Development of EST-SSR markers in Larix principis-rupprechtii Mayr and evaluation of their polymorphism and cross-species amplification

Key messageWe developed and validated a new set of polymorphic EST-SSR markers across Larix species and evaluated genetic diversity in a clonal seed orchard of Larix principis-rupprechtii Mayr.AbstractPrince Rupprecht's larch (Larix principis-rupprechtii Mayr) is an important deciduous conifer species that has been widely planted in North China due to its major ecological and commercial value. However, the paucity of genomic data and robust molecular markers has hampered genetic and genomic studies. Here, transcriptome sequencing of L. principis-rupprechtii callus was performed using the Illumina platform. By mining 43,753 assembled unigenes, 1418 expressed sequence tag-simple sequence repeats (EST-SSRs) derived from 1300 unigenes were identified. A total of 1065 primer pairs were designed and 240 of these selected at random for validation among 24L. principis-rupprechtii individuals. Of these, 52 primer pairs were scored as polymorphic, and 20 polymorphic EST-SSR markers were further selected to genotype 66 clones deployed in a clonal seed orchard of L. principis-rupprechtii; these exhibited a moderate level of genetic diversity, as reflected by the mean values of the number of alleles (Na=3.85) and polymorphism information content (PIC=0.424). Additionally, all of the 20 EST-SSR markers could amplify clear and stable bands across three related Larix species. A neighbor-joining (NJ) clustering tree uniquely distinguished 66 clones and distributed these into three main clusters, which was further validated by principal coordinate analysis (PCoA). The developed EST-SSR markers will serve as valuable tools for future genetics and breeding research in larch species. The evaluation of genetic diversity among 66 clones will provide important information for efficient management and utilization of genetic material in L. principis-rupprechtii breeding programs.