Comparison of a modified assay method for the endopeptidase marker Ep-D1b with the Sequence Tag Site marker XustSSR2001–7DL for strawbreaker foot rot resistance in wheat

The endopeptidase marker Ep‐D1b and Sequence Tag Site (STS) marker XustSSR2001–7DL were reported to be closely associated with the most effective resistance gene (Pch1) in wheat (Triticum aestivum L.) for strawbreaker foot rot [Pseudocercosporella herpotrichoides (Fron) Deighton]. Our objectives were to: (i) develop an efficient assay method for Ep‐D1b in wheat; (ii) correlate endopeptidase zymograms to strawbreaker foot rot reactions of various wheat genotypes; and (iii) compare the utility of Ep‐D1b and XustSSR2001–7DL for predicting disease response. An improved method of assaying for the Ep‐D1b marker using roots from a single seedling was developed, which is a 2.5‐fold improvement over the previous method. Thirty‐eight wheat genotypes with known reactions to strawbreaker foot rot were analysed for Ep‐D1b and the STS marker. Six distinct endopeptidase zymograms were identified among these 38 genotypes tested, and three of these patterns were novel. The endopeptidase marker was 100% accurate for predicting strawbreaker foot rot disease response, whereas the STS marker predicted the correct phenotype with approximately 90% accuracy. The endopeptidase marker Ep‐D1b was more effective and was more economical for use in marker‐assisted selection strategies for Pch1 in our laboratory compared with the STS marker.     

Linkage relations among eyespot resistance gene Pch2, endopeptidase Ep-A1b, and RFLP marker Xpsr121 on chromosome 7A of wheat

Marker-based selection of Ep-D1b has been used successfully to incorporate Pch1, the gene for eyespot resistance on chromosome 7D, into commercial wheat. However, attempts to transfer resistance conferred by Pch1 (on chromosome 7A) through selection for Ep-A1b have not always been successful. Linkage relations among eyespot resistance gene Pch2, a gene encoding for an isozyme of endopeptidase, Ep-A1b, and RFLP marker Xpsr121 on chromosome 7A were determined using 80 homozygous recombinant substitution lines. The recombinant lines were derived from eyespot susceptible ‘Chinese Spring’ hybridized with a resistant disomic substitution line of ‘Cappelle Desprez’ that has chromosome 7A substituted into ‘Chinese Spring’. Segregations of Pch2, Ep-A1b and Xpsr121 fit an expected 1:1 single-locus ratios based on χ² tests. Linkage analysis revealed that Pch2 was not tightly linked to Ep-Alb (15% recombination). However, close linkage (3.8% recombination) existed between Ep-A1b and Xpsr121. The order of these loci is Pch2-Xpsr121-Ep-A1b. Unlike Pch1 and Ep-D1b, where little or no recombination is found, Pch1 and Ep-A1b showed considerable recombination and therefore linkage cannot be utilized efficiently in marker-based selection.     

Location of a Gene for Resistance to Eyespot (Pseudocercosparella herpotrichoides) on Chromosome 7D of Bread Wheat

Chromosome 7D of the wheat line VPM1 derived from a cross of Aegilops ventricosa with wheat confers resistance to the facultative fungal parasite Pseudocercosporella herpotrichoides. To determine the number of genes responsible fur this resistance, homozygous recombinant lines were developed from an F₁ between the wheat variety ‘Hobbit sib’ and a substitution line carrying chromosome 7D of VPM1 in a ‘Hobbit sib’ background. Resistance to Pseudocercosporella herpotrichoides is shown to be determined by a single gene located distally on the long arm of chromosome 7D. EpD1b, a unique allele of a gene encoding the readily detectable isoenzyme — endopeptidase, maps without recombination to Pch1 suggesting for two separate genes a maximum recombination value of 0.03 (P 0.05). Resistance to Pherpotrichoides could alter-natively be a product of Ep-D1b. Pch1 is also mapped against a gene for adult plant resistance to brown rust (Puccinia recondita), to Rc3 which confers coleoptile colour, and to α-Amy-D2, an isozyme that encodes α-amylase production.     

Microsatellite monitoring of recombination around the Vrn-B1 locus of wheat during early backcross breeding

The length of chromosomal segments retained around the Vrn‐B1 gene controlling sensitivity to vernalization in wheat (Triticum aestivum L.) was studied in the first and third backcrosses by using microsatellite markers. Eleven polymorphic markers located on chromosome 5B were used for microsatellite analysis. It was shown in the first backcross that plants with a donor segment around the gene of interest not longer than 50% of chromosome 5B could be selected. When selection is not molecular‐marker assisted, the length of the chromosomal donor segment with the target gene may reach 94% of chromosome 5B even in plants of the third backcross generation. The considerable length differences in the 5B microsatellite loci between the winter and spring lines of wheat studied indicate that these markers are promising in marker‐assisted backcrossing or marker‐assisted selection for the Vrn‐B1 gene using different combinations of Spring and Winter genotypes.     

A PCR-based DNA marker for detection of mutant and normal alleles of the Wx-D1 gene of wheat

To assist waxy wheat breeding a DNA marker was developed to discriminate mutant and normal alleles at the Wx‐D1 locus. This polymerase chain reaction‐based marker distinguishes the mutant from the normal allele by targeting the previously reported deletion basis of the mutant. The marker codominantly identifies the normal allele of the Wx‐D1 gene from the mutant allele originated from the Chinese landrace ‘Baihoumai’. However, attempts with a number of primer combinations targeting this deletion failed to amplify the corresponding fragment from an unrelated wheat line (NP150) that has a mutant null allele at the same locus. This indicates that NP150 has a different mutant allele from that of ‘Baihoumai’. This marker is a useful tool to identify wheat cultivars with mutant and normal alleles of the Wx‐D1 gene, and is used in marker‐assisted selection of the Wx‐D1 gene in our waxy wheat breeding programme.     

Development of molecular markers linked to the wheat powdery mildew resistance gene Pm4b and marker validation for molecular breeding

Powdery mildew, caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici, is an important disease in wheat (Triticum aestivum L.). Bulk segregant analysis (BSA) was employed to identify SRAP (sequence‐related amplified polymorphism), sequence tagged site (STS) and simple sequence repeat (SSR) markers linked to the Pm4b gene, which confers good resistance to powdery mildew in wheat. Out of 240 SRAP primer combinations tested, primer combinations Me8/Em7 and Me12/Em7 yielded 220‐bp and 205‐bp band, respectively, each of them associated with Pm4b. STS‐₂₄₁ also linked to Pm4b with a genetic distance of 4.9 cM. Among the eight SSR markers located on wheat chromosome 2AL, Xgwm382 was found to be polymorphic and linked to Pm4b with a genetic distance of 11.8 cM. Further analysis was carried out using the four markers to investigate marker validation for marker‐assisted selection (MAS). The results showed that a combination of the linked markers STS_?241, Me8/Em7_?220 and Xgwm382 could be used for marker‐assisted selection of the resistance gene Pm4b in wheat breeding programmes.     

A polymorphic microsatellite in the 3'end of 'waxy' genes of wheat, Triticum aestivum

Potential polymorphism of an (AT)_N microsatellite at the 3’end of waxy genes in bread wheat was examined. Primers were designed from a published cDNA sequence of a wheat waxy gene. Polymerase chain reaction (PCR) amplification of genomic DNA from 135 mainly Australian cultivars revealed eight alleles on chromosome 7A. This polymorphic microsatellite is a potential codominant marker for the Wx-A1 locus in breeding programmes. A distinguishable fragment was also amplified from chromosome 7D. This fragment was absent where a plant was null for the waxy gene on chromosome 7D, being a dominant marker for the Wx-D1 locus. The primers were also useful for amplifying genomic DNA from barley, rye and triticale and can be used to detect potential polymorphism in these species.     

Inheritance of resistance of wheat to eyespot at the adult stage

Moderate resistance to eyespot was first incorporated in the variety Cappelle‐Desprez (CD). Later the gene Pch1, which could confer a higher level of resistance, was introduced from Aegilops ventricosa. However Pch1‐carrying varieties can sustain significant eyespot‐induced yield losses in severe attack situations. A strategy to further enhance the resistance of wheat is by pyramiding Pch1 and the genes for resistance in CD. The first requirement to achieve this is a better understanding of the genetics of resistance in CD. The resistance of the 21 Cappelle‐Desprez (Bezostaya) disomic substitution lines was evaluated. Chromosome 7A was confirmed as carrying a major gene for resistance to eyespot at the seedling stage. However, this study demonstrates that this chromosome has no effects at the adult stage. Chromosome 5A was shown to carry a major gene for resistance to eyespot at the adult stage, which was stably expressed each year of testing. Chromosomes 1A and 2B had significant effects for only two years among four.     

Identification of the Rf gene conferring fertility restoration of the CMS Dian-type 1 in rice by using simple sequence repeat markers and advanced inbred lines of restorer and maintainer

Cytoplasmic male sterility of Dian‐type 1 (CMS‐D1) was developed 30 years ago in Yunnan. A major gene conferring fertility restoration for the CMS‐D1 system was detected by microsatellite markers in advanced inbred lines consisting of 196 maintainers and 62 restorers developed in breeding programmes of hybrid rice involving the CMS‐D1 system. The gene was mapped between two simple sequence repeat markers, OSR33 and RM228, on chromosome 10, and was temporarily designated as Rf‐D1(t). The genetic distances of the gene to the two microsatellite markers were 3.4 and 5.0 cM, respectively. This linkage was confirmed by using an F2 population derived from a cross between a CMS‐D1 line and a restorer. This study also demonstrated that using OSR33 was reliable and efficient for identification of restoring lines in hybrid rice breeding with the CMS‐D1 system.     

Diagnostic value of molecular markers linked to the eyespot resistance gene <Emphasis Type="Italic">Pch1</Emphasis> in wheat

Eyespot is a major disease of common wheat (Triticum aestivum L.) in temperate climates and causes yield losses of up to 40%. The causal agents of eyespot are Oculimacula acuformis (syn. Tapesia acuformis; anamorph: Helgardia acuformis syn. Pseudocercosporella herpotrichoides var. acuformis) and O. yallundae (syn. T. yallundae; anamorph: H. yallundae syn. P. h. var. herpotrichoides). Pch1 located on chromosome 7DL is the most important and most effective resistance gene, but its use in practical breeding is limited because of difficulties in phenotyping and the fact that markers like XustSSR2001-7DL are often population-specific in German wheat cultivars. Therefore, based on results obtained for endopeptidase Ep-D1a, which is very closely linked to Pch1, molecular markers located in the terminal region of chromosome 7DL were analysed in three DH (double haploid) populations. In a next step these molecular markers were validated on a set of German winter wheat cultivars to obtain information on their usefulness for marker assisted selection (MAS). Based on the analysis of 127 DH-lines, linkage to Pch1 (Ep-D1) was obtained for Xorw1, Xorw5, Xorw6, Xcfd175, Xbarc76, Xwmc14, and Xcfa2040. Analyses of 104 German winter wheat cultivars showed that Xorw1, Xorw6 and the SSR Xcfd175 of these markers are well suited for MAS in German wheat breeding.     

Microsatellite variability and its use in the characterization of cultivated clones of Hevea brasiliensis

Microsatellite markers were developed and evaluated in Hevea brasiliensis, an important crop species producing natural rubber of commercial utility. Of eight microsatellite markers, four were found to be highly informative, amplifying a total of 19 alleles when evaluated against 27 cultivated Hevea clones/genotypes. Power of discrimination of the microsatellite loci was in the range of 0.62‐0.89, with a mean of 0.76 indicating these microsatellites could be valuable genetic markers for diversity characterization. A combination of four microsatellite markers was successfully used to discriminate uniquely all the 27 Hevea clones and some clone‐specific allelic profiles were generated. Cross‐species amplification of the markers developed in H. brasiliensis had also been demonstrated with two other Hevea species, H. benthamiana and H. spruceana, indicating a high degree of sequence homology at the flanking regions. Sequence analysis of the repeat region at the 3′‐UTR of the hydroxymethylglutaryl‐coenzyme A reductase gene, containing clusters of AG repeats in 15 clones, revealed the existence of two alleles based on the repeat length polymorphisms. Homozygosity as well as heterozygosity for both the alleles had also been detected among the clones. Frequency of homozygotes for the smaller allele (allele‐1) was found to be lower than the larger allele (allele‐2) among the primary clones of H. brasiliensis.     

Effect of the ph1b mutant on chromosome pairing in hybrids between Dasypyrum villosum and Triticum aestivum

Chromosome pairing was analysed in F₁ hybrids of the wheat cultivar ‘Chinese Spring’ (CS) and its ph1b mutant (CSphlb) with Dasypyrum villosum. On average, 1.61 chromosomes per cell paired in the hybrid CS ×D. villosum, but 14.43 in the hybrid CS ph1b×D. villosum. Genomic fluorescence in situ hybridization (GISH) revealed three types of homoeologous association between wheat (W) and D. villosum (D) chromosomes (W‐D, D‐W‐W and D‐W‐D) in pollen mother cells of the CS ph1b×D. villosum hybrid, and only one type (W‐W), in the CS ×D. villosum hybrid. Both F₁ hybrids were self‐sterile. The seed set of the backcross of CS ×D. villosum with CS was 6.67% and that of CS ph1b×D. villosum with CS or CS ph1b was only 0.45%. The chromosome number of BC₁ plants varied from 48 to 72. Translocations of chromosome segments or entire arms between wheat and D. villosum chromosomes were detected by GISH in the BC₁ plants from the backcross of CS ph1b×D. villosum to CS ph1b.     

Genetic mapping and inheritance of Russian wheat aphid resistance gene in accession IG 100695

The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is an important pest of small‐grain cereals, particularly wheat, worldwide. The most efficient strategy against the RWA is to identify sources of resistance and to introduce them into susceptible wheat genotypes. This study was conducted to determine the mode of inheritance of the RWA resistance found in ICARDA accession IG 100695, to identify wheat microsatellite markers closely linked to the gene and to map the chromosomal location of the gene. Simple sequence repeat (SSR) marker scores were identified in a mapping population of 190 F₂ individuals and compared, while phenotypic screening for resistance was performed in F_2 : 3 families derived from a cross between ‘Basribey’ (susceptible) and IG 100695 (resistant). Phenotypic segregation of leaf chlorosis and rolling displayed the effect of a single dominant gene, temporarily denoted Dn100695, in IG 100695. Dn100695 was mapped on the short arm of chromosome 7D with four linked SSR markers, Xgwm44, Xcfd14, Xcfd46 and Xbarc126. Dn100695 and linked SSR markers may be useful for improving resistance for RWA in wheat breeding.     

Characterization of dinucleotide and trinucleotide EST-derived microsatellites in the wheat genome

Over the past decade microsatellites or simple sequence repeats (SSRs) have attracted a considerable amount of attention from researchers. The aim of the present paper was to analyse expressed sequence tag-derived SSR (EST-SSR) marker variability in wheat and to investigate the relationships between the number and type of repeat units and the level of microsatellite polymorphism. Two hundred and forty-one new EST-SSR markers available in a public database () were characterized in eight durum wheat cultivars (Svevo, Ciccio, Primadur, Duilio, Meridiano, Claudio, Latino, Messapia), two accessions of Triticum turgidum var. dicoccoides (MG4343, MG29896), one accession of T. turgidum var. dicoccum (MG5323) and in the common wheat cv. Chinese Spring. Of these, 201 primer pairs (83.4%) amplified PCR products successfully, while the remaining 40 (16.6%) failed to amplify any product. Of the EST-SSRs analysed, 45.2% of the primer pairs amplified one or two PCR products. Multiple discrete PCR products were observed among both di- and trinucleotide EST-SSR markers (31.2 and 40.5%, respectively). Markers based on dinucleotide microsatellites were more polymorphic than those based on trinucleotide SSRs in the 12 wheat genotypes tested (68.9 and 52.7%, respectively). An average of 2.5 alleles for dinucleotide and 2.0 alleles for trinucleotide SSRs was observed. The data reported in the present work indicate the presence of a significant relationship between motif sequence types and polymorphism. The primer set based on the AG repeat motif showed the lowest percentage of polymorphism (55.0%), while the primer set based on the AC repeat motif showed t he highest percentage (85.0%). Among trinucleotide SSRs, the AGG microsatellite markers showed the highest percentage of polymorphism (70.0%), and the ACG motif the lowest value (25.0%). The characterization of these new EST-SSR markers and the results of our studyon the effect of repeat number and type of motifs could have important applications in the genetic analysis of agronomically important traits, quantitative trait locus discovery and marker-assisted selection.     

Evaluation of cold hardiness in two sets of near-isogenic lines of wheat (Triticum aestivum) with polymorphic vernalization alleles

In wheat, variation at the orthologus Vrn‐1 loci, located on each of the three genomes, A, B and D, is responsible for vernalization response. A dominant Vrn‐1a allele on any of the three wheat genomes results in spring habit and the presence of recessive Vrn‐1b alleles on all three genomes results in winter habit. Two sets of near‐isogenic lines (NILs) were evaluated for DNA polymorphisms at their Vrn‐A1, B1 and D1 loci and for cold hardiness. Two winter wheat cultivars, ‘Daws’ and ‘Wanser’ were used as recurrent parents and ‘Triple Dirk’ NILs were used as donor parents for orthologous Vrn‐1 alleles. The NILs were analysed using molecular markers specific for each allele. Only 26 of 32 ‘Daws’ NILs and 23 of 32 ‘Wanser’ NILs had a plant growth habit that corresponded to the marker genotype for the markers used. Freezing tests were conducted in growth chambers programmed to cool to ?21.5°C. Relative area under the death progress curve (AUDPC), with a maximum value of 100 was used as a measure of death due to freezing. The average relative AUDPC of the spring habit ‘Daws’Vrn‐A1a NILs was 86.15; significantly greater than the corresponding winter habit ‘Daws’Vrn‐A1b NILs (42.98). In contrast, all the ‘Daws’Vrn‐A1bVrn‐B1aVrn‐D1b and Vrn‐A1bVrn‐B1bVrn‐D1a NILs (spring habit) had relative AUDPC values equal to those of their ‘Daws’ sister genotypes with Vrn‐A1bVrn‐B1bVrn‐D1b NILs (winter habit). The average AUDPC of spring and winter habit ‘Wanser’ NILs differed at all three Vrn‐A1, Vrn‐B1 and Vrn‐D1 locus comparisons. We conclude that ‘Daws’ and ‘Wanser’ have different background genetic interactions with the Vrn‐1 loci influencing cold hardiness. The marker for Vrn‐A1 is diagnostic for growth habit and cold hardiness but there is no relationship between the Vrn‐B1 and Vrn‐D1 markers and the cold tolerance of the NILs used in this study.     

Genotype analysis of the wheat semidwarf Rht‐B1b and Rht‐D1b ancestral lineage

In wheat, semidwarfism resulting from reduced height (Rht)‐B1b and Rht‐D1b was integral to the ‘green revolution’. The principal donors of these alleles are ‘Norin 10’, ‘Seu Seun 27’ and ‘Suwon 92’ that, according to historical records, inherited semidwarfism from the Japanese landrace ‘Daruma’. The objective of this study was to examine the origins of Rht‐B1b and Rht‐D1b by growing multiple seed bank sources of cultivars comprising the historical pedigrees of the principal donor lines and scoring Rht‐1 genotype and plant height. This revealed that ‘Norin 10’ and ‘Suwon 92’ sources contained Rht‐B1b and Rht‐D1b, but the ‘Seu Seun 27’ source did not contain a semidwarf allele. Neither Rht‐B1b nor Rht‐D1b could be definitively traced back to ‘Daruma’, and both ‘Daruma’ sources contained only Rht‐B1b. However, ‘Daruma’ remains the most likely donor of Rht‐B1b and Rht‐D1b. We suggest that the disparity between historical pedigrees and Rht‐1 genotypes occurs because the genetic make‐up of seed bank sources differs from that of the cultivars actually used in the pedigrees. Some evidence also suggests that an alternative Rht‐D1b donor may exist.     

Analysis of diallel crosses between wheat genotypes with genetically different resistance to Pseudocercosporella herpotrichoides (Fron) Deighton

Two diallels were analysed for general combining ability (GCA) and specific combining ability (SCA) to study the resistance of crosses‐between wheat genotypes, advanced to the F₅ generation, to Pseudocer‐cosporella herpotrichoides. The parents either carried the resistance‐gene Pch‐1 or had different levels of quantitative resistance, one genotype was susceptible. At medium milk‐ripening, significant effects were‐found for GCA and SCA. GCA effects were the more important. Diallel crosses between genotypes, all carrying Pch‐1, revealed interactions‐of the gene with the genotypic background. Some combinations had a‐higher level of resistance than the best parent. In these populations'CH‐75417’ was involved as a parent. Both ‘CH‐75417’ and ‘F–210.13.4.42’ had significant GCA effects. Crosses between quantitatively resistant parents yielded populations that transgressed both parents. The increased resistance level was associated with ‘Cappelle‐Desprez’, distinguished by its high GCA. In some crosses SCA contributed significantly to an increase in resistance level. Selection for resistance within the best advanced populations is recommended since it‐takes advantage of additive gene action and the high heritability estimates based on ELISA values in plant progenies.     

Quantitative trait loci mapping of adult‐plant resistance to leaf rust in a Fundulea 900 × ‘Thatcher’ wheat cross

Wheat leaf rust (LR), caused by the obligate biotrophic fungus Puccinia triticina (Pt), is a destructive foliar disease of common wheat (Triticum aestivum L.) worldwide. The most effective, economic means to control the disease is resistant cultivars. The Romanian wheat line Fundulea 900 showed high resistance to LR in the field. To identify the basis of resistance to LR in Fundulea 900, a population of 188 F_2:3 lines from the cross Fundulea 900/‘Thatcher’ was phenotyped for LR severity during the 2010–2011, 2011–2012 and 2012–2013 cropping seasons in the field at Baoding, Hebei Province. Bulked segregant analysis and simple sequence repeat markers were used to identify the quantitative trait loci (QTLs) for LR adult‐plant resistance in the population. Three QTLs were detected and designated as QLr.hebau‐1BL, QLr.hebau‐2DS and QLr.hebau‐7DS. Based on the chromosome positions and molecular marker tests, QLr.hebau‐1BL is Lr46, and QLr.hebau‐7DS is Lr34. QLr.hebau‐2DS was derived from ‘Thatcher’ and was close to Lr22. This result suggests that Lr22b may confer residual resistance on field nurseries when challenged with isolates virulent on Lr22b, or another gene linked to Lr22b confers this resistance from ‘Thatcher’. This study confirms the value of Lr34 and Lr46 in breeding for LR resistance in China; the contribution of the QTL to chromosome 2D needs further validation.     

A microsatellite marker linked to leaf rust resistance transferred from Aegilops triuncialis into hexaploid wheat

Aegilops triuncialis (UUCC) is an excellent source of resistance to various wheat diseases, including leaf rust. Leaf rust‐resistant derivatives from a cross of a highly susceptible Triticum aestivum cv.‘WL711’ as the recurrent parent and Ae. triuncialis Ace.3549 as the donor and with and without a pair of acrocentric chromosomes were used for molecular tagging. The use of a set of sequence tagged microsatellite (STMS) markers already mapped to different wheat chromosomes unequivocally indicated that STMS marker gwm368 of chromosome 4BS was tightly linked to the Ae. triuncialis leaf rust resistance gene transferred to wheat. The presence of the Ae. Triuncialis‐specific STMS gwm368 homoeoallele along with the non‐polymorphic 4BS allele in the rust‐resistant derivatives with and without the acrocentric chromosome indicates that the resistance has been transferred from the acrocentric chromosome to either the A or the D genome of wheat. This alien leaf rust resistance gene has been temporarily named as LrTr.     

Mapping of the Vrn-B1 gene in Triticum aestivum using microsatellite markers

An F₂ population segregating for the dominant gene Vrn‐B1 was developed from the cross of the substitution line ‘Diamant/'Miro‐novskaya 808 5A’ and the winter wheat cultivar ‘Bezostaya 1′. Microsatellite markers (Xgwm and Xbarc) with known map locations on chromosome 5B of common wheat were used for mapping the gene Vrn‐B1. Polymorphism between parental varieties was observed for 28 out of 34 microsatellite markers (82%). Applying the quantitative trait loci mapping approach, the target gene was mapped on the long arm of chromosome 5B, closely linked to Xgwm408. The map position of Vrn‐B1 suggests that the gene is homoeologous to other vernalization response genes located on the homoeologous group 5 chromosomes of wheat, rye and barley.