Abstract
Encephalartos Lehm. (Zamiaceae) is the largest cycad genus (65 species) endemic
to Africa, and it is the most endangered of all cycads. Species within the genus are
under significant threat from habitat loss and poaching from wild populations. Due to
their slow growth rate and the recalcitrant nature of their seeds, living collections of
botanical gardens are one of the best places to help conserve these species. Since
cycads are dioecious, understanding the ratio between megasporangiate (♀) and
microsporangiate (♂) cycads within ex-situ and in-situ populations is important for
effective reproduction. It is also important to understand the balance between these
sexes for trade purposes, as megasporangiate plants are considered more valuable
than microsporangiate plants. Sex determination in the Cycas L. genus has been
proven to be successful by Liu et al. (2022), where it was discovered that two MADSbox
transcription factor genes could be used to determine the sex of cycads. The first
gene (CYCAS_034085) was identified on chromosome 8 of microsporangiate plants,
while the CYCAS_010388 gene was found to be on chromosome 2 of both
megasporangiate and microsporangiate plants. This study aims to determine if the
CYCAS_034085 gene can be used to identify the sex of Encephalartos individuals
using a simple PCR-based test. A total of 88 accessions representing six
Encephalartos species (Encephalartos altensteinii Lehm., Encephalartos latifrons
Lehm., Encephalartos lebomboensis Verd., Encephalartos princeps
R.A.Dyer, Encephalartos trispinosus R.A.Dyer, and Encephalartos woodii Sander.)
were collected from the living collection of Kirstenbosch National Botanical Garden,
South Africa. Among them were 43 known microsporangiate accessions, 30 known
megasporangiate accessions, and 15 accessions of undetermined sex. DNA
extraction followed the 10x CTAB method, with subsequent PCR amplification using
XIII
the primers developed to amplify the CYCAS_034085 and CYCAS_010388 genes.
Statistical analysis involved the recording of the sex data, descriptive statistics, t-tests
and regression tests. Performance metrics (accuracy, precision, specificity, and
sensitivity) were also calculated for the PCR-based sex identification method by
utilizing a 2 × 2 confusion matrix. The results revealed that primer combination 6265FZamia
+ 6745R-Zamia was able to amplify the CYCAS_034085 gene in the
Encephalartos microsporangiate accessions. It was also found that among the 73
known-sex accessions, 38 were identified as microsporangiate plants, and 25 were
determined to be megasporangiate plants. The accuracy of the PCR-based method
was calculated to be 86.3% – demonstrating that PCR could effectively differentiate
between microsporangiate and megasporangiate plants. In conclusion, PCR-based
sex determination can be a valuable tool for cycad conservation and management
efforts, aiding in the preservation of these endangered plants.