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Targeted sequencing of a complex locus within a polyploid genome using reduced representation libraries

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Abstract

Apospory is a form of gametophytic apomixis in which embryos develop from unreduced embryo sacs derived from aposporous initials formed from nucellar cells of ovules to produce offspring genetically identical to the female plant. Apospory in Pennisetum squamulatum (8X) and Cenchrus ciliaris (4X) is a dominant trait controlled by a physically large, hemizygous, heterochromatic chromosomal block called the apospory-specific genomic region (ASGR). Both apomictic species are polyploid, with genome sizes estimated at 2600 to 3000 Mbp for C. ciliaris and 9400 to 10,300 Mbp for P. squamulatum. A study was conducted to determine whether duplex-specific nuclease (DSN) normalization of DNA from apomictic and sexual genotypes would reduce repetitive sequences and allow bioinformatic analysis to predict sequence contigs derived from the ASGR. DSN libraries from four genotypes were sequenced using Illumina® HiSeq 2000 technology. 39 out of 44 tested sequence characterized amplified region (SCAR) markers from in silico predicted ASGR-specific contigs were mapped to the ASGR in a Pennisetum F1 mapping population. Eighteen SCARs showed apomict-specific amplification in C. ciliaris. The successful mapping of ~90 % of the SCAR markers to the ASGR in the Pennisetum F1 mapping population shows that DSN normalization and Illumina sequencing can be used as an effective strategy for targeted mapping of a physically large locus rich in repetitive sequences, like that of the ASGR.

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Acknowledgments

This study was supported by Pioneer Hi-Bred. We thank Gunawati Gunawan for providing technical support.

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Authors and Affiliations

  1. Department of Horticulture, University of Georgia – Tifton Campus, Tifton, GA, 31793, USA

    Joann Conner, Sirjan Sapkota & Peggy Ozias-Akins

  2. DuPont Pioneer, DuPont Experimental Station, Wilmington, DE, 19803, USA

    Stéphane Deschamps

  3. DuPont Pioneer, Johnston, IA, 50131, USA

    Kevin Fengler & Mark Cigan

Authors
  1. Joann Conner
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  2. Sirjan Sapkota
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  3. Stéphane Deschamps
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  4. Kevin Fengler
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  5. Mark Cigan
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  6. Peggy Ozias-Akins
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Corresponding author

Correspondence to Joann Conner.

Electronic supplementary material

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Supplementary Table 1 Summary of mapping results. Supplementary material 1 (PDF 65 kb)

Supplementary Table 2 Primer sequence information. Supplementary material 2 (PDF 39 kb)

Supplementary Table 3 BlastN results. Supplementary material 3 (PDF 196 kb)

11032_2016_482_MOESM4_ESM.pdf

Supplementary Table 4 Comparison of read alignment data between Velvet and SPAdes generated contigs. Supplementary material 4 (PDF 202 kb)

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Conner, J., Sapkota, S., Deschamps, S. et al. Targeted sequencing of a complex locus within a polyploid genome using reduced representation libraries. Mol Breeding 36, 60 (2016). https://doi.org/10.1007/s11032-016-0482-y

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