Whole Genome Amplification of Single Pollen Grains for Haplotype Determination in Citrus

Most sequencing studies do not reveal haplotypes at the whole chromosome level. However, relationships between DNA sequence and phenotype can be more fully understood with haplotype information. Reconstructing the haplotype of a diploid cell in heterozygous individuals has proven to be challenging and phasing entire genomes in GWAS data sets has been a computational bottleneck. Recently, Single-Cell Genomics (SCG) opened new opportunities to overcome the existing technological impediments to obtaining phase information. Genotyping of several haploid individuals (pollen grains) can reveal the haplotypes of the paternal plant. A multitude of methods for sample preparation have been introduced but, current WGA methods available are not suitable for plant cells unless they are protoplasts. Citrus mature pollen grains are bicellular and have a double wall. We developed an easy and reliable method for pollen grain isolation and lysis, and WGA of single pollen grains by optimizing the REPLI-g single cell kit (QIAGEN). We tested our method on different citrus genotypes and citrus relatives (Poncirus trifoliata) having haplotypes previous inferred by linkage mapping. To evaluate the amplification performance, we used SSR markers located on each linkage group (2 SSRs for each group) and a larger set of marker on one group. The mean call rate was 78% indicating effective whole-genome amplification.  In this poster we also demonstrate the effectiveness of our method in citrus haplotype reconstruction. We plan to analyze WGA DNA on SNP genotyping arrays.