Analysis of Published PCR Primers to Determine Sex of Industrial Hemp Seedlings

Industrial hemp refers to non‐intoxicating strains of Cannabis sativa that produce high levels of cannabidiolic acid (CBDA), the precursor to cannabidiol (CBD). CBD has known health benefits and is being tested for a wide range of potential medicinal uses. When growing hemp for CBD, unfertilized female plants are desired as they produce high amounts of CBDA in their flower organs. Therefore, early identification and elimination of male hemp plants is imperative and allows cultivators to spend fewer resources on plants that won’t produce flower and could possibly pollinate female plants. It has been reported that male cannabis plants can be identified weeks before they show any visual sex features using the polymerase chain reaction (PCR), however the results from reported studies have been inconsistent in identifying male plants. Here, genomic DNA from thirteen hemp seedlings was analyzed using male‐DNA‐specific PCR primer pairs described in the research literature. Three different primer pairs (SCAR323, SCAR119, and MADC2) were used in the analyses. SCAR323 primers have been reported to amplify a male‐specific amplicon of 323 base pairs (bp); SCAR119 have been reported to amplify a male‐associated amplicon of 119 bp; and MADC2 primers have been reported to amplify a male‐associated amplicon of 300 bp and a female‐associated amplicon of 450 bp. PCR analyses showed DNA from all 13 of our tested hemp seedlings produced a single SCAR323 amplicon of around 320 bp, suggesting this primer set might not be useful for sex determination. A SCAR119 amplicon of the expected size was seen using DNA from 6 of the 13 seedlings. The DNA from the same 6 plants produced an amplicon of about 400 bp using the MADC2 primers, while DNA from plants that lacked SCAR119 amplicon showed a MADC2 amplicon of about 550 bp. After seven weeks of growth, the hemp plants revealed the presence of male or female reproductive organs. The presence of male pollen sacs correlated with the SCAR119 amplicon and the MADC2 amplicon of 400 bp, indicating these primer sets could be a useful tools for determining plant sex at the seedling stage.