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naica® Droplet Chip Digital PCR System for High-throughput Determination of Meiotic Recombination Rate in Barley Pollen Nuclei
2024-05-07
In plants, homologous recombination can be detected by several techniques, such as cytological detection through meiotic chromosome analysis, genotyping by sequencing and molecular markers or fluorescently labeled lines (FTLs) in segregating populations. FTLs are a powerful tool for measuring meiotic recombination events in pollen or seeds in Arabidopsis. However, FTLs are not suitable for crops because it is laborious and expensive to generate FTLs in crops with particularly large genomes. In addition, different crops or certain genotypes are not suitable for genetic transformation. As an alternative, microspore (tetrad or pollen nucleus) genotyping or sequencing is used to directly detect the results of meiotic recombination in meiotic products. However, sequencing/genotyping of crop microspores is quite expensive, so the number of tests that can be performed is limited, especially for large genome species such as cereals.
Measuring the meiotic recombination rate of male gametes before fertilization has the advantages of large sample size, independent molecular marker analysis and immediate recombination exchange analysis, but the gamete DNA content is limited, and sequencing/genotyping methods usually rely on whole genome amplification (WGA). Genotyping of single gametes directly through PCR reactions is also impossible due to the low DNA content of haploid gametes. In barley, single pollen nucleus genotyping is completed by separating single haploid pollen nuclei from intraspecific hybrids by fluorescence activated cell sorting, followed by WGA and multi-locus KASP genotyping or single-cell genome sequencing. The DNA of a single haploid pollen nucleus is limited, and the price of WGA is high, resulting in a limited number of analyzed samples and the inability to complete high-throughput analysis.
Scientists from the Leibniz Institute of Plant Genetics and Crop Plant Research in Germany recently published a paper on meiotic recombination rate measurement in The Plant Journal. The article uses the naica® droplet chip digital PCR system to measure the meiotic recombination rate in gametes to achieve high-throughput and low-cost genotyping.
Using genotyping analysis based on the naica® droplet chip digital PCR system, high-throughput measurement of meiotic recombination rates in barley pollen nuclei can be completed without a large amount of pre-performed WGA. After obtaining pollen, the pollen nuclei in the pollen are removed and purified by flow cytometry. The obtained pollen nuclei are added to the Mix of the naica® droplet chip digital PCR system for detection to obtain the meiotic recombination rate. By genotyping a total of >42,000 individual pollen nuclei (up to 4900 nuclei per plant), the meiotic recombination rates within two centromeres and two distant chromosome intervals are measured in hybrid plants. The recombination frequency determined in the pollen nucleus is close to the frequency detected in the segregating population.
▲ Figure 1: Workflow for single pollen nucleus genotyping of barley using the naica® Droplet Chip Digital PCR System. (a) Anthers of hybrid plants; (b) Pollen and pollen nuclei are separated in suspension by using filters with different mesh sizes (100 and 20 μm). (c) Pollen nuclei are stained with propidium iodide and flow-sorted into digital PCR reaction mix. (d) 25 μl of digital PCR reaction mix (including sorted pollen nuclei) is loaded into one of the four chambers of the sapphire chip. (e) Droplet generation and thermal cycling are performed in Geode. (f) After thermal cycling, the sapphire chip was scanned in naica® Prism 3, and data analysis was performed in Crystal Miner software.
This article used a dual probe method to detect the meiotic recombination rate of pollen nuclei. For example, the interval Id 3-1 between InDel3118 and InDel3135 detected in the previous feasibility verification was labeled with HEX for the Barke (B) allele-specific probe (green) and with FAM for the Morex (M) allele-specific probe (blue) (Figure 2b). The researchers mixed pollen nuclei from parental genotypes in a 1:1 ratio and also detected pollen nuclei from hybrid plants heterozygous for Id 3-1. In the parental mixed sample test, the droplets of the two parental genotypes were equal, and the two markers showed the same fluorescence (HEX for B or FAM for M) (Figure 2b). In the detection of hybrid material samples, it is expected that different droplet groups representing recombination events will appear, that is, droplets showing two colors at the same time (HEX for InDel3118 and FAM for InDel3135, and vice versa) (Figure 2b). In actual detection, it was found that the parental genotypes obtained approximately equal numbers of droplets, which showed the same fluorescence for the two markers (Figure 2d, e, green and blue rectangles). In the detection of pollen nuclei of hybrid plants, droplets with two colors (HEX and FAM) were detected, indicating recombination events (Figure 2e, red rectangle). In addition, droplets in which only one marker was successfully amplified (Figure 2d, e, clusters I and iii) and droplets without any amplification (Figure 2d, e, cluster ii) can be distinguished. It shows that it is completely feasible to encapsulate and genotype single pollen nuclei using the naica® droplet chip digital PCR system.
▲ Figure 2. Genotyping of barley pollen nuclei using the naica® Droplet Chip Digital PCR System. (a) InDel or single nucleotide polymorphism (SNP) markers defining four chromosomal intervals on barley chromosomes 1 and 3. (b) Genotyping analysis of pollen nuclei based on the naica® Droplet Chip Digital PCR System using Id 3-1 as an example: possible combinations of two fluorescent probes enable discrimination between recombinant and non-recombinant pollen nuclei. (c) Raw view of the active droplet array. Each chamber typically contains approximately 25,000 stable active droplets. Droplets that have been successfully amplified in any channel (FAM or HEX) are light grey, while dark grey droplets are negative. (d, e) Genotyping data of pollen nuclei based on the naica® Droplet Chip Digital PCR System from the chip chambers are displayed in the software as dot plots of pollen nuclei from parental genotypes mixed in a 1:1 ratio (d) and pollen nuclei from hybrid plants heterozygous with Id 3-1. (e) Two non-recombinant parent populations were detected as droplets with successful genotyping by two HEX-labeled (green boxes) or FAM-labeled allele probes (blue boxes). In the dot plot of the parental genotype mixture (d), the HEX and FAM double-positive droplets are represented by gray boxes as false positives + noise. The HEX and FAM double-positive droplets in the hybrid plants are recombinant populations including false positives and noise, shown as red boxes (e). Clusters (I) and (iii) represent droplets that successfully amplified only one marker
The naica® Droplet Chip Digital PCR System has extremely high resolution, so in those droplets that successfully amplified the marker, the nuclei within the droplets can also be observed (Figure 2c). The researchers further optimized the experiment by analyzing the number of droplet-encapsulated nuclei, and improved the efficiency of genotyping by pre-treating pollen nuclei with thermostable restriction enzymes. Because the number of nuclei is positively correlated with the number of droplets that encapsulate a single nucleus, the optimal interval for loading nuclei is proposed (different nuclei of different sizes vary in different species).
The detection based on 2-color probes in this paper is very successful, and further through the 6-color platform, more groups of genotyping detection can be performed simultaneously, multiple genotyping data will be obtained, and the recombination rate of more than one chromosomal interval on the same or different chromosomes can be measured in parallel, or the intensity/presence of CO interference can be measured.
In general, the genotyping of single barley pollen nuclei based on the naica® droplet chip digital PCR system provides reliable, rapid and high-precision meiotic recombination measurements within the specified chromosomal interval of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclei and genome sizes shows that the proposed method is widely applicable to the genotyping of single nuclei.
Professor Stefan Heckmann and Dr. Yun-Jae Ahn from the Leibniz Institute of Plant Genetics and Crop Science (IPK) in Germany also shared their research results with us online. If you want to intuitively understand the details of this article, please click https://mp.weixin.qq.com/s/KNXVs6rOt8MYpBjzuKZZ9A to watch it.
naica® six-channel droplet chip digital PCR system
The naica® six-channel droplet chip digital PCR system from Stilla Technologies of France is based on the Crystal droplet chip digital PCR technology. It automates droplet generation and amplification. Each sample well can detect 6 fluorescent channels, intelligently identify droplets and perform quality control. The absolute copy number concentration of at least 6 target genes can be obtained within 3 hours.
