Chapter title |
Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA.
|
---|---|
Chapter number | 2 |
Book title |
Haplotyping
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6750-6_2 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6748-3, 978-1-4939-6750-6
|
Authors |
Choi, Kyuha, Yelina, Nataliya E, Serra, Heïdi, Henderson, Ian R, Yelina, Nataliya E., Henderson, Ian R., Kyuha Choi, Nataliya E. Yelina, Heïdi Serra, Ian R. Henderson |
Editors |
Irene Tiemann-Boege, Andrea Betancourt |
Abstract |
During meiosis, homologous chromosomes undergo recombination, which can result in formation of reciprocal crossover molecules. Crossover frequency is highly variable across the genome, typically occurring in narrow hotspots, which has a significant effect on patterns of genetic diversity. Here we describe methods to measure crossover frequency in plants at the hotspot scale (bp-kb), using allele-specific PCR amplification from genomic DNA extracted from the pollen of F1 heterozygous plants. We describe (1) titration methods that allow amplification, quantification and sequencing of single crossover molecules, (2) quantitative PCR methods to more rapidly measure crossover frequency, and (3) application of high-throughput sequencing for study of crossover distributions within hotspots. We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions. |
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Unknown | 1 | 50% |
Demographic breakdown
Type | Count | As % |
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Members of the public | 2 | 100% |
Mendeley readers
Geographical breakdown
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Unknown | 28 | 100% |
Demographic breakdown
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Student > Ph. D. Student | 4 | 14% |
Student > Bachelor | 3 | 11% |
Researcher | 3 | 11% |
Student > Master | 3 | 11% |
Professor | 2 | 7% |
Other | 4 | 14% |
Unknown | 9 | 32% |
Readers by discipline | Count | As % |
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Agricultural and Biological Sciences | 17 | 61% |
Biochemistry, Genetics and Molecular Biology | 4 | 14% |
Unknown | 7 | 25% |