Identification and Characterization of Random Amplified Polymorphic DNA Markers Linked to a Major Gene (Cr2) for Resistance to Cronartium ribicola in Pinus monticola

DNA markers tightly linked to resistance (R) genes provide a very powerful tool for both marker-assisted selection in plant breeding and positional cloning of R genes. In the present study, a linkage of random amplified polymorphic DNA (RAPD) markers to the single dominant gene (Cr2) for resistance to white pine blister rust fungus (Cronartium ribicola) was investigated in western white pine (Pinus monticola). A mapping population of 128 individual megagametophytes was generated from seeds of a heterozygous resistant tree (Cr2/cr2), and the corresponding seedlings of each megagametophyte were subjected to the test of phenotype segregation by inoculation with C. ribicola. Bulked segregant analysis and haploid segregation analysis identified eight robust RAPD markers linked to Cr2. This constitutes the first Cr2 genetic linkage map spanning 84.7 cM with four markers only 3.2 cM from Cr2. One sequence (U256-1385) of these linked markers was significantly similar to the Ty3/gypsy-like long terminal direct repeats retrotransposons. Another marker, U570-843, had no significant similarity to any entry in either GenBank or the loblolly genomics data bank. As presumed that the average physical distance per centimorgan is about 10 Mb in P. monticola, it is probably unrealistic to use these DNA markers for positional cloning of the Cr2 gene.

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Comparative Transcriptomics and RNA-Seq-Based Bulked Segregant Analysis Reveals Genomic Basis Underlying Cronartium ribicola vcr2 Virulence

Frontiers in Microbiology ◽

10.3389/fmicb.2021.602812 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jun-Jun Liu ◽

Richard A. Sniezko ◽

Arezoo Zamany ◽

Holly Williams ◽

Kangakola Omendja ◽

...

Keyword(s):

Allele Frequency ◽

Molecular Mechanisms ◽

Bulked Segregant Analysis ◽

Diagnostic Tools ◽

Cronartium Ribicola ◽

Pinus Monticola ◽

Rna Seq ◽

White Pine ◽

Fungal Virulence ◽

Genome Wide

Breeding programs of five-needle pines have documented both major gene resistance (MGR) and quantitative disease resistance (QDR) to Cronartium ribicola (Cri), a non-native, invasive fungal pathogen causing white pine blister rust (WPBR). WPBR is one of the most deadly forest diseases in North America. However, Cri virulent pathotypes have evolved and can successfully infect and kill trees carrying resistance (R) genes, including vcr2 that overcomes MGR conferred by the western white pine (WWP, Pinus monticola) R gene (Cr2). In the absence of a reference genome, the present study generated a vcr2 reference transcriptome, consisting of about 20,000 transcripts with 1,014 being predicted to encode secreted proteins (SPs). Comparative profiling of transcriptomes and secretomes revealed vcr2 was significantly enriched for several gene ontology (GO) terms relating to oxidation-reduction processes and detoxification, suggesting that multiple molecular mechanisms contribute to pathogenicity of the vcr2 pathotype for its overcoming Cr2. RNA-seq-based bulked segregant analysis (BSR-Seq) revealed genome-wide DNA variations, including about 65,617 single nucleotide polymorphism (SNP) loci in 7,749 polymorphic genes shared by vcr2 and avirulent (Avcr2) pathotypes. An examination of the distribution of minor allele frequency (MAF) uncovered a high level of genomic divergence between vcr2 and Avcr2 pathotypes. By integration of extreme-phenotypic genome-wide association (XP-GWAS) analysis and allele frequency directional difference (AFDD) mapping, we identified a set of vcr2-associated SNPs within functional genes, involved in fungal virulence and other molecular functions. These included six SPs that were top candidate effectors with putative activities of reticuline oxidase, proteins with common in several fungal extracellular membrane (CFEM) domain or ferritin-like domain, polysaccharide lyase, rds1p-like stress responsive protein, and two Cri-specific proteins without annotation. Candidate effectors and vcr2-associated genes provide valuable resources for further deciphering molecular mechanisms of virulence and pathogenicity by functional analysis and the subsequent development of diagnostic tools for monitoring the virulence landscape in the WPBR pathosystems.

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Genetic Mapping of PcDw Determining Pear Dwarf Trait

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.136.1.48 ◽

2011 ◽

Vol 136 (1) ◽

pp. 48-53 ◽

Cited By ~ 11

Author(s):

Caihong Wang ◽

Yike Tian ◽

Emily J. Buck ◽

Susan E. Gardiner ◽

Hongyi Dai ◽

...

Keyword(s):

Ssr Markers ◽

Rapd Markers ◽

Bulked Segregant Analysis ◽

Random Amplified Polymorphic Dna ◽

Dominant Gene ◽

Pyrus Pyrifolia ◽

Chi Square ◽

Sequence Characterized Amplified Regions ◽

Chi Square Analysis ◽

Simple Sequence

European pear (Pyrus communis) ‘Aihuali’ carrying the dwarf character originating from ‘Nain Vert’ was crossed with ‘Chili’ (Pyrus bretschneideri). A total of 352 F1 progenies was produced to investigate the inheritance of the dwarf trait, and 111 of these were used to develop molecular markers. Chi-square analysis showed that the character fitted a 1:1 ratio indicative of a single dominant gene, which we have named PcDw. Using a bulked segregant analysis approach with 500 random amplified polymorphic DNA (RAPD) and 51 simple sequence repeat (SSR) markers from pear (Pyrus pyrifolia and P. communis) and apple (Malus ×domestica), four markers were identified as cosegregating with the dwarf character. Two of these were fragments produced by the S1212 and S1172 RAPD primers, and the other two were the pear SSR markers KA14 and TsuENH022. The RAPD markers were converted into sequence-characterized amplified regions (SCARs) and designated S1212-SCAR318 and S1172-SCAR930 and, with the SSR markers KA14 and TsuENH022, were positioned 5.9, 9.5, 8.2, and 0.9 cM from the PcDw gene, respectively. Mapping of the KA14 and TsuENH022 markers enabled the location of the PcDw gene on LG 16 of the pear genetic linkage map.

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Assessment of genetic diversity among surian Toona sinensis Roem in progenies test using random amplified polymorphic DNA markers

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.25798 ◽

2018 ◽

Vol 22 (1) ◽

pp. 22

Author(s):

Jayusman Jayusman ◽

Muhammad Na’iem ◽

Sapto Indrioko ◽

Eko Bhakti Hardiyanto ◽

ILG Nurcahyaningsih

Keyword(s):

Genetic Diversity ◽

Dna Markers ◽

Rapd Markers ◽

Random Amplified Polymorphic Dna ◽

Management Strategies ◽

Genetic Distances ◽

Sampled Data ◽

Toona Sinensis ◽

The Mean ◽

Individual Trees

Surian Toona sinensis Roem is one of the most widely planted species in Indonesia. This study aimed to estimate the genetic diversity between a number of surian populations in a progeny test using RAPD markers, with the goal of proposing management strategies for a surian breeding program. Ninety-six individual trees from 8 populations of surian were chosen as samples for analysis. Eleven polymorphic primers (OP-B3, OP-B4, OP-B10, OP-H3, OP-Y6, OP-Y7, OP-Y8, OP-Y10, OP-Y11, OP-Y14, and OP-06) producing reproducible bands were analyzed for the 96 trees, with six trees per family sampled. Data were analyzed using GenAlEx 6.3, NTSYS 2.02. The observed percentage of polymorphic loci ranged from 18.2% to 50%. The mean level of genetic diversity among the surian populations was considered to be moderate (He 0.304). Cluster analysis grouped the genotypes into two main clusters, at similarity levels of 0.68 and 0.46. The first two axes of the PCoA explained 46.16% and 25.54% of the total variation, respectively. The grouping of samples into clusters and subclusters did not correspond with family and their distances, but the grouping was in line with the genetic distances of the samples.

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Origin of the Black Shank Resistance Gene, Ph, in Tobacco Cultivar Coker 371-Gold

Plant Disease ◽

10.1094/pdis.2002.86.10.1080 ◽

2002 ◽

Vol 86 (10) ◽

pp. 1080-1084 ◽

Cited By ~ 34

Author(s):

E. S. Johnson ◽

M. F. Wolff ◽

E. A. Wernsman ◽

W. R. Atchley ◽

H. D. Shew

Keyword(s):

Nicotiana Tabacum ◽

Rapd Markers ◽

Random Amplified Polymorphic Dna ◽

Dominant Gene ◽

Nicotiana Plumbaginifolia ◽

Black Shank ◽

Breeding Lines ◽

Tobacco Cultivar ◽

Qualitative Resistance ◽

Interspecific Transfer

Flue-cured tobacco (Nicotiana tabacum) cultivar Coker 371-Gold (C 371-G) possesses a dominant gene, Ph, that confers high resistance to black shank disease, caused by race 0 of the soil-borne pathogen Phytophthora parasitica var. nicotianae. The origin of this gene is unknown. Breeding lines homozygous for the Ph gene were hybridized with NC 1071 and L8, flue-cured and burley genotypes known to possess qualitative resistance genes from Nicotiana plumbaginifolia and N. longiflora, respectively. The F1 hybrids were out-crossed to susceptible testers and the progenies evaluated in field black shank nurseries and in greenhouse disease tests with P. parasitica var. nicotianae race 0. Results showed that Ph was allelic to Php from N. plumbaginifolia in NC 1071. Testcross populations of hybrids between burley lines homozygous for Ph and L8, possessing Phl from N. longiflora, showed that Ph and Phl integrated into the same tobacco chromosome during interspecific transfer. Nevertheless, the two loci were estimated to be 3 cM apart. Random amplified polymorphic DNA (RAPD) analyses of the testcross progenies confirmed that recombination between the two loci was occurring. Forty-eight RAPD markers linked to Ph in doubled haploid lines were used in cluster analyses with multiple accessions of N. longiflora and N. plumbaginifolia, breeding lines L8, NC 1071, and DH92-2770-40, and cultivars K 326, Hicks, and C 371-G. A cladogram or region tree confirmed the data obtained from field and greenhouse trials, that Ph, transferred from C 371-G to DH92-2770-40, and Php in NC 1071 were allelic and originated from N. plumbaginifolia.

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Segregation of random amplified polymorphic DNA markers and strategies for molecular mapping in tetraploid alfalfa

Genome ◽

10.1139/g93-112 ◽

1993 ◽

Vol 36 (5) ◽

pp. 844-851 ◽

Cited By ~ 23

Author(s):

K. F. Yu ◽

K. P. Pauls

Keyword(s):

Chromosome Segregation ◽

Dna Markers ◽

Rapd Markers ◽

Molecular Mapping ◽

Random Amplified Polymorphic Dna ◽

Rapd Marker ◽

Female Parent ◽

Linkage Groups ◽

Double Reduction ◽

Chromatid Segregation

An F1 population was used to analyze the inheritance of random amplified polymorphic DNA (RAPD) markers in tetraploid alfalfa. Of the 32 RAPD markers that were used for a segregation analysis in this study, 27 gave ratios that are consistent with random chromosome and random chromatid segregation at meiosis. However, among all of the RAPD markers (121) that were screened in this study, only one example of a double reduction, that is typical of chromatid segregation, was observed. These results indicate that random chromosome segregation is likely the predominant but not the exclusive mode of inheritance for tetraploid alfalfa. χ2 analyses of cosegregation for RAPD marker pairs derived from the female parent revealed nine linkages that fell into four linkage groups. The recombination fractions among linked marker pairs ranged from 1 to 37%. These are the first molecular linkage groups reported in tetraploid alfalfa. In addition, various strategies for molecular mapping in the tetraploid alfalfa genome are proposed that should be of interest to plant breeders who are planning to use molecular markers for alfalfa or other tetraploid species.Key words: RAPD markers, tetraploid alfalfa, segregation, linkage groups.

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Genetic mapping and non-Mendelian segregation of mating type loci in the oomycete, Phytophthora infestans.

Genetics ◽

10.1093/genetics/141.2.503 ◽

1995 ◽

Vol 141 (2) ◽

pp. 503-512 ◽

Cited By ~ 2

Author(s):

H S Judelson ◽

L J Spielman ◽

R C Shattock

Keyword(s):

Phytophthora Infestans ◽

Mating Type ◽

Dna Markers ◽

Bulked Segregant Analysis ◽

Random Amplified Polymorphic Dna ◽

Mating Types ◽

Mendelian Segregation ◽

Range Restriction ◽

Type Locus ◽

Mating Type Locus

Abstract DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in the normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balance lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude form progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.

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The relative susceptibility of needle- and stem-derived white pine tissue cultures to artificial inoculation with mycelium of Cronartium ribicola

Canadian Journal of Botany ◽

10.1139/b69-257 ◽

1969 ◽

Vol 47 (11) ◽

pp. 1789-1790 ◽

Cited By ~ 4

Author(s):

A. E. Harvey ◽

J. L. Grasham

Keyword(s):

Rust Fungus ◽

Cronartium Ribicola ◽

Tissue Cultures ◽

Pinus Monticola ◽

White Pine ◽

Relative Susceptibility ◽

Blister Rust ◽

Leaf Mesophyll ◽

Leaf Tissues ◽

Artificial Inoculations

Tissue cultures of Pinus monticola Dougl. derived from stem cortex and leaf tissues were found susceptible to artificial inoculations with mycelium from the blister rust fungus (Cronartium ribicola Fisch. ex Rabenh.). Tissue cultures from leaf mesophyll grew slower and were colonized more rapidly by this fungus than those derived from stem cortex.

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Identification of coupling and repulsion phase RAPD markers for powdery mildew resistance gene er-1 in pea

Genome ◽

10.1139/g98-014 ◽

1998 ◽

Vol 41 (3) ◽

pp. 440-444 ◽

Cited By ~ 18

Author(s):

K R Tiwari ◽

G A Penner ◽

T D Warkentin

Keyword(s):

Powdery Mildew ◽

Resistance Gene ◽

Rapd Markers ◽

Powdery Mildew Resistance ◽

Bulked Segregant Analysis ◽

Random Amplified Polymorphic Dna ◽

Powdery Mildew Resistance Gene ◽

Erysiphe Pisi ◽

Mildew Resistance ◽

Repulsion Phase

Powdery mildew is a serious disease of pea caused by the obligate parasite Erysiphe pisi Syd. Random amplified polymorphic DNA (RAPD) analysis has emerged as a cost-effective and efficient marker system. The objective of this study was to identify RAPD markers for powdery mildew resistance gene er-1. The resistant cultivar Highlight (carrying er-1) and the susceptible cultivar Radley were crossed, and F3 plants were screened with Operon (OP) and University of British Columbia (UBC) primers, using bulked segregant analysis. A total of 416 primers were screened, of which amplicons of three Operon primers, OPO-18, OPE-16, and OPL-6, were found to be linked to er-1. OPO-181200 was linked in coupling (trans to er-1) and no recombinants were found. OPE-161600 (4 ± 2 cM) and OPL-61900 (2 ± 2 cM) were linked in repulsion (cis to er-1). The fragments OPO-181200 and OPE-161600 were sequenced and specific primers designed. The specific primer pair Sc-OPO-181200 will be useful in identifying homozygous resistant individuals in F2 and subsequent segregating generations. Sc-OPE-161600 will have greatest utility in selecting heterozygous BC\dn6 nF1 individuals in backcross breeding programs.Key words: bulked segregant analysis,Erysiphe pisi, pea, RAPD.

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332 GENETIC SIMILARITIES AMONG CHINESE VEGETABLE BRASSICAS USING RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS

HortScience ◽

10.21273/hortsci.29.5.478b ◽

1994 ◽

Vol 29 (5) ◽

pp. 478b-478

Author(s):

Jianping Ren ◽

Warren F. Lamboy ◽

lames R. McFerson ◽

Stephen Kresovich ◽

Jianping Ren

Keyword(s):

Chinese Cabbage ◽

Dna Markers ◽

Rapd Markers ◽

Random Amplified Polymorphic Dna ◽

Chinese Kale ◽

Diversity Assessment ◽

Wide Range ◽

Germplasm Accessions ◽

Chinese Vegetable ◽

Vegetable Brassicas

Fifty-two germplasm accessions of Chinese vegetable Brassicas were analyzed using 112 random amplified polymorphic DNA (RAPD) markers. The array of material examined spanned a wide range of morphological, geographic, and genetic diversity, and included 30 accessions of Brassica rapa (Chinese cabbage, pakchoi, turnip, broccoletto), 18 accessions of B. juncea (leaf, stem, and root mustards), and 4 accessions of B. oleracea ssp.alboglabra (Chinese kale). The RAPD markers unambiguously identified all 52 accessions. Net and Li genetic similarities were computed and used in UPGMA cluster analyses. Accessions and subspecies clustered into groups corresponding to the three species, but some accessions of some subspecies were most closely related to accessions belonging to another subspecies. Using genetic similarities, it was found that Chinese cabbage is more. likely to have been produced by hybridization of turnip and pakchoi, than as a selection from either turnip or pakchoi alone. RAPD markers provide a fast, efficient technique for diversity assessment that complements methods currently in use in genetic resources collections.

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666 PB 110 RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS ARE INADEQUATE FOR FINGERPRINTING GRAPE ROOTSTOCKS

HortScience ◽

10.21273/hortsci.29.5.528c ◽

1994 ◽

Vol 29 (5) ◽

pp. 528c-528

Author(s):

Alan T. Bakalinsky ◽

Hong Xu ◽

Diane J. Wilson ◽

S. Arulsekar

Keyword(s):

Dna Markers ◽

Rapd Markers ◽

Restriction Site ◽

Random Amplified Polymorphic Dna ◽

Rapd Marker ◽

Base Length ◽

Specific Primer ◽

Annealing Temperatures ◽

Individual Reaction ◽

Length Variant

A total of eight random amplified polymorphic DNA (RAPD) markers were generated in a screen of 77 primers of 10-base length and were detected reproducibly among nine different grape (Vitis) rootstocks. Occasional failed amplifications could not be explained rationally nor easily corrected by systematic replacement of individual reaction components. In an effort to improve their reliability, the RAPD markers were cloned, their termini sequenced, and new sequence-specific primer pairs were synthesized based on addition of 10 to 14 bases to the 3' termini of the original 10-mers. Six pairs of the new primers were evaluated at their optimal and higher-than optimal annealing temperatures. One primer pair amplified a product the same size as the original RAPD marker in all rootstocks, resulting in loss of polymorphism. Post-amplification digestion with 7 different restriction endonucleases failed to reveal restriction site differences. Three primer pairs amplified an unexpected length variant in some accessions. Two other pairs of primers amplified a number of unexpected bands. Better approaches for exploiting the sequence differences that account for the RAPD phenomenon will be discussed.

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