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Title: High-Throughput resequencing of maize landraces at genomic regions associated with flowering time

item JAMANN, TIFFANY - University Of Illinois
item SOOD, SHILPA - North Carolina State University
item WISSER, RANDALL - University Of Delaware
item Holland, Jim - Jim

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/8/2016
Publication Date: 1/3/2017
Citation: Jamann, T., Sood, S., Wisser, R., Holland, J.B. 2017. High-Throughput resequencing of maize landraces at genomic regions associated with flowering time. PLoS One. 12:e0168910.

Interpretive Summary: Modern DNA sequencing has advanced rapidly, such that relatively inexpensive, low coverage sequencing is feasible for large numbers of plant samples. A problem with such low coverage sequencing, however, is that it provides a sample of sequence information from each individual. If a researcher is most interested in sequence variation at a set of specific genes, the low coverage sequencing approach will not be sufficient. Higher coverage whole genome sequence is prohibitively expensive for large samples of plants, however. We developed an alternative strategy to obtain high coverage sequence data on a set of target genes from a large sample of maize landraces from the Americas. This method provided excellent quality data and we were able to identify sequence variation in both copies of each target gene in each plant. A high percentage of the sequence variants identified in these landrace samples were novel, and were not previously included in the current gold standard set of variants identified in maize inbred lines.

Technical Abstract: Despite the reduction in the price of sequencing, it remains expensive to sequence and assemble whole, complex genomes of multiple samples for population studies, particularly for large genomes like those of many crop species. Enrichment of target genome regions coupled with next generation sequencing is a cost-effective strategy to obtain sequence information for loci of interest across many individuals, providing a less expensive approach to evaluating sequence variation at the population scale. Here we evaluate amplicon-based enrichment coupled with semi-conductor sequencing on a validation set consisting of five maize inbred lines, two hybrids and 19 landrace accessions. We report the use of a multiplexed panel of 319 PCR assays that target candidate loci associated with photoperiod sensitivity in maize while requiring 25 ng or less of starting DNA per sample. Enriched regions had an average on-target sequence read depth of 105 with 99% of the sequence data mapping to the maize ‘B73’ reference. Alignment with B73 and variant calling using SAMtools and GATK identified 1,308 variants and 1,532 variants, respectively. Of the variants called by both SAMtools and GATK, 32.0% were not previously reported in maize. Due to the high sequence read depth, heterozygote genotypes could be called with at least 96.5% accuracy in hybrid materials using GATK. We found that the genetic data corroborated historical descriptions of relationships between maize races. In conclusion, amplicon-based resequencing using highly multiplexed PCR coupled with semi-conductor sequencing is a cost-effective strategy for sequencing targeted genomic loci in diverse maize materials.