Analysis of the NMI01 Marker for a Population Database of Cannabis Seeds

Abstract

We have analyzed the distribution of genotypes at a single hexanucleotide short tandem repeat (STR) locus in a Cannabis sativa seed database along with seed-packaging information. This STR locus is defined by the polymerase chain reaction amplification primers CS1F and CS1R and is referred to as NMI01 (for National Marijuana Initiative) in our study. The population database consists of seed seizures of two categories: seed samples from labeled and unlabeled packages regarding seed bank source. Of a population database of 93 processed seeds including 12 labeled Cannabis varieties, the observed genotypes generated from single seeds exhibited between one and three peaks (potentially six alleles if in homozygous state). The total number of observed genotypes was 54 making this marker highly specific and highly individualizing even among seeds of common lineage. Cluster analysis associated many but not all of the handwritten labeled seed varieties tested to date as well as the National Park seizure to our known reference database containing Mr. Nice Seedbank and Sensi Seeds commercially packaged reference samples.

Genetic structure of Cannabis sativa var. indica cultivars based on genomic SSR (gSSR) markers: Implications for breeding and germplasm management

“…Many studies have been performed with different types of molecular markers to evaluate the diversity among accessions in C. sativa, revealing that the crop is genetically very diverse (e.g., Shirley et al, 2013;Gao et al, 2014;Sawler et al, 2015). Amazingly, there are very few studies evaluating the intra-varietal diversity of C. sativa, which is very important for selection, breeding and developing uniform cultivars.…”

“…indica cultivars had four or more (up to eight) individuals, which is a limited number of cultivars to draw general conclusions. Shirley et al (2013) using a single SSR marker found several genetic profiles in DNA extracted from individual seeds of each of several C. sativa var. indica accessions, with an important level of heterozygous loci.…”

“…Among the genetic markers used to evaluate genetic diversity in Cannabis, SSRs (also called short tandem repeats, STS) have proved to be highly polymorphic (Gilmore et al, 2003;Howard et al, 2008;Allgeier et al, 2011;Shirley et al, 2013;Gao et al, 2014;Soler et al, 2016). Cannabis sativa genomic SSRs, which in general are more polymorphic than expressed sequence tag SSRs (EST-SSRs) (Eujayl et al, 2011;Muñoz-Falcón et al, 2011), can be obtained from previously published reports (Gilmore et al, 2003;Soler et al, 2016) or developed de novo from the available genome sequence of C. sativa (van Bakel et al, 2011).…”

Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications

“…The use of DNA from embryos extracted via the developed protocol enabled successful SSR characterization from 2n zygotes. Herein, we found that C. sativa presents a certain degree of heterozygosis; thus, DNA extraction from whole seeds (achenes) could theoretically result in the amplification of three alleles when the maternal plant is heterozygous and the allele carried by the male gamete is different from the maternal alleles . However, we did not find any SSR profiles with three alleles, indicating a lack of contamination in the embryo tissues resulting from the presence of tissue from the seed testa or achene pericarp.…”

“…The 11 genomic SSR primers used in this study have previously been used to detect many alleles and represent an addition to the available SSR markers for the genetic characterization of Cannabis . The fact that genomic SSR markers are usually highly polymorphic compared with EST‐SSRs is likely a primary factor in this high degree of polymorphism and suggests that the development of further SSRs for identification purposes could benefit from the availability of the genome sequence .…”