Comparison of SNP Calling Pipelines and NGS Platforms to Predict the Genomic Regions Harboring Candidate Genes for Nodulation in Cultivated Peanut

Abstract

Cultivated peanut (Arachis hypogaea L.) forms root nodules to enable a symbiotic relationship with rhizobia for biological nitrogen fixation. To understand the genetic factors of peanut nodulation, it is fundamental to genetically map and clone the genes involved in nodulation. For genetic mapping, high throughput genotyping with a large number of polymorphic markers is critical. In this study, two sets of sister recombinant inbred lines (RILs), each containing a nodulating (Nod+) and non-nodulating (Nod-) line, and their Nod+ parental lines were extensively genotyped. Several next generation sequencing (NGS) methods including target enrichment sequencing (TES), RNAsequencing (RNA-seq), genotyping by sequencing (GBS), and the 48K Axiom Arachis2 SNP array, and various analysis pipelines were applied to identify single nucleotide polymorphisms (SNP) among the two sets of RILs and their parents. TES revealed the largest number of homozygous SNPs (15,947) between the original parental lines, followed by the Axiom Arachis2 SNP array (1,887), RNA-seq (1,633), and GBS (312). Among the five SNP analysis pipelines applied, the alignment to A/B genome followed by HAPLOSWEEP revealed the largest number of homozygous SNPs and highest concordance rate (79%) with the array. A total of 222 and 1,200 homozygous SNPs were polymorphic between the Nod+ and Nod− sister RILs and between their parents, respectively. A graphical genotype map of the sister RILs was constructed with these SNPs, which demonstrated the candidate genomic regions harboring genes controlling nodulation across the whole genome. Results of this study mainly provide the pros and cons of NGS and SNP genotyping platforms for genetic mapping in peanut, and also provide potential genetic resources to narrow down the genomic regions controlling peanut nodulation, which would lay the foundation for gene cloning and improvement of nitrogen fixation in peanut.

CAPG: Comprehensive Allopolyploid Genotyper

“…SWEEP (Clevenger et al ., 2015) and HAPLOSWEEP (Clevenger et al ., 2018), the latter recommended over SWEEP (Peng et al ., 2020), use these references to identify allelic SNPs via homozygous individuals at variable sites bracketed by likely homoeologous loci. However, the most common subgenome reference aware method (Peng et al ., 2017; Zhou et al ., 2014, and M2 in Peng et al ., 2020) aligns reads to both subgenomes, keeps uniquely aligned reads, and applies a diploid genotyper to each subgenome.…”

“…Increasingly, however, available subgenome references (Bertioli et al ., 2016; Lu et al ., 2019; Wang et al ., 2019) or genomes of closely related diploid ancestral species (Bertioli et al ., 2016; Du et al ., 2018) provide direct information for assigning reads. SWEEP (Clevenger et al ., 2015) and HAPLOSWEEP (Clevenger et al ., 2018), the latter recommended over SWEEP (Peng et al ., 2020), use these references to identify allelic SNPs via homozygous individuals at variable sites bracketed by likely homoeologous loci. However, the most common subgenome reference aware method (Peng et al ., 2017; Zhou et al ., 2014, and M2 in Peng et al ., 2020) aligns reads to both subgenomes, keeps uniquely aligned reads, and applies a diploid genotyper to each subgenome.…”

CAPG: comprehensive allopolyploid genotyper

“…Increasingly, however, available subgenome references ( Bertioli et al , 2016 ; Lu et al , 2019 ; Wang et al , 2019 ) or genomes of closely related diploid ancestral species ( Bertioli et al , 2016 ; Du et al , 2018 ) provide direct information for assigning reads. SWEEP ( Clevenger et al , 2015 ) and HAPLOSWEEP ( Clevenger et al , 2018 ), the latter recommended over SWEEP ( Peng et al , 2020 ), use these references to identify allelic SNPs via homozygous individuals at variable sites bracketed by likely homoeologous sites. However, the most common subgenome reference aware method ( Peng et al , 2017 ; Zhou et al , 2014 , and M2 in Peng et al , 2020 ) aligns reads to both subgenomes, keeps uniquely aligned reads and applies a diploid genotyper to each subgenome.…”

“…SWEEP ( Clevenger et al , 2015 ) and HAPLOSWEEP ( Clevenger et al , 2018 ), the latter recommended over SWEEP ( Peng et al , 2020 ), use these references to identify allelic SNPs via homozygous individuals at variable sites bracketed by likely homoeologous sites. However, the most common subgenome reference aware method ( Peng et al , 2017 ; Zhou et al , 2014 , and M2 in Peng et al , 2020 ) aligns reads to both subgenomes, keeps uniquely aligned reads and applies a diploid genotyper to each subgenome.…”

Genetic Diversity and Genome-Wide Association Study of Seed Aspect Ratio Using a High-Density SNP Array in Peanut (Arachis hypogaea L.)

“…A total of 2882 polymorphic SNPs retained from the second edition of the Axiom_Arachis array (Axiom_Arachis2) were used to identify loci controlling pod construction trait using 195 F7 recombinant inbred lines (RILs) [ 43 ]. The 48K Axiom Arachis2 SNP array was applied to identify single nucleotide polymorphisms (SNP) among the two sets of RILs and the two original Nod+ parental lines to explore the genetic factors and genetic regions controlling nodulation in peanut [ 44 ].…”