Lifeasible, as a leading company in plant breeding technologies, offers tailored positional cloning service for our customers worldwide.
Positional cloning, also known as map-based cloning, is a technique for the positioning of a trait-associated gene in the genome and involves methods such as linkage analysis, association mapping, and bioinformatics. Due to its high efficiency and accuracy, the positional cloning technique has shed light on modern plant breeding for the identification of genes or quantitative trait loci (QTLs) associated desired traits, and can potentially accelerate marker-assisted selection as well as introduction of valuable traits.
The principle of positional cloning is to isolate a particular allele from a cDNA library with specific primers, using chromosome waking or thermal asymmetric interlaced PCR (tail-PCR), which are used in the identification of unknown regions flanking a known DNA sequence.
| Initial Positioning | Isolating populations: F2, BC1, DH, etc. Molecular markers: SSR, CAPS AFLP, RFLP, RAPD, etc. Generally, flanking markers 5-10 cm away from the target gene can be screened out through preliminary positioning. |
| Build A Linkage Map | Integrate existing genetic maps and integrate different types of molecular markers For species that have been sequenced, new analysis markers are designed based on DNA sequences near the gene of interest Use the collinearity of comparative genomes to obtain molecular markers from other organisms in the same family and genus |
| Fine Positioning | Use flanking markers to screen out recombinant individual plants, then use all polymorphic markers to analyze the recombinant individual plants, and combine the phenotypic identification results to locate the target gene within a range of 0.5 cm or less. |
| Physical Map Construction | The difference in recombination values of different chromosome segments will affect the size of the genetic distance, so the distance between the molecular marker and the target gene should be calculated based on the actual number of bases. |
| Chromosome Walking | Use the molecular markers most closely linked to both sides of the target gene to screen the positive clones in the large fragment DNA library and find the corresponding large fragment clones. Chromosome walking is then performed from both ends until a large fragment clone or spanning stack with molecular markers on both sides of the target gene is obtained. |
| Identify Candidate Genes | Use large fragment clones containing the target gene, such as BAC or YAC clones, to screen the cDNA library, and query the biological data information database to determine candidate genes. Then, (1) Whether cDNA and target gene are co-segregated (2) Whether the spatiotemporal expression of cDNA is consistent with the phenotype (3) Determine the cDNA sequence and query the database to understand the function of the gene (4) Screen the mutant library to find out the changes in DNA sequence and their relationship with function (5) Perform functional complementary verification |
| Functional Complementation Verification | Functional complementation testing (transgenicity) is the most direct and conclusive way to identify genes RNA interference (RNAi) can also be used to effectively identify target genes |
While cloning of QTLs requires information of both genetic and physical maps, and is performed following a series of steps:
At Lifeasible, our teams of geneticists, statisticians, and software engineers will work closely toward the most optimized solvent for any given project, providing both scientific and technical insights for population type, sample size, marker collection, and other critical aspects of the positional cloning program, to ensure the success of your projects.
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Lifeasible provides comprehensive plant breeding services to support customers' related projects, including traditional breeding techniques and modern molecular breeding techniques. We also provide diversified plant analysis services.
Lifeasible has established a one-stop service platform for plants. In addition to obtaining customized solutions for plant genetic engineering, customers can also conduct follow-up analysis and research on plants through our analysis platform. The analytical services we provide include but are not limited to the following:
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