Genic simple sequence repeat markers for measuring genetic diversity in a native food crop: a case study of Australian Kunzea pomifera F.Muell. (muntries)

Kunzea pomifera F.Muell. (muntries) is a native food crop in Australia that produces spicy apple-flavoured berries. Orchards were established in the 1990s using a variety of wild-collected selections. No published records are currently available of formal plant breeding although the Rivoli Bay selection is the most widely planted. DNA markers are needed to reliably assess genetic diversity and validate plant selections, however genomic resources of K. pomifera are not available. Initially, based on the large MYB gene family, eight MYB gene fragments were amplified and cloned using a degenerate PCR primer strategy. Targeted amplicon sequencing was subsequently used to evaluate diversity of 19 K. pomifera plants and this revealed the presence of single nucleotide polymorphisms and simple sequence repeats (SSRs). Three genic SSR markers were developed from K. pomifera MYB gene fragments and another three genic SSRs were successfully transferred from K. pulchella (Lindl.) A. S. George. A high transfer rate (75%) was obtained due to selecting SSRs in genic regions (introns or intergenic regions) and using Eucalyptus grandis W.Hill sequences to identify conserved regions. The six SSRs were highly polymorphic with observed heterozygosity (Ho) (average Ho = 0.63) and polymorphic information content (PIC) (average PIC = 0.54). Using the six genic SSRs, 15 unique genotypes were identified from the K. pomifera collections in two orchards. Importantly, plants in a new diversity panel were genotyped and suspected mis-labeling events confirmed. The genic SSRs and approach developed will benefit marker assisted selection programs by increasing the proportion of genic markers that could be linked to important horticultural traits.