Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome

Abstract

Background: Rapid acquisition of accurate genotyping information is essential for all genetic marker-based studies. For species with relatively small genomes, complete genome resequencing is a feasible approach for genotyping; however, for species with large and highly repetitive genomes, the acquisition of whole genome sequences for the purpose of genotyping is still relatively inefficient and too expensive to be carried out on a high-throughput basis. Sorghum bicolor is a C 4 grass with a sequenced genome size of~730 Mb, of which~80% is highly repetitive. We have developed a restriction enzyme targeted genome resequencing method for genetic analysis, termed Digital Genotyping (DG), to be applied to sorghum and other grass species with large repeat-rich genomes. Results: DG templates are generated using one of three methylation sensitive restriction enzymes that recognize a nested set of 4, 6 or 8 bp GC-rich sequences, enabling varying depth of analysis and integration of results among assays. Variation in sequencing efficiency among DG markers was correlated with template GC-content and length. The expected DG allele sequence was obtained 97.3% of the time with a ratio of expected to alternative allele sequence acquisition of >20:1. A genetic map aligned to the sorghum genome sequence with an average resolution of 1.47 cM was constructed using 1,772 DG markers from 137 recombinant inbred lines. The DG map enhanced the detection of QTL for variation in plant height and precisely aligned QTL such as Dw3 to underlying genes/alleles. Higher-resolution NgoMIV-based DG haplotypes were used to trace the origin of DNA on SBI-06, spanning Ma1 and Dw2 from progenitors to BTx623 and IS3620C. DG marker analysis identified the correct location of two miss-assembled regions and located seven super contigs in the sorghum reference genome sequence.

Sorghum Phytochrome B Inhibits Flowering in Long Days by Activating Expression of SbPRR37 and SbGHD7, Repressors of SbEHD1, SbCN8 and SbCN12

“…Template for sequencing on an Illumina GAIIx sequencer was generated following the standard Digital Genotyping (DG) protocol [33]. Genotypes of all individuals from both populations were identified.…”

3D sorghum reconstructions from depth images identify QTL regulating shoot architecture

“…Genotypes for members of the BTx623 3 IS3620C RIL population were obtained previously and available to construct a genetic map for mapping QTLs for the image-based phenotypes across multiple developmental time points (Morishige et al, 2013;Truong et al, 2014;McCormick et al, 2015). Measurements obtained from plant meshes were grouped into two categories: organ-level measurements and composite measurements.…”

“…Genotypes for the BTx623 3 IS3620C RIL population were generated previously using Digital Genotyping, a restriction enzyme-based, reducedrepresentation sequencing assay (Morishige et al, 2013). Genotypes were called using the naive pipeline of the RIG workflow with the GATK, and the genetic map was constructed as described previously with marker orderings relative to the version 3 assembly of the sorghum reference genome, Sbi3 (Department of Energy-Joint Genome Institute [http://phytozome.jgi.doe.…”

QTL mapping and loci dissection for leaf epicuticular wax load and canopy temperature depression and their association with QTL for staygreen in Sorghum bicolor under stress

“…Digital genotyping (DG) (Morishige et al 2013) was accomplished using the high throughput Illumina Genome Analyzer Platform II (Illumina.com 2014), a next generation sequencer previously described (Quail et al 2001). The Illumina sequencer is hosted at the Texas A&M AgriLife Genomics and Bioinformatics Services in College Station, Texas, USA.…”