa allele. The PPO-2Ab and PPO-2Da alleles were associated with the low grain PPO activity and the PPO-2Aa and PPO-2Db alleles associated with the high PPO activity. The genotypes carrying both PPO-2Ab and PPO-2Da showed the lowest PPO activity, while the genotypes carrying both PPO-2Aa and PPO-2Db showed the highest PPO activity. Comparison of PPO05 and STS01 with the STS markers PPO18 and PPO29 showed that the larger and small fragments of PPO05 were equivalent to the 876- and 685-bp fragments of PPO18, respectively, and that STS01 was the complementary marker of PPO29. Thus, the STS markers PPO05 and STS01 along with PPO18 and PPO29 are the efficient and reliable markers for the evaluation of PPO activity and can be used in wheat breeding programs to improve the quality of noodles and other end products.     

Genetic mapping of new seed-expressed polyphenol oxidase genes in wheat (Triticum aestivum L.)

Polyphenol oxidase (PPO) enzymatic activity is a major cause in time-dependent discoloration in wheat dough products. The PPO-A1 and PPO-D1 genes have been shown to contribute to wheat kernel PPO activity. Recently a novel PPO gene family consisting of the PPO-A2, PPO-B2, and PPO-D2 genes has been identified and shown to be expressed in wheat kernels. In this study, the sequences of these five kernel PPO genes were determined for the spring wheat cultivars Louise and Penawawa. The two cultivars were found to be polymorphic at each of the PPO loci. Three novel alleles were isolated from Louise. The Louise X Penawawa mapping population was used to genetically map all five PPO genes. All map to the long arm of homeologous group 2 chromosomes. PPO-A2 was found to be located 8.9 cM proximal to PPO-A1 on the long arm of chromosome 2A. Similarly, PPO-D1 and PPO-D2 were separated by 10.7 cM on the long arm of chromosome 2D. PPO-B2 mapped to the long arm of chromosome 2B and was the site of a novel QTL for polyphenol oxidase activity. Five other PPO QTL were identified in this study. One QTL corresponds to the previously described PPO-D1 locus, one QTL corresponds to the PPO-D2 locus, whereas the remaining three are located on chromosome 2B.     

Distribution of puroindoline alleles in bread wheat cultivars of the Yellow and Huai valley of China and discovery of a novel puroindoline a allele without PINA protein

Kernel hardness is one of the most important factors determining the milling and processing quality of bread wheat (Triticum aestivum L.). In the present study, 267 wheat cultivars and advanced lines from the Yellow and Huai Valley of China, CIMMYT, Russia and Ukraine were used for identification of SKCS (Single Kernel Characterization System) hardness and puroindoline alleles. Results indicated that Pinb-D1b is the most popular genotype in wheat cultivars from the Yellow and Huai Valley, Russia and Ukraine, whereas PINA null is a predominant genotype in wheat cultivars and advanced lines from CIMMYT. Molecular characterization of PINA-null alleles indicated that one Chinese landrace Chiyacao had the allele Pina-D1l with a single nucleotide C deletion at position 265 in Pina coding region based on sequencing results, and 35 of 39 PINA-null alleles belonged to Pina-D1b according to PCR amplification with the sequence-tagged site (STS) marker Pina-N developed previously. The remaining three cultivars (Jiangdongmen, Heshangtou and Hongquanmang from China) with PINA-null alleles were characterized at the DNA level by a primer walking strategy, and the results showed that all three cultivars with PINA-null alleles possessed a uniform 10,415-bp deletion from −5,117 bp to +5,298 bp (ATG codon references zero), designated as Pina-D1r. Correspondingly, an STS marker Pina-N2 with an expected fragment size of 436-bp spanning the 10,415-bp deletion was developed for detection of the Pina-D1r allele. This study provided a useful molecular marker for straightforward detection of one of the PINA-null alleles and would also be helpful to further understand the molecular and genetic basis of kernel hardness in bread wheat.     

New DNA markers for high molecular weight glutenin subunits in wheat

End-use quality is one of the priorities of modern wheat (Triticum aestivum L.) breeding. Even though quality is a complex trait, high molecular weight (HMW) glutenins play a major role in determining the bread making quality of wheat. DNA markers developed from the sequences of HMW glutenin genes were reported in several previous studies to facilitate marker-assisted selection (MAS). However, most of the previously available markers are dominant and amplify large DNA fragments, and thus are not ideal for high throughput genotyping using modern equipment. The objective of this study was to develop and validate co-dominant markers suitable for high throughput MAS for HMW glutenin subunits encoded at the Glu-A1 and Glu-D1 loci. Indels were identified by sequence alignment of allelic HMW glutenin genes, and were targeted to develop locus-specific co-dominant markers. Marker UMN19 was developed by targeting an 18-bp deletion in the coding sequence of subunit Ax2* of Glu-A1. A single DNA fragment was amplified by marker UMN19, and was placed onto chromosome 1AL. Sixteen wheat cultivars with known HMW glutenin subunits were used to validate marker UMN19. The cultivars with subunit Ax2* amplified the 362-bp fragment as expected, and a 344-bp fragment was observed for cultivars with subunit Ax1 or the Ax-null allele. Two co-dominant markers, UMN25 and UMN26, were developed for Glu-D1 by targeting the fragment size polymorphic sites between subunits Dx2 and Dx5, and between Dy10 and Dy12, respectively. The 16 wheat cultivars with known HMW glutenin subunit composition were genotyped with markers UMN25 and UMN26, and the genotypes perfectly matched their subunit types. Using an Applied Biosystems 3130xl Genetic Analyzer, four F₂ populations segregating for the Glu-A1 or Glu-D1 locus were successfully genotyped with primers UMN19, UMN25 and UMN26 labeled with fluorescent dyes.     

小麦PPO活性基因等位变异的区域分布研究

为了解中国不同生态区小麦种质资源籽粒多酚氧化酶(PPO)活性基因的等位变异的差异与分布,利用小麦PPO活性基因的功能标记PPO16、PPO29与PPO18,检测了来自中国7个不同生态麦区的379份小麦种质资源的等位变异和分布差异。结果表明:(1)在2AL染色体该基因位点有2种等位变异类型:Ppo-A1a(高PPO)和Ppo-A1b(低PPO),其频率分别为51.5%和48.5%。(2)在2DL染色体该基因位点有3种等位变异类型:Ppo-D1a(低PPO)、Ppo-D1b(高PPO)和Ppo-D1ab(中间型),其频率分别为57.8%、32.5%和9.8%。(3)该基因在2AL和2DL染色体上的位点变异有6种不同类型的组合:Ppo-A1a/D1a(中间型)、Ppo-A1a/D1b(高PPO)、Ppo-A1a/Ppo-D1ab(中间型)、Ppo-A1b/D1a(低PPO)、Ppo-A1b/D1b(中间型)和Ppo-A1b/Ppo-D1ab(中间型),其中,与低PPO活性相关的基因型组合Ppo-A1b/D1a的频率为25.6%。(4)小麦PPO活性基因不同变异类型在各生态区的分布存在明显差异,基因型Ppo-A1b在北部冬麦区和西南冬麦区的比例较大,基因型Ppo-D1a在黄淮冬麦区和北部冬麦区的比例较大,基因型组合Ppo-A1b/D1a在北部冬麦区的比例较大。研究认为,结合采用分子标记辅助选择(MAS),有利于小麦籽粒外观品质的遗传改良和新品种选育。     

Inheritance of grain polyphenol oxidase (PPO) activity in multiple wheat (Triticum aestivum L.) genetic backgrounds

Grain polyphenol oxidase (PPO) activity can cause discoloration of wheat (Triticum aestivum L.) food products. Five crosses (PI 117635/Antelope; Fielder/NW03681; Fielder/Antelope; NW07OR1070/Antelope; NW07OR1066/OR2050272H) were selected to study the genetic inheritance of PPO activity. STS markers, PPO18, PPO29 and STS01, were used to identify lines with putative alleles at the Ppo-A1 and Ppo-D1 loci conditioning low or high PPO activity. ANOVA showed significant genotypic effects on PPO activity (P?<?0.0001) in all populations. The generations and generation?×?genotype effects were not significant in any population. A putative third (null) genotype at Ppo-A1 (no PCR fragments for PPO18) was discovered in NW07OR1066 and NW07OR1070 derived populations, and these had the lowest mean PPO activities. Results demonstrated that both Ppo-A1 and Ppo-D1 loci affect the kernel PPO activity, but the Ppo-A1 has the major effect. In three populations, contrary results were observed to those predicted from previous work with Ppo-D1 alleles, suggesting the markers for Ppo-D1 allele might give erroneous results in some genetic backgrounds or lineages. Results suggest that selection for low or null alleles only at Ppo-A1 might allow development of low PPO wheat cultivars.     

Genetic analysis of the dwarfing gene (Rht8) in wheat. Part I. Molecular mapping of Rht8 on the short arm of chromosome 2D of bread wheat (Triticum aestivum L.)

 Two sets of single chromosome recombinant lines comparing 2D chromosomes from the wheat varieties ‘Ciano 67’ and ‘Mara’ with the common 2D chromosome of ‘Cappelle-Desprez’ in a ‘Cappelle-Desprez’ background were used to detect a diagnostic wheat microsatellite marker for the dwarfing gene Rht8. The genetic linkage maps place the wheat microsatellite marker WMS 261 0.6 cM distal to Rht8 on the short arm of chromosome 2D. By PCR analysis the WMS 261 alleles of ‘Mara’, ‘Cappelle-Desprez’ and ‘Ciano 67’ could be distinguished by different fragment sizes of 192 bp, 174 bp and 165 bp, respectively. A screen of over 100 international varieties of wheat showed that the three allelic variants were all widespread. It also demonstrated that a limited number of varieties carried novel WMS 261 variants of over 200 bp. Following classification of the individual recombinant lines for allelic variants at the WMS 261 locus it was possible to attribute a 7- to 8-cm reduction in plant height with the WMS 261-192-bp allele compared to the WMS 261-174-bp allele in the set of recombinant lines comparing 2D chromosomes of ‘Mara’ and ‘Cappelle-Desprez’. A height reduction of around 3 cm was detected between the WMS 261-174-bp allele and the WMS 261-165-bp allele in the recombinant lines comparing 2D chromosomes of ‘Cappelle-Desprez’ and ‘Ciano 67’. Received: 17 October 1997 / Accepted: 12 November 1997     

Characterization of phytoene synthase 1 gene (<Emphasis Type="Italic">Psy1</Emphasis>) located on common wheat chromosome 7A and development of a functional marker

Phytoene synthase (Psy), a critical enzyme in the carotenoid biosynthetic pathway, demonstrated high association with the yellow pigment (YP) content in wheat grain. Characterization of Psy genes and the development of functional markers for them are of importance for marker-assisted selection in wheat breeding. In this study, the full-length genomic DNA sequence of a Psy gene (Psy-A1) located on chromosome 7A, was characterized by in silico cloning and experimental validation. The cloned Psy-A1 comprises six exons and five introns, 4,175 bp in total, and an ORF of 1,284 bp. A co-dominant marker, YP7A, was developed based on polymorphisms of two haplotypes of Psy-A1, yielding 194 and 231-bp fragments in cultivars with high and low YP content, respectively. The marker YP7A was mapped on chromosome 7AL using an RIL population from cross PH82-2/Neixing 188, and a set of Chinese Spring nullisomic–tetrasomic lines and ditelosomic line 7AS. Psy-A1, co-segregating with the STS marker YP7A, was linked to SSR marker Xwmc809 on chromosome 7AL with a genetic distance of 5.8 cM, and explained 20–28% of the phenotypic variance for YP content across three environments. A total of 217 Chinese wheat cultivars and advanced lines were used to validate the association between the polymorphic band pattern and grain YP content. The results showed that the functional marker YP7A was closely related to grain YP content and, therefore, could be used in wheat breeding programs targeting of YP content for various wheat-based products. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

AFLP and STS tagging of Lr19, a gene conferring resistance to leaf rust in wheat

Amplified fragment length polymorphism (AFLP) markers were used to enrich the map of the wheat chromosomal region containing the Thinopyrum-derived Lr19 leaf rust resistance gene. The region closest to Lr19 was targeted through the use of deletion and recombinant lines of the translocated segment. One of the AFLP bands thus identified was converted into a sequence-tagged-site (STS) marker. This assay generated a 130-bp PCR fragment in all Lr19-carrying lines tested, except for one deletion mutant, while non-carrier template failed to amplify any product. This sequence represents the first marker to map on the distal side of Lr19 on chromosome 7el₁. The conversion process of AFLP fragments to STS markers was technically difficult, mainly because of the presence of contaminating fragments. Various approaches were taken to reduce the frequency of false positives and to identify the correct clone. We were able to formulate a general verification strategy prior to clone sequencing. Various other factors causing problems with converting AFLP bands to an STS assays are also discussed. Received: 15 September 2000 / Accepted: 5 January 2001     

Use of RAPD- and STS-analysis for marking genes of a homeologic group from chromosome 5 of common wheat

The search for STS (sequence-tagged site) and RAPD (random amplified polymorphic DNA) markers tightly linked to some genes of homeologous group 5 chromosomes of common wheat Triticum aestivum L., more specifically, awns inhibitor genes (B1), vernalization response gene (Vrn1), and homeologous chromosome pairing gene (Ph1), was conducted. To estimate the linkage of the gene with the marker, wheat lines marked with recessive alleles b1 and vrn1 were used. RELP (restriction fragment length polymorphism) and SSR (simple sequence repeat) analyses of isogenic wheat lines were conducted to characterize the chromosomal region transferred to the isogenic line from the donor parent. In RAPD analysis of isogenic wheat lines marked with recessive alleles b1 and vrn1, 95 arbitrary primers were used. To develop STS markers, analysis of the primary structure of RELP markers Xpsr426 and Xcdo504, tightly linked to the Vrn1 gene, and the Xpsr1201 marker, located at the Ph1 locus, was carried out. Two markers that are tightly linked to the Vrn1 gene (5AL)--RAPD marker Xr405 and STS marker Xsts426--were obtained in this work. In addition, there is every reason to believe that Xsts426 can be used as a PCR marker of genes Vrn2 (5BL) and Vrn3 (5DL), while Xsts1201, of the gene Ph1 (5BL).     

Molecular Analysis of Leaf Rust-Resistant Introgression Lines Obtained by Crossing of Hexaploid Wheat Triticum aestivum with Tetraploid Wheat Triticum timopheevii

Twenty-four Triticum aestivum×T. timopheevii hybrid lines developed on the basis of five varieties of common wheat and resistant to leaf rust were analyzed by the use of microsatellite markers specific for hexaploid wheat T. aestivum. Investigation of intervarietal polymorphism of the markers showed that the number of alleles per locus ranged from 1 to 4, depending on the marker (2.5 on average). InT. timopheevii, amplification fragments are produced by 80, 55, and 30% of primers specific to the A, B, and D common wheat genomes, respectively. Microsatellite analysis revealed two major areas of introgression of the T. timopheevii genome: chromosomes of homoeological groups 2 and 5. Translocations were detected in the 2A and 2B chromosomes simultaneously in 11 lines of 24. The length of the translocated fragment in the 2B chromosome was virtually identical in all hybrid lines and did not depend on the parental wheat variety. In 15 lines developed on the basis of the Saratovskaya-29, Irtyshanka, and Tselinnaya-20, changes occurred in the telomeric region of the long arm of the 5A chromosome. Analysis with markers specific to the D genome suggested that introgressions of the T. timopheevii genome occurred in chromosomes of the D genome. However, the location of these markers on T. timopheevii chromosomes is unknown. Our data suggest that the genes for leaf rust resistance transferred from T. timopheevii to T. aestivum are located on chromosomes of homoeological group 2.     

High-resolution cytological mapping of the long arm of chromosome 5A in common wheat using a series of deletion lines induced by gametocidal (Gc) genes of Aegilops speltoides

Gametocidal (Gc) genes of Aegilops in the background of the wheat genome lead to breakage of wheat chromosomes. The Q gene of wheat was used as a marker to select 19 deletion lines for the long arm of chromosome 5A of common wheat, Triticum aestivum cv. Chinese Spring (CS). The extents of deleted segments were cytologically estimated by the C-banding technique. The DNAs of deletion lines were hybridized with 22 DNA probes recognizing sites on the long arm of the chromosome (5AL) to determine their physical order. Based on the breeding behavior of the deletion lines, the location of a novel gene (Pv, pollen viability) affecting the viability of the male gamete was deduced. The segment translocated from 4AL to 5AL in CS was cytologically estimated to represent 13% of the total length of 5AL. Although DNA markers were almost randomly distributed along the chromosome arm, DNA markers located around the centromere and C-banded regions were obtained only rarely. Some deletion lines were highly rearranged in chromosome structure due to the effect(s) of the Gc gene. Applications of Gc genes for manipulating wheat chromosomes are discussed.     

Chromosomal location of PCR fragments as a source of DNA markers linked to aluminium tolerance genes in rye

 To identify and locate rye DNA sequences homologous to three wheat c-DNAs (wali1, wali2 and wali5) whose expression is induced by aluminium (Al) stress, we designed three pairs of specific primers. They were used in the amplification of genomic DNA from wheat-rye disomic addition lines. The wali2 pair of primers amplified a 878-bp rye DNA fragment (rali2) located on chromosomes 4R and 7R that showed 79.37% homology with the corresponding wheat c-DNA. RAPD fragments were also used as genetic markers. We located 22 different RAPDs distributed on 11 different rye chromosome arms using wheat-rye disomic and ditelocentric addition lines. Thirteen of these markers were located on the chromosomes 3R, 4R and 6R, which also carry aluminium-tolerance genes. The OPA08 ₄₁₅ and OPR01 ₆₀₀ RAPD markers, located on the 6RL and 6RS chromosome arms, respectively, were converted to SCAR markers (SCA08 ₄₁₅ and SCR01 ₆₀₀ ) and linked to Alt1 gene (SCR01 ₆₀₀ -2.1 cM-Alt1-33.5 cM-SCA08 ₄₁₅ ). We propose that the chromosomal location of RAPDs and SCARs using wheat-rye addition lines is a source of DNA markers linked to aluminium-tolerance loci and offers a valuable strategy in marker-assisted selection for the introgression of tolerance genes in wheat. Received: 9 June 1997 / Accepted: 19 September 1997     

Dreb1 genes in wheat (Triticum aestivum L.): development of functional markers and gene mapping based on SNPs

The Dreb genes are involved in abiotic stress tolerances, such as drought, salinity, low temperature and ABA. The purpose of the present research was to establish protocols for the development of genome-specific and allele specific markers in common wheat (Triticum aestivum L.) using the Dreb1 genes as an example. Based on the available sequences of Dreb1 genes in common wheat and related species, five primer pairs were designed using Primer Premier 5.0. Two primers, P25F/PR and P21F/P21R, amplified 596- and 1113-bp fragments, respectively, from the A genome, P18F/P18R amplified a 717-bp fragment from the B genome, and primers P22F/PR and P20F/P20R amplified 596- and 1193-bp fragments, respectively, from the D genome. Using these genome-specific primers and the Chinese Spring using nulli-tetrasomic lines, the Dreb1 genes were located on chromosomes 3A, 3B and 3D. Two SNPs (S646 and S770) in Dreb-B1 distinguished the Opata 85 and W7984 parents of the ITMI mapping population, but there was no polymorphism between the orthologous Dreb-A1 and Dreb-D1 sequences. By assaying the genotypes of 115 RILs with the allele-specific primer P40 based on SNP S770, Dreb-B1 was mapped between markers Xmwg818 and Xfbb117 on chromosome 3BL. This genetic mapping of Dreb-B1 on chromosome 3B may be helpful in wheat breeding programs aimed at improving drought tolerance.     

Microsatellite markers linked to six Russian wheat aphid resistance genes in wheat

The Russian wheat aphid (RWA), Diuraphis noxia Mordvilko, is a serious economic pest of wheat and barley in North America, South America, and South Africa. Using aphid-resistant cultivars has proven to be a viable tactic for RWA management. Several dominant resistance genes have been identified in wheat, Triticum aestivum, including Dn1 in PI 137739, Dn2 in PI 262660, and at least three resistance genes (Dn5+) in PI 294994. The identification of RWA-resistant genes and the development of resistant cultivars may be accelerated through the use of molecular markers. DNA of wheat from near-isogenic lines and segregating F₂ populations was amplified with microsatellite primers via PCR. Results revealed that the locus for wheat microsatellite GWM111 (Xgwm111), located on wheat chromosome 7DS (short arm), is tightly linked to Dn1, Dn2 and Dn5, as well as Dnx in PI 220127. Segregation data indicate RWA resistance in wheat PI 220127 is also conferred by a single dominant resistance gene (Dnx). These results confirm that Dn1, Dn2 and Dn5 are tightly linked to each other, and provide new information about their location, being 7DS, near the centromere, instead of as previously reported on 7DL. Xgwm635 (near the distal end of 7DS) clearly marked the location of the previously suggested resistance gene in PI 294994, here designated as Dn8. Xgwm642 (located on 1DL) marked and identified another new gene Dn9, which is located in a defense gene-rich region of wheat chromosome 1DL. The locations of markers and the linked genes were confirmed by di-telosomic and nulli-tetrasomic analyses. Genetic linkage maps of the above RWA resistance genes and markers have been constructed for wheat chromosomes 1D and 7D. These markers will be useful in marker-assisted breeding for RWA-resistant wheat. Received: 17 May 2000 / Accepted: 13 June 2000     

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     

Genetic and in silico comparative mapping of the polyphenol oxidase gene in bread wheat (Triticum aestivum L.)

Polyphenol oxidases (PPOs) are involved in the time-dependent darkening and discolouration of Asian noodles and other wheat end products. In this study, a doubled haploid (DH) population derived from Chara (moderately high PPO activity)/WW2449 (low PPO activity) was screened for PPO activity based on l-DOPA and l-tyrosine assays using whole seeds. Both these assays were significantly genetically correlated (r=0.91) in measuring the PPO activity in this DH population. Quantitative trait loci (QTLs) analysis utilising a skeleton map enabled us to identify a major QTL controlling PPO activity based on l-DOPA and l-tyrosine on the long arm of chromosome 2A. The simple sequence repeat (SSR) marker GWM294b explained over 82% of the line mean phenotypic variation from samples collected in both 2000 and 2003. Four SSR markers were validated for PPO linkage in genetically diverse backgrounds and proven to correctly predict the PPO activity in more than 92% of wheat lines. Physical mapping using deletion lines of Chinese Spring has confirmed the location of the GWM294b, GWM312 and WMC170 on chromosome 2AL, between deletion breakpoints 2AL-C to 0.85. In order to identify functional gene markers, data searches for alignments between rice BAC/PAC clones assembled on chromosome 1 and 4, chromosome 7, and (1) the wheat expressed sequence tags mapped in deletion bin (2AL-C to 0.85) and (2) the coding sequence of a previously cloned wheat PPO gene were made and found significant sequence similarities with the PPO gene or common central domain of tyrosinase. Available PPO gene sequences in the National Centre for Biotechnology Information (NCBI) database have revealed that there is a significant molecular diversity at the nucleotide and amino acid level in the wheat PPO genes.Electronic Supplementary Material Supplementary material is available for this article at .