Sequencing of Single Pollen Nuclei Reveals Meiotic Recombination Events at Megabase Resolution and Circumvents Segregation Distortion Caused by Postmeiotic Processes

Abstract

Meiotic recombination is a fundamental mechanism to generate novel allelic combinations which can be harnessed by breeders to achieve crop improvement. The recombination landscape of many crop species, including the major crop barley, is characterized by a dearth of recombination in 65% of the genome. In addition, segregation distortion caused by selection on genetically linked loci is a frequent and undesirable phenomenon in double haploid populations which hampers genetic mapping and breeding. Here, we present an approach to directly investigate recombination at the DNA sequence level by combining flow-sorting of haploid pollen nuclei of barley with single-cell genome sequencing. We confirm the skewed distribution of recombination events toward distal chromosomal regions at megabase resolution and show that segregation distortion is almost absent if directly measured in pollen. Furthermore, we show a bimodal distribution of inter-crossover distances, which supports the existence of two classes of crossovers which are sensitive or less sensitive to physical interference. We conclude that single pollen nuclei sequencing is an approach capable of revealing recombination patterns in the absence of segregation distortion.

Keywords: crossover; crossover interference; homologous recombination; meiosis; pollen; segregation distortion; single-cell genomics.

Figure 1

Graphical genotypes of individual pollen nuclei revealed by single-cell genome sequencing. Recombination events were detected in 40 individual pollen nuclei. The two parental barley genotypes are shown in red (“Morex”) and blue (“Barke”). Consensus genotypes were mapped to the physical reference genome of barley at 1 Mbp resolution. Centromere positions are indicated by dashed black lines. White gaps which consistently occur in all samples are regions where no genetic polymorphisms exist between “Morex” and “Barke.”

Figure 2

Frequency of recombination events in pollen and DH plants. Relative frequency of the average number of recombination events per chromosome is shown for the pollen (blue) and DH population (red) in classes ranging from 0 to 4. Error bars represent the standard deviations based on measurements conducted on all seven barley chromosomes.

Figure 3

Elevated recombination frequencies in distal regions of barley chromosome 5H. Recombination frequency in pollen (blue) and DH plants (red) was calculated in 5 Mbp sliding windows along chromosome 5H and plotted along the physical map. The position of the centromere is marked by a black diamond.

Figure 4

Inter-crossover distance reveals positive crossover interference and supports the existence of two crossover classes in barley. The frequency of the distance between crossovers on the same chromatid (inter-crossover distance) in pollen (blue) and DH plants (red) was determined in 100 Mbp classes ranging from < 100 to >700 Mbp. The relative frequency of nearby crossovers present in each class was plotted. Error bars represent the standard deviation based on measurements conducted on all seven barley chromosomes.

Figure 5

Physical distribution of first and second crossover positions. Physical positions of first and second crossover event for all samples showing more than two crossovers in the pollen (blue) and DH (red) population. Approximate centromeric regions are marked by gray boxes. Strong physical interference is shown by dots accumulated in the top left quarter. Weak physical interference is shown by dots accumulated in the bottom left and top right quarter.

Figure 6

Segregation distortion is almost absent if measured in pollen but abundant in DH plants. Allele frequencies for “Morex” (black) and “Barke” (gray) measured in pollen (dashed line) and DH plants (straight line) are shown as 10 Mbp moving averages for (A) chromosome 2H and (B) chromosome 3H of barley. Dashed red lines represent the significance threshold of distorted segregation ratios (χ²-test, P < 0.05). Pollen or DH allele frequencies above the significance threshold mark genomic regions of distorted segregation ratios.