Developing and Testing Molecular Markers in Cannabis sativa (Hemp) for Their Use in Variety and Dioecy Assessments

Cannabis sativa (2n = 2x = 20) is a popular species belonging to the Cannabaceae family. Despite its use for medical, recreational, and industrial purposes as well as its long history, the genetic research on this species is in its infancy due to the legal implications and the prohibition campaigns. The recent legalization of Cannabis in many countries along with the use of genomics boosted the approaches aimed at marker-assisted selection, germplasm management, genetic discrimination, and authentication of cultivars. Nonetheless, the exploitation of molecular markers for the development of commercial varieties through marker-assisted breeding schemes is still rare. The present study aimed to develop an informative panel of simple sequence repeat markers to be used for the genotyping of high breeding value C. sativa lines. Starting from 41 nuclear SSR designated by in silico analyses, we selected 20 highly polymorphic and discriminant loci that were tested in 104 individuals belonging to 11 distinct hemp varieties. The selected markers were successful in accessing homozygosity, genetic uniformity, and genetic variation within and among varieties. Population structure analysis identified eight genetic groups, clustering individuals based on sexual behaviors (dioecious and monoecious) and geographical origins. Overall, this study provides important tools for the genetic characterization, authentication, conservation of biodiversity, genetic improvement and traceability of this increasingly important plant species.

Insights into opium poppy (Papaver spp.) genetic diversity from genotyping-by-sequencing analysis

Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and a versatile model system to study secondary metabolism. However, our knowledge of its genetic diversity is limited, restricting utilization of the available germplasm for research and crop improvement. We used genotyping-by-sequencing to investigate the level of genetic diversity and population structure in a collection of poppy germplasm consisting of 91 accessions originating in 30 countries of Europe, North Africa, America, and Asia. We identified five genetically distinct subpopulations. Although the accessions were from geographically diverse regions, no strong association was detected between geographic origin and subpopulation at regional and sub-regional levels. However, most accessions obtained from the same country were grouped together as genetically distinct, likely a consequence of the restriction on movement of poppy germplasm. Phylogenetic analysis identified clades that were largely consistent with the subpopulations detected. Clades could be differentiated broadly based on capsule dehiscence, plant stature and seed weight, traits believed to be associated with poppy domestication. Our study determined that the genetically diverse collection was likely composed of both wild and cultivated forms. This has direct implications for germplasm management and utilization of the available diversity for genetic improvement of poppy.

Genetic diversity of eucalypts for germplasm conservation in Forest Area with the Special Purpose of Mount Bromo, Karanganyar, Indonesia

Rahayu, Fatimah, Wiwoho J, Firdaus SU, Pujiyono, Marimin, Arianto DP, Pramono A. 2021. Genetic diversity of eucalypts for germplasm conservation in Forest Area with the Special Purpose of Mount Bromo, Karanganyar, Indonesia. Biodiversitas 22: 4223-4235. As a repository of a gene pool, eucalypts germplasm enriches biodiversity, maintains ecosystem sustainability, and aids in conservation. Therefore, this study aims to analyze the genetic diversity of eucalypts (Corymbia and Eucalyptus) for the development of germplasm conservation in Forest Area with the Special Purpose (KHDTK) Bromo Forest, Karanganyar, Indonesia. In this study, 14 simple sequence repeat (SSR) markers were used to assess the genetic diversity among 20 accessions (Corymbia and 5 Eucalyptus species) from Central and West Java. Subsequently, the genetic parameters were measured and a phylogenetic tree was constructed. The result showed that the SSR markers have high variability, although they belong to different genera. Furthermore, the genetic diversity showed 49 alleles with an average of 3 alleles per locus, while the polymorphism information content (PIC) values were 0.55. There were 4 SSR markers (EMBRA13, EMBRA8, EMCRC11, and EMBRA2) with high PIC value, while the gene diversity (He) of Corymbia and 5 Eucalyptus showed a low level of genetic diversity. The genetic relationship and population structure were divided into genera Corymbia and Eucalyptus. For further application, the eucalypt cultivated in the KHDTK Bromo Forest can contribute as a reference set and 14 SSR markers as a potential marker in combination with morphological characterization to generate a database for germplasm management and conservation.

Patterns of Genetic Variation in a Soybean Germplasm Collection as Characterized with Genotyping-by-Sequencing

Genomic characterization is playing an increasing role in plant germplasm conservation and utilization, as it can provide higher resolution with genome-wide SNP markers than before to identify and analyze genetic variation. A genotyping-by-sequencing technique was applied to genotype 541 soybean accessions conserved at Plant Gene Resources of Canada and 30 soybean cultivars and breeding lines developed by the Ottawa soybean breeding program of Agriculture and Agri-Food Canada. The sequencing generated an average of 952,074 raw sequence reads per sample. SNP calling identified 43,891 SNPs across 20 soybean chromosomes and 69 scaffolds with variable levels of missing values. Based on 19,898 SNPs with up to 50% missing values, three distinct genetic groups were found in the assayed samples. These groups were a mixture of the samples that originated from different countries and the samples of known maturity groups. The samples that originated from Canada were clustered into all three distinct groups, but 30 Ottawa breeding lines fell into two groups only. Based on the average pairwise dissimilarity estimates, 40 samples with the most genetic distinctness were identified from three genetic groups with diverse sample origin and known maturity. Additionally, 40 samples with the highest genetic redundancy were detected and they consisted of different sample origins and maturity groups, largely from one genetic group. Moreover, some genetically duplicated samples were identified, but the overall level of genetic duplication was relatively low in the collection. These findings are useful for soybean germplasm management and utilization.

Quick Identification of Genetic Provenances and Best Practice Woodland Management of Chinese Pine (Pinus tabuliformis)

Chinese pine (Pinus tabuliformis) is one of the most widespread, ecologically and economically important tree species in North China. In this study, we analyzed and compared the genetic diversity and population structure of 158 individuals from 17 populations of P. tabuliformis, by group III, a new mitochondrial marker system (nad1-2, nad4-3, nad5-1 and nad7-1) with two other groups of marker systems. In contrast to the conservation in the evolution of the mitochondrial sequence, the new mitochondrial marker system of P. tabuliformis shows as extremely high polymorphism as 25, whose haplotypes are more than four times of the group I marker system (nad1-2, nad4-3, and nad5-1) as 8 haplotypes. Although the group II, nad7-1 (19 haplotypes), showed high resolution in the provenance identification of P. tabuliformis, the new mitochondrial marker system is more accurate for detection of specific groups like HL, WT and NS and powerful to differentiate populations between GD and SS. The results suggested that the new mitochondrial marker system is as high as the resolution of GBS (genotype by seqencing). It is much more available and will be of great help to provenance identification and molecular assisted breeding of P. tabuliformis. This study will make theoretical foundation for the following studies on the evaluation, cultivation and germplasm management of P. tabuliformis populations and aid the breeding, biodiversity and conservation programs of forest species.

Genetic diversity assessment of ancient mulberry (Morus spp.) in Lebanon using morphological, chemical and molecular markers (SSR and ISSR)

Lebanon has ancient mulberry trees which are the remnants of the abundant orchards that dominated its lands during the nineteenth century. Lebanese mulberry germplasm has not been assessed yet. This study aims to collect local old rainfed mulberry accessions from different geographical regions and assess their diversity by using morphological and molecular markers (SSR and ISSR). Genetic diversity of 70 accessions of mulberry were evaluated by using 27 morphological traits. The dendrogram based on the morphological attributes showed a relative separation of the different accessions based on fruits color and taste. Molecular analysis was performed for the accessions by using selected SSR and ISSR primers. The primers marked a high discriminating power (0.7 to 0.89). The dendrogram constructed on the base of UPGMA method showed 13 different groups. The clustering patterns indicated no location nor local name specificity among mulberry accessions. The combination of SSR and ISSR primers was informative for estimating the extent of mulberry genetic diversity. It can be concluded that there is a high level of genetic diversity within mulberry trees in Lebanon. These results will be useful for mulberry germplasm management in terms of biodiversity protection and as a valuable source of gene pool for crop improvement.

Genetic characterisation and population structure analysis of Anatolian figs (Ficus carica L.) by SSR markers

The common fig (Ficus carica L.) is a tree species and is one of the oldest fruit trees cultivated in Turkey. The Western Anatolian region of Turkey produces nearly a quarter of the total dried fig production of the world. This region also harbours a rich fig germplasm. However, so far this germplasm has remained largely uncharacterised. In this study, using 14 simple sequence repeat (SSR) primer pairs, we analysed a total of 310 fig accessions from six different regions of Anatolia. In structure analyses, Western Anatolian accessions formed a group, which was correlated with their geographical distribution. In addition, 7 identical, 36 synonymous, and 22 homonymous fig accessions were identified. In multilocus lineages (MLLs) analysis a total of 54 accessions were matched to different accessions as clone assignment. The results will facilitate future germplasm management and breeding efforts in this economically important tree species by identifying genetic diversity, genetic relations and characterising the structure of studied populations and accessions.

Comprehensive Characterization and Validation of Chromosome-Specific Highly Polymorphic SSR Markers From Pomegranate (Punica granatum L.) cv. Tunisia Genome

The simple sequence repeat (SSR) survey of ‘Tunisia’ genome (296.85 Mb) identified a total of 365,279 perfect SSRs spanning eight chromosomes, with a mean marker density of 1,230.6 SSRs/Mb. We found a positive trend in chromosome length and the SSR abundance as marker density enhanced with a shorter chromosome length. The highest number of SSRs (60,708) was mined from chromosome 1 (55.56 Mb), whereas the highest marker density (1,294.62 SSRs/Mb) was recorded for the shortest chromosome 8 (27.99 Mb). Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. Across the eight chromosomes, the class III had maximum number of SSR motifs (301,684, 82.59%), followed by the class II (31,056, 8.50%) and the class I (5,003, 1.37%). Examination of the distribution of SSR motif types within a chromosome suggested the abundance of hexanucleotide repeats in each chromosome followed by dinucleotides, and these results are consistent with ‘Tunisia’ genome features as a whole. Concerning major repeat types, AT/AG was the most frequent (14.16%), followed by AAAAAT/AAAAAG (7.89%), A/C (7.54%), AAT/AAG (5.23%), AAAT/AAAG (4.37%), and AAAAT/AAAAG (1.2%) types. We designed and validated a total of 3,839 class I SSRs in the ‘Tunisia’ genome through electronic polymerase chain reaction (ePCR) and found 1,165 (30.34%) SSRs producing a single amplicon. Then, we selected 906 highly variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across multiple draft genomes of pomegranate, which provided us a subset of 265 highly polymorphic SSRs. Of these, 235 primers were validated on six pomegranate genotypes through wet-lab experiment. We found 221 (94%) polymorphic SSRs on six genotypes, and 187 of these SSRs had ≥ 0.5 PIC values. The utility of the developed SSRs was demonstrated by analyzing genetic diversity of 30 pomegranate genotypes using 16 HvSSRs spanning eight pomegranate chromosomes. In summary, we developed a comprehensive set of highly polymorphic genome-wide SSRs. These chromosome-specific SSRs will serve as a powerful genomic tool to leverage future genetic studies, germplasm management, and genomics-assisted breeding in pomegranate.

Conversion of Metabolomic Data to Genomic Marker for Genetic Characterization of Piper betle L. Chemotypes: A Review

The Indian perfumery industry is shifting towards natural product. In India including West Bengal betel leaves produces high quality essential oil as well contribute to Indian fresh vegetable export. The crop is cultivated from stem cutting and suffers from authenticity problem of cultivars with redundant names. The genetic screening and characterization of cultivars were not initiated due to unavailability of reliable markers. The essential oil metabolomic study identified some polar and non-polar volatile signature compounds. Metabolomic profiling of cultivars is not consistent due to seasonal variation in the production of secondary metabolites and ignorance in marking of unique trace discriminatory compounds. In this paper gene ontogeny study was made on major signature compounds to obtain the complete coding sequence (CDS) of the aroma-genes. The CDS information of aroma-genes could be utilized to construct robust DNA markers to eradicate authentication problem and germplasm management of Piper. The direct genomic analysis could supersede the metabolome profiling. Information available in NCBI, DDBJ and EMBL database were searched for gene ontogeny study utilizing available metabolomic data. The information and method depicted could be efficiently utilized for Piper genomics. Aroma-scientists could apply this technique to validate promising cultivars and competent germplasm management.