Conservation of biological resources has become imperative due to the accelerated climate change that challenges the survival of many species of organisms essential to maintain the balance in various ecosystems. Exploration of genetic resources and its diversity is one of the best methods to gain insights to develop a suitable program for conservation and utilization of resources at the brink of extinction and subjected to endangerment. For plants, molecular marker technologies like Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeat (SSR), Inter Simple Sequence Repeat (ISSR) and other such methods can be used to characterize genotypes and acquire information on germplasm and useful traits that can be applied for designing conservation strategies (Odong et al., 2011; Zeinalabedini et al., 2012). Chrysanthemum morifolium, a member of Asteraceae family, is a flowering plant that finds multiple uses as an economically and medicinally important crop – it is the second most cut flower after rose (Kobeissi et al., 2019) and possesses anti-bacterial, anti-viral, anti-inflammatory, anti-mutagenic, anti-neoplastic activities (Deng et al., 2010; Teixeira da Silva, 2003, Liu et al., 2007) attributed to the presence of phenolic and flavonoid components. It is also used in the treatment of common cold, headache, dizziness, wind-heat, etc. Genetic improvement of this allohexaploid plant has been challenging due to its genetic complexity, high levels of heterozygosity, presence of inbreeding depression and self-incompatibility (Anderson, 2006). Also, there are very limited sources of genetic knowledge about the various genotypes that can be put to fruitful use (Feng et al., 2016; Kobeissi et al., 2019). Many genotypes have been developed by cultivation and breeding (Anderson, 2006). According to (Liu et al., 2012), selection of appropriate parents which are distinct genotypes is essential for improvement of suitable cultivars. Continuous asexual propagation by cuttings has led to loss in genetic diversity and has negatively influenced the stability of chemical constituents (Shao et al., 2010). In this article, an effort has been made to understand the use of different molecular markers to assess the genetic diversity of various genotypes in China and Iran.
Assessment using SSR markers (Feng et al., 2016)
In this study, Expressed Sequence Tag (EST) datasets (7300 from GenBank) were used to identify the SSR markers in 32 different genotypes of C.morifolium grown in China. SSR markers are advantageous for detection of genetic diversity as they exhibit genetic co-dominance, they are multi-allelic, spread across the genome and their ease of scorability (Powell et al., 1996). An in-silico method was performed to identify suitable SSR loci using MicroSAtellite (MISA) software. This resulted in the identification of hexa-, penta-, tetra-, tri-, and di nucleotide repeats with a tandem array of core repeats of not less than 4, 4, 5, 7 and 10 respectively. Primers were generated for the loci with proper flanking sequences and PCR amplification enabled the identification of 218 microsatellites from 207 ESTs, out of which 10 ESTs contained more than one loci (Table 1). The primer pairs that produced stable amplification and gave rise to clear separated bands with polymorphism were used for rest of the analysis. This comprised of 17 novel EST-SSR markers and 38 markers that were used in other studies. 98.9% (1306 out of 1319) of the bands amplified exhibited polymorphism. Polymorphic Information Content (PIC) was found to be 0.972 on an average. This suggested that high levels of genetic diversity exists among the genotypes used in the study and that this can be used for creating breeding strategies for the improvement of cultivars for various characteristics. Unweighted Pair Group Method Analysis (UPGMA) with arithmetic mean dendogram divided the genotypes into two groups with significant similarity index (0.584) and Principal Coordinate Analysis (PCoA) was also performed which confirmed the dendogram generated. The clustering was based on the origin and ecological distribution of the genotypes which indicated that adopting in situ conservation strategies would be the best choice for the conservation of germplasm of C.morifolium genotypes as their populations are on decline due to rapid urbanization and a few populations have become rare.
Assessment using morphology, ISSR and SRAP markers (Shao et al., 2010)
In this study, 29 populations of C.morifolium, and 1 each of C.indicum and C.nankingense were used to study 20 different morphological traits and identify ISSR and Sequence-Related Amplified Polymorphism (SRAP). Generally, morphological traits are used to quantify genetic variation and assess the performance of genotypes in their environment though studying these is a labor-intensive process and the data could be affected due to the environmental influences (Fu et al., 2008). Here, the data gathered was standardized and used to determine the Euclidean distance between the different genotypes. Nine traits exhibited high Co-efficient of Variation (CV) values which signified the existence of high levels of genetic diversity in the population being studied. 182 ISSR marker fragments were produced and 81.87% were polymorphic. UPGMA method was used to cluster them and it yielded 3 clusters. Genotypes in northern and southern regions were grouped separately and two outgroup species were grouped in a separate cluster. 243 SRAP marker fragments were amplified and 75.72% were polymorphic. Again, application of UPGMA resulted in three clusters though these were random and not based on regions. These results suggested that in situ conservation areas need to be established for conservation of core populations that possess high levels of genetic diversity.
Assessment using AFLP markers and phenotypic traits (Roein et al., 2014)
This study used 48 germplasms of C.morifolium to assess genetic diversity and population structure of these plants in Iran to design breeding programs to improve various characteristics of the plant. 15 phenotypic traits were studied and Phenotypic Co-efficient of Variation (PCV) and Genotypic Co-efficient of Variation (GCV) were determined. As PCV was greater than GCV, it indicated that phenotypic characters can determine reliable phenotypic and genotypic variances in the study population. These were used for construction of dendogram using Ward’s method which grouped them into four clusters based on different aspects of flower. Further, 2114 AFLP marker loci were identified of which 2099 were polymorphic. Based on this data, Neighbor-Joining method was used to generate a dendogram. This resulted in clustering of genotypes into 6 groups which was confirmed by PCoA. It also showed very low correlation between primer combinations which in turn signified that the markers represent the entire genome and that they can be used in future population studies. Population structure analysis classified the genotypes into 4 clusters that represented a mixed population which could be attributed to history of domestication, breeding, resource exchange and heterozygosity levels and self-incompatibility. (Anderson 2006, Zhang et al., 2010; Zhao et al., 2010). As the clustering obtained by phenotyping and molecular analysis were different, it shows that interspecific hybridization could be explored to come up with new varieties with remarkable characteristics (Cheng et al., 2011). Also, the high levels of polymorphism identified, suggest that this data can be utilized for germplasm conservation.
Assessment using SCoT and SSR markers (Kobeissi et al., 2019)
This study used 32 genotypes to study SSR and Start Codon Targeted (SCoT) polymorphism. Of the 30 SSR primers used, 25 were polymorphic. It resulted in the amplification of 64 alleles with an average PIC value of 0.37. Primers with more number of alleles had a greater PIC value which indicated high levels of genetic variation. 7 out of 8 SCoT primers were polymorphic and produced 63 bands of which 61 were polymorphic with an average PIC value of 0.34. SplitsTree software was used for clustering analysis of both the results which gave rise to 4 groups. From the PIC values obtained, it was inferred that the efficiency of either methods is almost equivalent. The cophenetic coefficient, which determines whether the dendogram preserves the pairwise distances between the original data points or not, is also equal (SSR – 0.71 and SCoT – 0.68). Thus, the data suggests that conservation of the genotypes can be performed in addition to identification of genes responsible for horticulturally important traits like flower color and shape, flowering time and disease and insect resistance.
It is evident from the above described studies that in-situ conservation of various genotypes of C.morifolium would be the best strategy to conserve the germplasm of this economically and medicinally important plant. The pursuit of finding ways to conserve the germplasm started with methods like Random Amplification of Polymorphic DNA (RAPD) and AFLP which yielded results giving only a minimal understanding of the genetic diversity. The field has hence progressed to the use of relatively high-throughput methods like SCoT which gave knowledge about the specific conserved regions of DNA and detected polymorphism of higher levels (Collard and Mackill, 2008; Hajibarat et al., 2015). Conservation genetics goes hand-in-hand with better understanding of the environmental factors which influence the survival of the species, phenotypes that correlate with the changes in the biological state of the members of the species and finally but most importantly understanding and cataloging of all genotypes that are associated with these phenotypes and other traits that govern or control diversity. For this purpose, large scale genome-wide association studies (GWAS) to screen and map for critical genotypes would be essential. Such large-scale projects could be achieved by fostering international collaborations under frameworks or regional consortia to enhance the utilization of genetic resources as laid down by the Convention on Biological Diversity (CBD). This would also benefit academia and industry by better communication between scientists and breeders worldwide.
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