A microsatellite sequence in the fifth intron provides a broad-spectrum SSR marker for multiple alleles of the <Emphasis Type="Italic">er1</Emphasis>/<Emphasis Type="Italic">PsMLO1</Emphasis> powdery mildew resistance gene in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Powdery mildew caused by the biotrophic ascomycete fungus Erysiphe pisi Syd. is one the most devastating diseases of peas (Pisum sativum L.) with enormous impact in seed production. The most efficient genetic resistance to this disease, so far identified, is conferred by the naturally occurring or experimentally induced by chemical mutagenesis recessive state of the locus er1. Genetically mapped over 2 decades ago, this gene was recently identified as a homolog of the barley (Hordeum sativum L.) powdery mildew resistance gene MLO, and renamed as PsMLO1. The broad wide resistance conferred by the er1/PsMLO1 locus was found to be a consequence of the loss of function of the encoded PsMLO1 protein. After the publication of the expressed sequence of this gene by another research group, we published the genomic sequences of this gene which harbors a relatively long (TA) microsatellite sequence (SSR) in the fifth intron. SSR markers based on this highly polymorphic microsatellite can be used for marker-assisted selection in multiple pea powdery mildew resistance breeding programs involving the er1/PsMLO1 resistance, except in the rare circumstances where the progenitor lines are monomorphic for the microsatellite sequence.     

Identification of a complete set of functional markers for the selection of er1 powdery mildew resistance in Pisum sativum L.

Powdery mildew is the most widespread disease of pea (Pisum sativum L.) and causes severe economic losses worldwide. Recessively inherited er1 powdery mildew resistance, successfully used for decades in pea breeding programs, has recently been shown to originate from the loss of function of the PsMLO1 gene. Five er1 alleles, each corresponding to a different PsMLO1 null mutation, have been characterized to date in pea germplasm. In order to aid er1 selection, we aimed to identify functional markers which target PsMLO1 polymorphisms directly responsible for the resistant phenotype. Highly informative cleaved amplified polymorphic sequence (CAPS), derived cleaved amplified polymorphic sequence (dCAPS), sequence tagged site (STS) and high-resolution melting (HRM) markers were developed which enable the selection of each of the five er1 alleles. Taken together, the results described here provide a powerful tool for breeders, overcoming limitations of previously reported er1-linked markers due to the occurrence of recombination with the resistance locus and/or the lack of polymorphism between parental genotypes. The HRM marker er1-5/HRM54 reported here, targeting a mutagenesis-induced er1 allele recently described by us, does not require manual processing after PCR amplification, and is therefore suitable for large-scale breeding programs based on high-throughput automated screening.     

加拿大豌豆品种（系）抗白粉病表型和基因型鉴定

对36个引自加拿大的豌豆品种(系)进行抗白粉病表型和标记基因型鉴定,明确了豌豆品种Cooper和Tara白粉病抗性等位基因。苗期接种了2个不同地理来源的豌豆白粉病菌分离物,32个品种(系)对2个分离物均表现为免疫;品系MP1818-2对云南白粉菌分离物EPYN免疫,但对北京分离物EPBJ感病;其余3个品种对2个分离物均感病。4个与豌豆抗白粉病基因er1连锁的SCAR标记将36个豌豆品种(系)区分为5个标记基因型。与野生型PsMLO1基因序列比较发现,豌豆品种Cooper和Tara的PsMLO1候选基因均在680 bp处发生C变G的单核苷酸突变。     

Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1

Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.     

The ENU-induced powdery mildew resistant mutant pea (Pisum sativum L.) lines S(er1mut1) and F(er1mut2) harbour early stop codons in the PsMLO1 gene

Two pea (Pisum sativum L.) powdery mildew-resistant mutant lines, S(er1mut1) and F(er1mut2), were previously obtained by experimental chemical mutagenesis with ethylnitrosourea. Identification and subsequent analysis of the genomic sequence of the PsMLO1 gene revealed one single nucleotide mutation in each mutant line that leads to either a transversion or a transition, respectively, resulting in premature stop codons that drastically truncate the protein product of this gene in these two mutant lines. These results confirm the previous findings that PsMLO1 is the powdery mildew resistance gene er1. Only one additional mutation (transition) was observed in the S(er1mut1), downstream of the protein-truncating stop codon. Mutations were not identified in the intron regions of the gene. Specific molecular markers (cleaved amplified polymorphic sequences and sequence-tagged sites) were generated for the protein-truncating mutations, and these provide breeders with very efficient tools for marker-assisted selection when either of the two mutated lines are used in plant breeding programmes.     

Pea powdery mildew <Emphasis Type="Italic">er1</Emphasis> resistance is associated to loss-of-function mutations at a <Emphasis Type="Italic">MLO</Emphasis> homologous locus

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F₂ individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.     

Development and validation of breeder-friendly KASPar markers for <Emphasis Type="Italic">er1</Emphasis>, a powdery mildew resistance gene in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Powdery mildew of pea is caused by Erysiphe pisi DC and is a serious threat to pea (Pisum sativum L.) production throughout much of the world. Development and utilization of genetic resistance to powdery mildew is considered an effective and sustainable strategy to manage this disease. One gene, er1, conferring powdery mildew resistance, was previously cloned and sequenced, and the functional markers for each resistance allele were reported. Allele-specific DNA markers are efficient and powerful tools to facilitate crop improvement and new cultivar development in breeding programs. However, extensive application of these markers is limited by gel-associated obstacles. In this study, eight breeder-friendly kompetitive allele-specific PCR (KASPar) markers were developed to overcome the problems of gel-based markers and increase the efficiency of genotypic screening. In order to identify additional pea germplasm with powdery mildew resistance, these KASPar markers were deployed and used to genotype a pea collection derived from the USDA pea single-plant (PSP) collection. Simultaneously, a phenotypic screening and a genotypic validation using the corresponding gel-based functional markers were conducted on the PSP collection. One pea accession, PI 142775, was identified by both phenotyping and genotyping to carry the allele er1-1 for powdery mildew resistance, indicating that the KASPar assay is an efficient and robust tool for breeding for powdery mildew resistance.     

Molecular characterization of a novel vernalization allele <Emphasis Type="Italic">Vrn-B1d</Emphasis> and its effect on heading time in Chinese wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) landrace Hongchunmai

Flowering time of wheat cultivars contributes greatly to the adaptability to environmental conditions and it is largely controlled by vernalization genes. In this study, 262 Chinese mini-core wheat cultivars were used to identify the allelic variation at VRN-B1 locus. A novel dominant allele Vrn-B1d was found in Chinese spring wheat landrace cultivar Hongchunmai. This allele contained several genetic divergence within the first intron comparing to the recessive allele vrn-B1, including one large 6850-bp deletion (670–7519 bp), one small 187-bp deletion (7851–8037 bp), one unique SNP (T to C, 7845 bp), and one 4-bp mutation (TTTT to ACAA, 7847–7850 bp). Meanwhile, it was also different from the three known dominant alleles at VRN-B1 locus. Two pairs of primers were designed to identify the novel allele Vrn-B1d and other four known alleles of VRN-B1. A multiplex PCR was established to discriminate all five alleles simultaneously. The greenhouse experiment with high temperature (non-vernalizing condition) and long light showed that F₂ plants containing Vrn-B1d allele headed significantly earlier than those with recessive vrn-B1 allele, suggesting that Vrn-B1d is a dominant allele conferring the spring growth habit. This study provides a useful germplasm and molecular markers for wheat breeding.     

Identification and validation of RAPD and SCAR markers linked to the gene <Emphasis Type="Italic">Er3</Emphasis> conferring resistance to <Emphasis Type="Italic">Erysiphe pisi</Emphasis> DC in pea

Three genes, er1, er2 and Er3, conferring resistance to powdery mildew (Erysiphe pisi) in pea have been described so far. Because single gene-controlled resistance tends to be overcome by evolution of pathogen virulence, accumulation of several resistance genes into a single cultivar should enhance the durability of the resistance. Molecular markers linked to genes controlling resistance to E. pisi may facilitate gene pyramiding in pea breeding programs. Molecular markers linked to er1 and er2 are available. In the present study, molecular markers linked to Er3 have been obtained. A segregating F₂ population derived from the cross between a breeding line carrying the Er3 gene, and the susceptible cultivar ‘Messire’ was developed and genotyped. Bulk Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to Er3. Four RAPD markers linked in coupling phase (OPW04_637, OPC04_640, OPF14_1103, and OPAH06_539) and two in repulsion phase (OPAB01_874 and OPAG05_1240), were identified. Two of these, flanking Er3, were converted to Sequence Characterized Amplified Region (SCAR) markers. The SCAR marker SCW4₆₃₇ co-segregated with the resistant gene, allowing the detection of all the resistant individuals. The SCAR marker SCAB1₈₇₄, in repulsion phase with Er3, was located at 2.8 cM from the gene and, in combination with SCW4₆₃₇, was capable to distinguish homozygous resistant individuals from heterozygous with a high efficiency. In addition, the validation for polymorphism in different genetic backgrounds and advanced breeding material confirmed the utility of both markers in marker-assisted selection.     

Molecular mapping of pea powdery mildew resistance gene <Emphasis Type="Italic">er2</Emphasis> to pea linkage group III

Powdery mildew caused by Erysiphe pisi D.C. is one of the most serious diseases that inflict heavy losses to pea crop world-wide. Identification of resistance sources and their incorporation into susceptible cultivars remains the most effective method of controlling the disease. The present study investigated the resistance phenotype, inheritance, and genomic location of gene(s) controlling resistance to powdery mildew in pea genotype ‘JI2480’. The powdery mildew resistance in ‘JI2480’ appeared to be a spatial phenomenon showing expression only in leaf tissues. By segregation analysis of an F₂ progeny of cross ‘Lincoln/JI2480’, the leaf resistance of ‘JI2480’ was shown to be controlled by a single recessive gene, presumed to be er2. Through linkage analysis of 111 resistant F₂ progeny plants with simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) markers adopted from the published linkage maps, the er2 gene was localized on pea linkage group III (LGIII). The assignment of er2 to LGIII, a position different from that reported for er1, has resolved the long standing controversy in the literature regarding the existence and genomic location of er2 gene. A RAPD marker OPX-17_1400, exhibiting cis phase linkage (2.6 cM) to er2 was successfully converted to a sequence characterized amplified region (SCAR) marker, ScX17_1400. The SCAR marker ScX17_1400 will ensure speedy and precise introgression of er2 into susceptible cultivars by permitting selection of er2 heterozygotes amongst BC _n F₁s without progeny tests and resistance screening.     

Resistance of bread wheat (Triticum aestivum L.) to preharvest sprouting: An association analysis

A statistical analysis of the data about 1422 bread wheat accessions with estimated preharvest sprouting was carried out. Close associations of preharvest sprouting resistance with the grain color and with resistance to Fusarium head blight were revealed, as well as weak, but statistically significant, associations with the habit, awnedness, and reduced height genes Rht-B1 and Rht-D1 (insensitive to gibberellin GA3). The pedigree analysis showed that the cluster structures of the gene pools of the North American red-grained and white-grained varieties are practically identical. In both groups, varieties that are resistant to preharvest sprouting differ from susceptible ones in the percentage of the contributions of the Crimean and Mediterranean landraces. Resistance is associated with a high contribution by the Crimean landrace and susceptibility is associated with a high contribution by the Mediterranean landrace.     

Genetic diversity and relationship of Fritillaria thunbergii Miq. landraces and related taxa

Genetic diversity and differentiation of four landraces of Fritillaria thunbergii Miq. and two congeners Fritillaria cirrhosa D. Don and Fritillaria anhuiensis S. C. Chen et S. F. Yin were evaluated using ISSR markers. The results showed that the genetic diversity of F. thunbergii was high at the specie level but relatively lower at the landrace level. A high level of genetic differentiation among four F. thunbergii landraces was detected based on the gene differentiation coefficient and the AMOVA, in line with the low inter-landrace gene flow. MS-tree analysis showed that the four F. thunbergii landraces were clustered on adjacent positions of the tree, and that Kuanye (Ft-KY) landrace was relatively distantly related to other landraces. In line with the MS-tree analysis, PCoA revealed that the three species of Fritillaria can be divided into two groups and three subgroups, among which there occurred a remarkable genetic differentiation.     

Marker assisted pea breeding: <Emphasis Type="Italic">eIF4E</Emphasis> allele specific markers to <Emphasis Type="Italic">pea seed-borne mosaic virus</Emphasis> (PSbMV) resistance

Traditional plant breeding relies upon crosses and subsequent selection of genotypes to meet desirable traits. The incorporation of marker-assisted selection into breeding strategies would result in a reduction in the number of offspring to be propagated, selected and tested. In the case of pea (Pisum sativum L.), the testing of resistance to viral pathogens such as pea seed-borne mosaic virus (PSbMV) is included in the breeding process. Resistance to the common strains of PSbMV is conferred by a single recessive gene (eIF4E), localized on LG VI (sbm-1 locus). We have analyzed for variation in the eIF4E genomic sequences from 43 pea varieties and breeding lines, reported as donors of resistance. This enabled a comprehensive investigation of the eIF4E gene structure and mutations responsible for PSbMV resistance were identified. Subsequently, PCR-based and gene-specific single nucleotide polymorphism and co-dominant amplicon length polymorphism markers were developed. All together 60 accessions were analyzed using sequence data and/or allele specific DNA markers. Developed allele specific markers were reproducibly amplified across a broad spectra of pea varieties and breeding lines. These were found to be 100% accurate in detecting the presence of the respective alleles when compared to symptomology and ELISA, testing (74% reliable). Hence, these molecular markers will substantially speed-up PSbMV diagnosis and resistance breeding processes in pea.     

Genetic mapping of a leaf rust resistance gene in the former Yugoslavian barley landrace MBR1012

Leaf rust, caused by Puccinia hordei Otth, is an important disease of barley (Hordeum vulgare L.) in many areas of the world. The appearance of new virulent races necessitates the identification of new resistance genes in barley. Screening of spring barley landraces from former Yugoslavia led to the identification of an accession (MBR1012) carrying resistance to the most widespread virulent leaf rust pathotypes in Europe. Ninety-one doubled haploid lines derived from a cross between landrace MBR1012 and the susceptible German cultivar Scarlett were evaluated for resistance to P. hordei isolate I-80 and segregated 48 resistant : 43 susceptible ( $ \chi_{1:1}^{2} = \, 0.29, $ p?=?0.6), indicating a monogenic inheritance of resistance. Using simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) markers, the resistance gene in MBR1012 was mapped to the telomeric region of chromosome 1HS. This gene is assigned the temporary locus designation of Rph _MBR1012 until it can be unequivocally determined to be different from all previously reported resistance genes. The closest flanking markers for Rph _MBR1012 are located 0.8?cM distal (SNP marker GBS546 and SSR marker GBMS187) and 6.0?cM proximal (SSR marker GMS21). The diagnostic value of the closest linked markers was assessed in a genetically diverse collection of 51 susceptible and resistant barley lines and cultivars. The SSR GBMS187 predicted the presence of Rph _MBR1012 with 100% accuracy. However, this marker could not be used singly for the rapid incorporation of resistance into high yielding barley cultivars, since it detects a null allele in MBR1012. Therefore, simultaneous use of the markers closely linked to Rph _MBR1012 is needed for transferring Rph _MBR1012 into adapted cultivars.     

Pea genes associated with non-host disease resistance to Fusarium are also active in race-specific disease resistance to Pseudomonas

A given plant species is able to resist most of the potentially pathogenic microorganisms with which it comes in contact. This phenomenon, known as non-host resistance, can be overcome only by a very small number of true pathogens which can use that plant as a host. In some cases, plants have developed mechanisms for overcoming infection by specific races or strains of a true pathogen. This race-specific resistance can be easily manipulated into agronomically important cultivars by plant breeders. We have previously described nine cDNA clones which represent pea genes active during non-host resistance against the fungus Fusarium solani f. sp. phaseoli. In the present work, we have used these cDNAs as probes to compare non-host resistance with race-specific responses of peas against three races of Pseudomonas syringae pv. pisi. Five of the genes most active during non-host resistance were also active in direct correlation with the phenotypic expression of resistance in race-specific reactions of five differential pea cultivars against three races of Pseudomonas syringae pv. pisi.     

Isolation of Race-and Cultivar-Specific Elicitors from Intercellular Fluids of Compatible Interactions of Pseudomonas syringae pv. pisi and Pea

Intercellular fluids obtained by an in vacuo infiltration technique from compatible race-cultivar interactions of five races of Pseudomonas syringae pv. pisi and pea induced extensive light brown necrotic (hypersensitive type of) lesions in resistant but not susceptible cultivars. In susceptible cultivars the intercellular fluids induced extetensive water soaking symptoms. The intercellular fluids elicited intermediate reactions in pea cultivars of moderate resistance. The intensity of the light brown necrotic reactions in pea appears to be positively correlated with the degree of resistance.     

Microsatellite marker-based diversity and population genetic analysis of selected lowland and mid-altitude maize landrace accessions of India

Maize (Zea mays L.) harbours significant genetic diversity not only in its centre of origin (Mexico) but also in several countries worldwide, including India, in the form of landraces. In this study, DNA fingerprinting of 48 landrace accessions from diverse regions of India was undertaken using 42 fluorescent dye-labeled Simple Sequence Repeat (SSR) markers, followed by allele resolution using DNA sequencer and analysis of molecular diversity within and among these landraces. The study revealed a large number of alleles (550), with high mean number of alleles per locus (13.1), and Polymorphism Information Content (PIC) of 0.60, reflecting the level of diversity in the landrace accessions. Besides identification of 174 unique alleles in 44 accessions, six highly frequent SSR alleles were detected at six loci (phi014, phi090, phi112, umc1367, phi062 and umc1266) with individual frequencies greater than 0.75, indicating that chromosomal regions harboring these SSR alleles are not selectively neutral. F statistics revealed very high genetic differentiation, population subdivision and varying levels of inbreeding in the landraces. Analysis of Molecular Variance showed that 63 % of the total variation in the accessions could be attributed to within-population diversity, and 37 % represented between population diversity. Cluster analysis of SSR data using Nei’s genetic distance and UPGMA revealed considerable genetic diversity in these populations, although no clear separation of accessions was observed based on their geographic origin.     

Genetic variation of Vp1 in Sichuan wheat accessions and its association with pre-harvest sprouting response

Pre-harvest sprouting (PHS) in bread wheat is a major abiotic constraint reducing yield and influencing the production of high quality grain. In China both spring and winter wheat regions are affected by PHS. Sichuan lies in southwest China, where the most of rainfall occurs during April to September when wheat is harvested. The present investigation was conducted to identify the allelic variability of Vp1, a gene that plays a role in maintenance and induction of dormancy, among Sichuan landraces and recent cultivars with different dormancy levels and to find potential sources of PHS resistance for breeding. Sichuan landrace and cultivar wheat accessions had a wide range of dormancy levels. The average germination index (GI) of Sichuan landrace accessions was 0.232, whereas at 0.674 it was much higher for cultivars. The different dormancy levels between landraces and cultivars indicated that pre-harvest sprouting resistance might have been neglected in recent Sichuan wheat breeding programs. The average GI of white grained accessions was higher than for red grained accessions. Particular Vp-1B gene fragments were specific in landraces or cultivars and in white or red grained accessions. The results indicated that Vp-1B markers could be used to distinguish cultivars and landraces. Significant relationships between certain Vp-1B allelesand GI of Sichuan wheat accessions were shown by Spearman’s rank correlation analysis.     

Validation of SSR markers associated with rust (Uromyces fabae) resistance in pea (Pisum sativum L.)

Pea rust is a devastating disease of peas especially in the sub-tropical regions of the world and greatly influenced by the environmental conditions during disease development. Molecular markers associated with pea rust resistance would be useful in marker assisted selection (MAS). Utility of molecular markers associated with the pea rust resistance were evaluated in 30 diverse pea genotypes using four SSR markers (AA446 and AA505 flanking the major QTL Qruf; AD146 and AA416 flanking the minor QTL, Qruf1). QTL, Qruf flanking markers were able to identify all the resistant genotypes when used together, except Pant P 31. While, SSR markers AD146 and AA416 flanking the minor QTL, Qruf1 were able to identify all the pea resistant genotypes used for validation, except for HUDP-11 by AD146 and Pant P 31 by AA416. Similarly, SSR markers AA446 and AA505 were able to identify all the susceptible pea genotypes, except IPFD 99–13, HFP 9415 and S- 143. SSR markers AD146 and AA416 were together able to identify all the pea susceptible genotypes used for validation, except KPMR 526, KPMR 632 and IPFD 99–13. On the basis of marker allele analysis it may be concluded that SSR markers (AA446, AA505, AD146 and AA416) can be used in MAS of pea rust resistance.