Pik Locus SNP Markers for Rapid Rice Blast Allele Screening
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Solution Overview
Problem
Current methods for identifying alleles at the rice-blast-resistant Pik locus are time-consuming, costly, and lack specificity, making them inefficient for accurate and rapid screening of rice germplasm resources.
Innovation Solution
A set of molecular markers comprising 12 primers is designed to detect nucleotides at specific SNP sites (806, 901, and 4210) in the Pik locus, allowing for the rapid and accurate identification of Pik, Pike, Pikm, Piks, and Pil alleles through PCR and electrophoresis detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional resistance spectrum analysis methods are used to identify Pik alleles, then comprehensive resistance spectrum data can be obtained, but the process is time-consuming and cannot accurately reflect genotypes
Solution Approach 1:
The invention extracts specific SNP sites (806, 901, and 4210) from the entire Pik locus sequence as diagnostic markers. By focusing only on these critical polymorphic positions rather than analyzing the complete gene sequence or conducting full resistance spectrum assays, the method achieves rapid and accurate allele identification while dramatically reducing time and computational resources required.
Solution Approach 2:
The invention creates simplified molecular marker copies (SNP-specific primers and probes) that represent the full Pik allele information. These marker copies enable direct genotyping through PCR and sequencing, providing accurate allele identification without needing to perform time-consuming resistance spectrum analysis with multiple Magnaporthe oryzae isolates.
2Measurement precision
If complete Pik locus sequencing is performed to identify alleles, then high accuracy can be achieved, but the process is costly and inefficient
Solution Approach 1:
The invention applies local quality by designing primers and probes that specifically target and amplify only the critical SNP regions (806, 901, 4210) within the Pik locus. This localized approach allows high-resolution allele differentiation at these key positions without the need for complete locus sequencing, thereby reducing costs and increasing screening throughput while maintaining diagnostic accuracy.
Solution Approach 2:
The invention segments the Pik locus analysis into discrete SNP position assessments. By dividing the continuous sequencing problem into targeted analysis of specific polymorphic sites, the method enables parallel processing of multiple samples through PCR-based approaches, significantly improving productivity compared to complete sequencing of each sample.
3Ease of operation
If existing dCAPS markers are used for Pik allele identification, then some alleles can be detected, but the process requires multiple markers and complex procedures
Solution Approach 1:
The invention develops a universal set of primers and probes that can simultaneously detect all major Pik allele types (Pik, Pike, Pikm, Piks, Pil) through a single PCR amplification and sequencing reaction. This multi-functional marker system eliminates the need to switch between multiple allele-specific markers, simplifying the detection procedure while comprehensively identifying any Pik allele present in the sample.
Solution Approach 2:
The invention merges multiple allele-specific detection capabilities into a unified molecular marker system. By combining universal primers that amplify the entire variable region with position-specific probes for the three diagnostic SNP sites, the method achieves comprehensive allele identification in a single integrated assay, reducing procedural complexity compared to using separate dCAPS markers for each allele.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The molecular markers provide high-throughput, low-cost, and specific identification of Pik alleles, reducing the time and cost associated with traditional screening methods and improving the efficiency of rice germplasm resource evaluation.
Implementation Method 1
allowing for the rapid and accurate identification of Pik, Pike, Pikm, Piks, and Pil alleles through PCR and electrophoresis detection
Implementation Method 2
through PCR and electrophoresis detection
Data Source
AI summary
For molecular genetic breeding of crops, molecular markers for identifying an allele at a rice-blast-resistant Pik locus of rice and use thereof are provided. The Pik gene is re-sequenced, and through sequence polymorphism analysis, identity codes, 3 SNPs, composed of nucleotide types at reverse sites 806, 901 and 4210 of the Pik-1 start codon have been found, which effectively identify Pik, Pike, Pil, Pikm, and Piks. The molecular markers quickly screen the allele type at the Pik locus, obtain an accurate result just by PCR (Polymerase Chain Reaction) and electrophoresis detection, and has high throughput and low cost.