Development of allele-specific PCR and RT–PCR assays for clustered resistance genes using a potato late blight resistance transgene as a model

2006 ◽  
Vol 114 (3) ◽  
pp. 501-513 ◽  
Author(s):  
B. P. Millett ◽  
J. M. Bradeen
Keyword(s):  
Late Blight ◽  
Resistance Genes ◽  
Late Blight Resistance ◽  
Potato Late Blight ◽  
Blight Resistance ◽  
Rt Pcr ◽  
Specific Pcr ◽  
Allele Specific ◽  
Pcr Assays ◽  
Allele Specific Pcr

Two distinct potato late blight resistance genes from Solanum berthaultii are located on chromosome 10

Euphytica ◽  
2008 ◽  
Vol 165 (2) ◽  
pp. 269-278 ◽  
Author(s):  
Tae-Ho Park ◽  
Simon Foster ◽  
Gianinna Brigneti ◽  
Jonathan D. G. Jones
Keyword(s):  
Late Blight ◽  
Resistance Genes ◽  
Late Blight Resistance ◽  
Potato Late Blight ◽  
Blight Resistance ◽  
Solanum Berthaultii ◽  
Chromosome 10

Transcriptome analysis of scions grafted to potato rootstock for improving late blight resistance

2020 ◽  
Author(s):  
Yuexin Li ◽  
Degang Zhao
Keyword(s):  
Disease Resistance ◽  
Late Blight ◽  
Protein Kinases ◽  
Molecular Mechanisms ◽  
Late Blight Resistance ◽  
Potato Late Blight ◽  
Susceptible Variety ◽  
Resistant Variety ◽  
Blight Resistance ◽  
Expression Levels

Abstract Background: Late blight seriously threatens potato cultivation worldwide. The severe and widespread damage caused by the fungal pathogen can lead to drastic decreases in potato yield. Although grafting technology has been widely used to improve crop resistance, the effects of grafting on potato late blight resistance as well as the associated molecular mechanisms remain unclear. Therefore, we performed RNA transcriptome sequencing analysis and the late blight resistance testing of the scion when the potato late blight-resistant variety Qingshu 9 and the susceptible variety Favorita were used as the rootstock and scion, respectively, and vice versa. The objective of this study was to evaluate the influence of the rootstock on scion disease resistance and to clarify the related molecular mechanisms.Results: A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that the expression levels of genes related to plant–pathogen interactions, plant mitogen-activated protein kinase (MAPK) signaling pathways, and plant hormone signal transduction pathways were significantly up-regulated in the scion when Qingshu 9 was used as the rootstock. These genes included late blight response genes encoding calcium-dependent protein kinases (CDPKs), chitin elicitor receptor kinases (CERKs), LRR receptor serine/threonine protein kinases (LRR-LRKs), NPR family proteins in the salicylic acid synthesis pathway, and MAPKs. When Favorita was used as the rootstock, the expression levels of the late blight response genes were not up-regulated in the Qingshu 9 scion, but the expression levels of the genes related to proline metabolism, fatty acid chain elongation, and diterpenoid biosynthesis pathways were down-regulated. Resistance results showed that self-grafting of the susceptible variety and grafting with the resistant variety as the rootstock increased the resistance of the susceptible scion to late blight. However, the resistance was stronger after grafting with the resistant variety as the rootstock. Using the susceptible variety as the rootstock decreased the late blight resistance of the resistant scion.Conclusions: Our results showed that changes to the expression of disease resistance genes in the scion after grafting are associated with late blight resistance. The results provide the basis for exploring the molecular mechanism underlying the effects of rootstocks on scion disease resistance.

scholarly journals Detection of Novel QTLs for Late Blight Resistance Derived from the Wild Potato Species Solanum microdontum and Solanum pampasense

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 732
Author(s):  
Fergus Meade ◽  
Ronald Hutten ◽  
Silke Wagener ◽  
Vanessa Prigge ◽  
Emmet Dalton ◽  
...  
Keyword(s):  
Late Blight ◽  
Resistance Genes ◽  
Late Blight Resistance ◽  
Gene Clusters ◽  
Genetic Maps ◽  
Blight Resistance ◽  
Wild Potato ◽  
Wild Potato Species ◽  
Potato Species ◽  
Solanum Microdontum

Wild potato species continue to be a rich source of genes for resistance to late blight in potato breeding. Whilst many dominant resistance genes from such sources have been characterised and used in breeding, quantitative resistance also offers potential for breeding when the loci underlying the resistance can be identified and tagged using molecular markers. In this study, F1 populations were created from crosses between blight susceptible parents and lines exhibiting strong partial resistance to late blight derived from the South American wild species Solanum microdontum and Solanum pampasense. Both populations exhibited continuous variation for resistance to late blight over multiple field-testing seasons. High density genetic maps were created using single nucleotide polymorphism (SNP) markers, enabling mapping of quantitative trait loci (QTLs) for late blight resistance that were consistently expressed over multiple years in both populations. In the population created with the S. microdontum source, QTLs for resistance consistently expressed over three years and explaining a large portion (21–47%) of the phenotypic variation were found on chromosomes 5 and 6, and a further resistance QTL on chromosome 10, apparently related to foliar development, was discovered in 2016 only. In the population created with the S. pampasense source, QTLs for resistance were found in over two years on chromosomes 11 and 12. For all loci detected consistently across years, the QTLs span known R gene clusters and so they likely represent novel late blight resistance genes. Simple genetic models following the effect of the presence or absence of SNPs associated with consistently effective loci in both populations demonstrated that marker assisted selection (MAS) strategies to introgress and pyramid these loci have potential in resistance breeding strategies.

scholarly journals Successful optimization of CRISPR/Cas9-mediated defined point mutation knock-in using allele-specific PCR assays in zebrafish

2017 ◽  
Author(s):  
Sergey V. Prykhozhij ◽  
Charlotte Fuller ◽  
Shelby L. Steele ◽  
Chansey J. Veinotte ◽  
Babak Razaghi ◽  
...  
Keyword(s):  
Point Mutation ◽  
Point Mutations ◽  
Mutation Site ◽  
Transmission Rates ◽  
Specific Pcr ◽  
Allele Specific ◽  
Pcr Assays ◽  
Combination Of Methods ◽  
Mutation Strategies ◽  
Allele Specific Pcr

AbstractSingle-stranded oligodeoxynucleotides (ssODN) are donor templates for homology-directed repair-based knock-in of point mutations using CRISPR/Cas9. To optimize the efficiency of ssODN-based knock-ins in zebrafish, we developed allele-specific PCR (AS-PCR) assays for introducing point mutations in tp53, cdh5 and lmna as case studies. In these point mutation strategies we introduced the codon mutations, sgRNA site mutations and restriction sites which can be detected by AS-PCR with the primers matching their respective alleles in combination with a common primer. We employed the anti-sense asymmetric oligo design as the main optimization as well as phosphorothioate oligo modification and also observed that proximity of the mutation site to the Cas9 cut site improves the efficiency when knock-ins into different genes were compared. We improved the efficiencies of two tp53 knock-ins using anti-sense asymmetric ultramer oligos (126-nt in length with homology arms of 36 and 90 nucleotides, anti-sense to the sgRNA) by 3-10 fold, the optimizations which resulted in successful founders for both tp53 knock-ins with transmission rates of 20-40 %. The initially low knock-in efficiency for tp53 mutants was likely due to the distance between the Cas9 cut site and mutations since cdh5 G767S knock-in located at the cut site had much higher founder identification and germline transmission rates. The phosphorothioate oligo modifications was used for a lamin A/C (lmna) knock-in strategy and it resulted in 40 % overall improvement in knock-in efficiency and greater knock-in consistency. We also determined that AS-PCR detected false-positive knock-ins which constituted 25-80 % of total in different strategies and developed a workflow to screen out the founders and F1 zebrafish carrying these undesirable modifications. In summary, we provide a complementary set of optimizations for CRISPR/Cas9-based ssODN knock-ins in zebrafish using a novel combination of methods.

Interspecific potato hybrids as a resource for late blight resistance genes

2014 ◽  
Vol 40 (1) ◽  
pp. 10-13
Author(s):  
E. V. Rogozina ◽  
V. A. Kolobaev ◽  
E. E. Khavkin ◽  
M. A. Kuznetsova ◽  
M. P. Beketova ◽  
...  
Keyword(s):  
Late Blight ◽  
Resistance Genes ◽  
Late Blight Resistance ◽  
Blight Resistance ◽  
Interspecific Potato Hybrids

Validation of SNP markers associated with ascochyta blight resistance in pea

2019 ◽  
Vol 99 (2) ◽  
pp. 243-249
Author(s):  
Ambuj B. Jha ◽  
Krishna K. Gali ◽  
Sabine Banniza ◽  
Thomas D. Warkentin
Keyword(s):  
Association Studies ◽  
Ascochyta Blight ◽  
Snp Markers ◽  
Blight Resistance ◽  
Maturity Stage ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Important Disease

Ascochyta blight of pea is an important disease that can cause severe yield loss. Our previous studies identified several closely linked single nucleotide polymorphism (SNP) markers associated with ascochyta blight resistance. The objective of this study was to validate SNP markers in 36 cultivars from the Saskatchewan pea regional variety trial. Ascochyta blight scores ranged from 1.0 to 9.0 at the physiological maturity stage under field conditions across the 25 site–years in Saskatchewan from 2013 to 2017. Based on Kompetitive Allele-Specific PCR assays, six SNP markers were used for an association study. SNP markers RGA-G3Ap103, PsC8780p118, and PsC22609p103 were significantly (P < 0.05) associated with ascochyta blight scores in 2013 and 2016 at Saskatoon. PsC8780p118 was significantly associated with ascochyta blight scores at Milden in 2014 and Rosthern in 2017. Furthermore, RGA-G3Ap103 showed significant association at Milden in 2014. Based on association studies, RGA-G3Ap103 and PsC8780p118 may have some potential as markers for pea breeding.

scholarly journals Higher Copy Numbers of the Potato RB Transgene Correspond to Enhanced Transcript and Late Blight Resistance Levels

2009 ◽  
Vol 22 (4) ◽  
pp. 437-446 ◽  
Author(s):  
James M. Bradeen ◽  
Massimo Iorizzo ◽  
Dimitre S. Mollov ◽  
John Raasch ◽  
Lara Colton Kramer ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Late Blight ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Rna Silencing ◽  
Late Blight Resistance ◽  
Blight Resistance ◽  
Transcript Levels ◽  
Cultivated Potato ◽  
Copy Numbers

Late blight of potato ranks among the costliest of crop diseases worldwide. Host resistance offers the best means for controlling late blight, but previously deployed single resistance genes have been short-lived in their effectiveness. The foliar blight resistance gene RB, previously cloned from the wild potato Solanum bulbocastanum, has proven effective in greenhouse tests of transgenic cultivated potato. In this study, we examined the effects of the RB transgene on foliar late blight resistance in transgenic cultivated potato under field production conditions. In a two-year replicated trial, the RB transgene, under the control of its endogenous promoter, provided effective disease resistance in various genetic backgrounds, including commercially prominent potato cultivars, without fungicides. RB copy numbers and transcript levels were estimated with transgene-specific assays. Disease resistance was enhanced as copy numbers and transcript levels increased. The RB gene, like many other disease resistance genes, is constitutively transcribed at low levels. Transgenic potato lines with an estimated 15 copies of the RB transgene maintain high RB transcript levels and were ranked among the most resistant of 57 lines tested. We conclude that even in these ultra–high copy number lines, innate RNA silencing mechanisms have not been fully activated. Our findings suggest resistance-gene transcript levels may have to surpass a threshold before triggering RNA silencing. Strategies for the deployment of RB are discussed in light of the current research.

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