Additional file 1 of High-throughput methods to identify male Cannabis sativa using various genotyping methods

Additional file 1: Supplemental Figure 1. High resolution melting profiles on selected Cannabis sativa male and not male samples from the SexID Steep Hill high throughput testing sample set of MADC2 primer set showing multiple non-male melt profile genotypes. The samples that are shown in red represent male calls with a Tm melting peak at 82°C, whereas the other colors (purple, blue, maroon) represent non-male genotypes with variable target melting peak. Supplemental Figure 2. High resolution melting profiles of a region upstream of MADC2. This analysis was performed using primers designated, -403 Fwd and -237 Rev, which target an upstream region of MADC2 on the proposed Y-chromosome. Curves with an inflection point at 78°C are indicative of a male genotype, and the other curves present represent the various non-male genotypes of the homologous region. Supplemental Figure 3. Genomic Scaffold and Amplicon Alignment on JL_Father’s Y-Chromosome. The above alignment shows the location of where the MADC2 region lies on a 1Mbp region of the JL_Father’s Y-chromosome scaffold. The highlighted region near 523Mbp shows the MADC2 probe binding site and the black bar represents the amplicon region. Supplemental Figure 4. Alignment of 42 male amplicon sequences to the MADC2 genomic region in the JL_Father genome that shows a high conservation between male genotypes of diverse drug-type and fiber-type cultivars. The highlighted region represents the probe binding site that is also conserved between all the genotypes. 011623 contig from PBBK aligned to 1Mbp Y_000295F contig from JL father with TaqMan probe highlighted 2) MADC2 aligned to Y contig from Jamaican Lion Father with TaqMan probe highlighted examined. Supplemental Figure 5. A. Preliminary Analysis of Loop mediated Isothermal PCR for Male (yellow) and not Male (Pink) identification. B. Twist Dx Assay of Male and Not Male DNA samples. Supplemental Figure 6. Example phenotypes of representative cannabis plants from the 158 European cultivars germinated, propagated, and tested at the CRAG. A. Male Cannabis plant expressing a male sex phenotype of immature staminate bearing flowers. B. Selected female trial plants grown in greenhouse post sex testing. C. Non male Cannabis plant expressing an alternative female sex phenotype of maturing pistallate bearing flowers with partial staminate bearing. Supplemental Table 1. SexID qPCR Results for 8595 Cannabis Samples – This table shows the Cycle quoient values observed for the SexID assay using the MADC2 Fwd + MADC2 Rev + MADC2 Taqman Probe. The sample IDs are representative of the Genkit lots that the samples as they were received. Supplemental Table 2. Results of 158 European Cultivars tested by HRM using the MADC2 primers. 71 out of 158 or 45% of the population was measured with the melting peak at 82°C indicative of the male MADC2 genotype, and variable target melting peaks were observed for the remaining 186 test samples indicative of a not-male MADC2 genotype. Supplemental Table 3. Cycle Thresholds/Quotients for MADC2 and the TC Cannabinoid Synthase Positive Control qPCR Primers. The average Cq Value describes the cycle at which a positive detection of the target amplification occurred. The Population standard deviation describes the standard deviation observed in that Cq value when ran on 5047 non-male samples for the MADC2 target and 8200 samples for the positive control. On analyzing our qPCR results we see an avg. Ct value of 26 with a standard deviation of 3 for our tissue control and MADC2 qPCR assays suggesting they are reproducible and within our acceptable range of median Cq +/- 10 for a specific and reproducible qPCR assay. Supplemental Table 4. A Validation set of 20 samples (M1-20) with two template controls (M21-22) and a no template control was tested with the SexID qPCR assay as a part of early assay validation. The validation plants were grown from seed, and phenotyped (Faux et al. 2014). The observed sex phenotype results were recorded and compared against the results obtained by the SexID assay. Plants with a positve MADC2 signal corresponded with plants with an observed male phenotype (staminate bearing flowers).

附加文件1：补充图1。本图展示了基于SexID Steep Hill高通量检测样本库中MADC2引物组的部分大麻（Cannabis sativa）雄性与非雄性样本的高分辨率熔解曲线（High Resolution Melting，HRM），可见多种非雄性熔解曲线基因型。红色样本为雄性判定样本，其解链温度（Tm）熔解峰位于82℃；其余颜色（紫色、蓝色、栗色）样本为非雄性基因型，其靶序列熔解峰存在差异。补充图2：MADC2上游区域的高分辨率熔解曲线。本分析使用命名为-403 Fwd和-237 Rev的引物，靶向拟议Y染色体上MADC2的上游区域。熔解曲线在78℃处出现拐点的样本为雄性基因型，其余曲线则对应该同源区域的各类非雄性基因型。补充图3：JL_Father的Y染色体基因组支架与扩增子比对。本比对展示了MADC2区域在JL_Father Y染色体支架1Mbp区段上的位置。523Mbp附近的高亮区域为MADC2探针结合位点，黑色条带代表扩增子区域。补充图4：42条雄性扩增子序列与JL_Father基因组中MADC2基因组区域的比对结果，显示不同药用型和纤维型大麻品种的雄性基因型之间具有高度保守性。高亮区域为探针结合位点，在所有基因型中均保守。本次比对包含：① 来自PBBK的011623重叠群与JL_Father的1Mbp Y_000295F重叠群的比对（TaqMan探针已高亮标注）；② MADC2序列与Jamaican Lion Father的Y染色体重叠群的比对（TaqMan探针已高亮标注）。补充图5：A. 用于雄性（黄色曲线）与非雄性（粉色曲线）鉴定的环介导等温扩增（Loop-mediated Isothermal Amplification，LAMP）初步分析结果；B. 雄性与非雄性DNA样本的Twist Dx检测结果。补充图6：从在CRAG完成萌发、扩繁与检测的158份欧洲栽培大麻品种中选取的代表性植株表型。A. 雄性大麻植株，表现为未成熟的雄花（具雄蕊群）表型；B. 经性别鉴定后在温室中培育的选定雌性试验植株；C. 非雄性大麻植株，表现为兼具部分雄花的成熟雌花（具雌蕊群）表型。补充表1：8595份大麻样本的SexID定量PCR（quantitative Polymerase Chain Reaction，qPCR）结果。本表格展示了使用MADC2 Fwd + MADC2 Rev + MADC2 TaqMan探针进行SexID检测时得到的循环阈值（Cq）值。样本ID对应样本接收时所属的Genkit批次。补充表2：使用MADC2引物通过高分辨率熔解（HRM）检测158份欧洲栽培大麻品种的结果。158份样本中有71份（占比45%）的熔解峰位于82℃，对应雄性MADC2基因型；其余186份测试样本的靶序列熔解峰存在差异，对应非雄性MADC2基因型。补充表3：MADC2与TC大麻素合酶阳性对照qPCR引物的循环阈值（Cq）。平均Cq值指靶序列扩增阳性检测出现时的循环数；群体标准差指针对MADC2靶序列检测5047份非雄性样本、针对阳性对照检测8200份样本时，所得Cq值的标准差。对本研究qPCR结果的分析显示，组织对照与MADC2 qPCR检测的平均Cq值为26，标准差为3，表明该检测具有良好重复性，且处于我们设定的特异性与重复性qPCR检测可接受的中位Cq±10范围内。补充表4：作为早期检测验证的一部分，使用SexID qPCR检测了一组验证样本（共20份，编号M1-M20），同时设置2份模板对照（M21-M22）与1份无模板对照。验证植株均由种子萌发培育，并完成表型鉴定（Faux等，2014）。记录观测到的性别表型结果，并与SexID检测结果进行比对。MADC2信号阳性的植株与观测到的雄性表型（具雄花）植株完全对应。