Microhaplotype genotyping-by-sequencing of 98 highly polymorphic markers in three chestnut tree species
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TECHNICAL NOTE
Microhaplotype genotyping‑by‑sequencing of 98 highly polymorphic markers in three chestnut tree species Benoit Laurent1 · Clément Larue1,2 · Emilie Chancerel1 · Erwan Guichoux1 · Rémy J. Petit1 · Teresa Barreneche3 · Cécile Robin1 · Olivier Lepais1 Received: 27 January 2020 / Accepted: 4 June 2020 © Springer Nature B.V. 2020
Abstract Chestnut species have large ecological, cultural and economic importance. Developing genetic markers for these species is of interest for conservation, breeding or evolutionary studies. We designed 192 primer pairs targeting microsatellites detected in the Castanea mollissima reference genome and tested them on C. sativa and C. crenata. We PCR amplified 3 × 50 microsatellites in 106 chestnut trees. Microhaplotype calling accounting for all polymorphisms resulted in a total of 98 high confidence polymorphic markers. Mean number of haplotypes per marker was 9.05 with respectively 71%, 12% and 16% of the variation corresponding to microsatellite variation in repeats number, SNP within the repeat motif and SNP or INDEL in the flanking sequence. Overall, the simple protocol described here generated a powerful multilocus genetic dataset for chestnut genetic investigations. Keywords Castanea sativa · C. crenata · C. mollissima · Microsatellites · SNP · INDEL · SSR-seq
Introduction The genus Castanea Mill. (chestnuts) comprises at least eight interfertile species (The Plant List 2013) including Castanea dentata (Marshall) Borkh from North America, C. sativa Mill. from Europe, and C. crenata Siebold & Zucc and C. mollissima Blume from Asia (Pereira-Lorenzo et al. 2012). Very closely related to oaks (Kremer et al 2012), another Fagaceae genus, chestnuts are keystone multipurpose trees that provide habitat for wildlife, edible nuts for both wildlife and human, timber and tannins, and play important cultural roles in some human societies (PereiraLorenzo et al. 2012; Powell et al. 2019). But chestnuts have endured one of the greatest environmental disasters that led to the functional extinction of C. dentata in North America and to important reduction of C. sativa population in Europe as a consequence of invasive pathogens (Desprez-Loustau * Benoit Laurent [email protected] 1
Univ. Bordeaux, INRAE, BIOGECO, 33610 Cestas, France
2
INVENIO, Maison Jeannette, 24140 Douville, France
3
Univ. Bordeaux, INRAE, BFP, 33140 Villenave d’Ornon, France
et al. 2007; Powell et al. 2019). In contrast, Asian chestnuts harbor quantitative resistance to these pathogens, allowing resistance breeding (Barreneche et al. 2019). Improved knowledge on the genetic diversity of chestnuts requires the development of numerous, reliable and polymorphic molecular markers. SSRs have remained popular markers given their high polymorphism, reproducibility, transferability and ease of detection (Guichoux et al. 2011; Lepais and Bacles 2011). Hence, the recent development of sequence-based microsatellite genotyping approaches (e.g. Vartia et al. 2016) has raised much interest. The aim of this
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