Universal Rice Primer (URP) and Start Codon Target (SCoT) Markers in Studying Population Structures and Genetic Variation in Ferula AssafoetidaL. Accessions

Ferula assafoetidais an herbaceous, annual and monocarpic genus of the Apiaceae family. So far, there has been common usage of Ferula oleo resinous gum in food and herbal medicines.The origin of F.assafoetidais can be traced back into the steppes of Iran and some reigns of Afghanistan with an extended distribution. Despite the economic value and therapeutic importance of F.assafoetida, only a few studies have reported on the genetic capacity of this herb.The present study was carried out on a set of 90 individual plants belonging to different populations of Ferula assafoetida L. via the start codon target marker (SCoT) and the universal rice primer (URP) markers. Twelve SCoT and twelve URP primers generated 192 and 149 polymorphic fragments, while having 16 and 12.41 fragments respectively on average per primer. The Polymorphism information content (PIC) for URP primers and ScoT ranged from 0.31 to 0.43 and 0.34 to 0.44 respectively, which indicated a good eciency forboth markers. The diversity indices including heterozygosity (He), percentage of polymorphic bands (PPB), Shannon’s information index (I) and marker index (MI) were calculated based on the SCoT and URP data.The results revealed that SCoT primers were more ecient than URP primers in identifying genetic diversity within populations. Neighbor joining (Nj), as a base for clustering, classied 90 accessions into 5 and 6 groups using SCoT and URP data respectively. Moreover, the combined data (SCoT+URP) succeeded in classifying all accessions into 6 groups, although this did not correspond with the geographical distribution of accessions. Structure analysis divided 90 genotypesinto 5 subpopulations using SCoT and URP markers, whereas the combined data (SCoT+URP) divided the accessions into 6 subpopulations, which conrmed the classication achieved by the Nj method. Principal coordinate analysis (PCoA) corroborated these conclusions. According to the analysis of molecular variance) AMOVA(, a high percentage of genetic diversity was found within the species, suggesting a rich diversity of germplasm

The only way to regenerate Asafoetida is through the production and distribution of seeds. Each owering stem of Asafoetida produces a lot of seeds in umbrella-like clusters. Asafoetida seeds are light and can be scattered well by the wind. Asafoetida seed length is 9 to 12 mm and its width is 5 to 7.5 mm. Its 1000seed weight is 12.5 grams, and its hectoliter weight is 196.4 grams per cubic centimeter (Zare et al. 2011).
Since this plant is a valuable source of income for a large number of villagers and farmers in Iran, local people collect the seeds and sprinkle it on land in pastures. This act is supported by organizations which safeguard biodiversity not only to avoid its extinction but also to allow sensible harvests from its populations (Zare et al. 2011).
The plant Asafoetida (Ferula assafoetida L.) is used to make dried latex (gum oleo resin), which is exuded from the rhizome and stems of this plant. In Iran, Asafoetidaoleo gum resin is locally called Khorakoma, Anghouzeh or Anguzakoma (Iranshahy and Iranshahi 2011). Asafetida has 3 primary parts, i.e. gum (25%), resin (40-64%) and essential oil (10-17%) (Takeoka 2001). Incisions on the roots or the cutting of the plant stems are the most common methods to obtain oleo-gum-resin. Exudates (oleo-gum-resin) are usually left to dry, are processed and then prepared for export. Assafoetida occurs in two principal forms, mass and tears. The mass type is the most popular form on the market, however (Upadhyay 2017). As a rangeland plant,it is important because of the substances that are extracted from the roots of this plant, i.e. compounds which have medicinal and pharmaceutical properties (Iranshahy and Iranshahi 2011(. For millennia, Assafoetida has been used to cure a wide variety of ailments, including urinary, gastrointestinal, and respiratory diseases, epilepsy, asthma, stomachache, atulence, intestinal parasite, poor metabolism, and in uenza, as well as an aphrodisiac, an emmenagogue and to treat snake and insect bites, although the best documented folk usage has been on intestinal worm infections. Antiviral (HSV, HRV, H1N1, HIV), antispasmodic, hypotensive and anti-diabetic behaviors of Assafoetida have resulted from several trials (Bahramia et al. 2013; Iranshahy and Iranshahi 2011). In Iranian traditional medicine, F.assafoetida is considered to be sedative, analgesic, carminative, antispasmodic, diuretic, anthelmintic, expectorant digestive, expectorant, laxative and aphrodisiac (Bagheri et al. 2011;Sadraei et al. 2003;Khajeh et al. 2004). Iran has a wide variety of natural ecosystems that make it one of the world's most diverse sources of plant genetic resources. Its wide range of diversity illustrates its signi cant capacity in selection, conservation and utilization of genetic resources. While this herb is widely spread in Iran, the extent of its genetic diversity remains a question. Relevant ndings in this area can pave the way for germplasm management initiatives and changes in plant characteristics (Kalia et al. 2014). Molecular markers that are DNA-based happen to be the most useful means of describing genetic variance. Different parameters exist for molecular marker methods and assist in structural analysis as well as in the study of genetic variation. In this context, molecular marker approaches have improved through time and developed further capacities to better determine genetic diversity, population composition and phylogenetic relationships. Advances in genomic tools have produced a wide variety of new marker approaches in recent years, including several new DNA-based marker systems and gene-targeted markers (Poczai et al. 2013). DNA markers are also commonly used as a valid way of measuring genetic variation (Rahimmalek et al. 2009).
SCoT (Start Codon Targeted) and URP (Universal rice Primers) are gene-targeted markers that are starting to replace others among the different PCR-based DNA markers. They are considered to have several advantages such as their degree of durability, informativeness, high polymorphism and low cost. The Start Codon Targeted (SCoT) polymorphism mechanism is known for its accuracy and simplicity. Its translation initiation codononboth DNA strands (ATG) comprises a short conserved region (Collard and Mackill 2009). SCoT markers are typically repeatable, polymorphic, and cost-effective. This method has been successfully used in establishing the genetic association of several species of plants, including cumin, due to its numerous advantages. The SCoT primer has been reported in many molecular studies of various plants such as fennel, orchid, coconut, F. assafetida and durum wheat (Yadav and (Kang et al. 2002). The URP marker has also gauged genetic diversity in Vigna species Dikshit et al. (2007) and it could be used on many other species as well. Although Iranian F.assafoetida has been studied by RAPD markers Sarhaddipour et al. (2014), there have been no efforts so far to identify the genetic diversity of this species by targeting speci c regions of the genome via gene-based markers (Tajbakht et al. 2018). The purpose of this research was to consider the application of URP and SCoT markers in order to identify the genetic composition of naturally-occurring genotypes in different geographical regions of Iran. This research also aimed to analyze the genetic relationship between and within the Iranian F. assafoetida populations. To the best of our knowledge, this is the rst time that URP and SCoT markers are studied for applications on the genetic diversity of F. assafoetida.

Materials And Methods
Plant materials and DNA extraction Ninety F. Assafoetida accessions were collected as representatives of intra-species diversity. These were from 30 populations that originated in different eco-geographical habitats among mountainous regions of Iran. Sample collection occurred in September and December. The sample position and geographical coordinates are shown in Fig. 1. After seed germination and development, a total genomic DNA of 90 ferula genotypes was extracted from young leaves. A modi ed CTAB protocol was used for isolating genomic DNA. The consistency and quantity of DNA were measured using an electrophoresis of agarose gel (0.8%).

SCoT-PCR ampli cation
In this study, 12 primers were designed for the SCoT analysis, according to Collard and Machill ) Table 1).
A total reaction volume of 20µl SCoT-PCR ampli cations were performed in a total reaction volume of 20µl, including 6.5 µl double distilled water, 10µl master mix 2XPCR (ready to use PCR master mix 2X; Ampliqon) and2 µlthe template DNA and 1.5 µl of each primer. PCR ampli cation occurred by having pre denaturation at 94°C for 5 min, followed by 34 cycles of 94°C for 1 min, annealing at 48.9-55°C (different for each primer) for 45 seconds and extension at 72°C for 1.5 min. The nal extension occurred for 5 min at 72°C. Eight temperature gradients were used for the ampli cation process of the PCR(i.e. 48°C, 49°C, 50°C, 50.5°C, 52°C, 53.5°C, 54°C, and 55°C). A speci c annealing temperature was identi ed for each of the 12 primers. PCR products were detected bya 1.5% agarose gel. They were photographed under UV light after being stained. The ampli ed SCoT fragment was scored as 1 (for the presence) or 0 (absence) of the bands.

URP-PCR ampli cation
For URP analysis,12 primers were designed for URP-PCR ampli cations which involved a total reaction volume of 20µl, including 6.5µl double distilled water,1.5µl of each primer, 2µl of the template DNA and 10µl of the master mix 2XPCR.URP PCR ampli cation involved predenaturation at 95°C for 10 min, then 34 cycles of denaturation at 95°C for 1 min, annealing at 42-48°C (different for each primer) for 45 seconds and extension at 72°C. The nal extension occurred at 72°C for 7 minutes. A gradient of seven temperatures (i.e. 42°C, 43°C, 44°C, 45°C, 46°C, 47°C and 48°C) was considered when amplifying for the PCR. A speci c annealing temperature was identi ed for each of the 12 primers. PCR products were detected by a 1.5% agarose gel. They were photographed under UV light after being stained. The ampli ed SCoT fragment was scored as 1 (for the presence) or 0 (absence) of the bands ) Table 1).

Data analysis
PCR products were scored independently in SCoT and the URP pro les were scored as absent (0)

SCoT and URP polymorphism
In the present analysis, 24 SCoT and URP primers were used for estimating the genetic diversity of F. assafoetida. Anoverview of the calculation of the URP and SCoT primers information can be outlined according to relevant parameters ( Table 1)

Genetic variationand diversity analysis
The results of AMOVA demonstrated that the major portion of genetic variation occurred within populations (91%based on SCoT markers, 88% based on URP markers and 90% based on pooled data ( Table 2). The ndings revealed thatmolecular variance (%) was much higher within populations than among populations (SCoT = 9%, URP = 12%, pooled data = 10%). Gene ow (Nm) and inter-population differentiation (Gst) con rmed this nding. The genetic differentiation coe cient of (Gst)/gene ow for  (Table 3). In URP, SCoT and pooled data analysis, the lowest parametric values of genetic diversity were observed in the Yazd population.

Principle coordinate analysis
According to SCoT, URP and their integrated results, the Principle Coordinate Analysis (PCoA) was carried out. Through the SCoT, the rst two axes described 37.37 (coord1 = 13.08 and coord2 = 24.29) of the overall genetic variation (Fig. 3-A). The fan-dendrogram resembled the results of principal coordinate analysis and cluster analysis. Likewise, the results of URP molecular markers entered into the principal coordinate analysis and indicated that the rst to the third principal coordinates accounted for 17.49%, 25.79% and 33.88% of the overall genetic variation (Fig. 3-B). In sum, these results accounted for 77.16% of the total genetic diversity and approved the cluster study. According to the results of the PCoA for pooled data, the rst, second and third principal coordinates comprised 11.19, 20.07, 27.52% of total molecular variation, thereby corresponding to 58.78 % of the total genetic variation ( Fig. 3-C). When PCoA was based on pooled data, it indicated on eco-geographical segregation for different populations as seen in Kerman and Hormozgan populations are both located in the same origin on the biplot. These observations were validated by cluster analysis (Fig. 3).

Discussion
Genetic diversity is an essential element of plant populations in facing environmental stimuli. The frequency of high genetic diversity within the population has been recorded in various species of plantsand theoutcrossing nature of these species contributes to diversity (Sheidai et al. 2013). Studying genetic diversity is a mechanism that describes species or individuals using speci c statistical methods or a combination of methods based on morphological characters or molecular properties of individuals and DNA-based marker data that make a more reliable distinction from genotypes ).
The genetic structure of a plant population depends not only on its genetic background, but also on especially in F. assafoetida L relatives. F. assafoetida has a particular distribution and it is di cult to nd samples.These data could be used for future programs that require genetic variation in ferula species. In this research, 12 SCoT primer and 12 URP primers were used for molecular characterization of 90 individual plantsof the 30 populations .There was a signi cant genetic variation within the populations.
This research demonstrated that SCoT and URP markers explained 98.37% and 97.91% of polymorphism (Table 1) and structure than other molecular marker techniques. Nevertheless, the best marker for a genus such as F. assafoetida would require a wide variety of genomes for assessment.Analysis of molecular variance (AMOVA) revealed a wide distribution of genetic variation within the F. assafoetida populations ( Table 2).
The results of AMOVA revealed a higher level of molecular variance within populations (URP = 88%, SCoT = 91%, pooleddata = 90%) than among population (URP = 12%, SCoT = 9%, pooled data = 10%). The difference between the populations at the p probability level was signi cant. The integration of SCoT and URP can be seen as good markers for a more precise evaluation of genetic diversity. The results con rmedthat SCoT primers show signi cant genetic diversity within medicinal plant populations.
These results indicate that the diversity within the populations is high and, in fact, there is no signi cant difference among the populations. It can be stated that they are a subset of a larger population.The markers nature can partly explain the exact isolation of samples, whereas the high polymorphism of SCoT and URP markers can greatly represent variations. High, medium and low degrees of genetic Overall, in the case of wild species, the genetic distance is determined by the geographical distance and the gene ow between species. In crosspollination species, due to a high rate of gene ow, the genetic gap between populations is low and genetic variation is distributed within populations (Pfeifer and Jetschke 2006). Hamrick and Godt (1996) stated that if the genetic ow between habitats is interrupted due to peculiar factors such as habitat destruction or improper harvesting, there will be an increase in genetic distance among populations, and genetic erosion will commence because of homogeneity.
SCoT, URP and SCoT + URP cluster analysis suggesting, genotypes that are related to different geographical areas and sometimes far apart are located in close branches. These results indicated a very high degree of gene migration among populations. In addition, F. assafoetida is cross-pollinated in nature and insects serve as its pollinator agents. The plants develop seeds that have large wings, thereby increasing the chances of seed dispersal further (Reddy et al. 2007). The pooled data (SCoT + URP) con rmed the ndings of the Beyesian structure. Population structure applies to any of the genetic patterns of individuals within the population. The list of possible subpopulations withina particular population can be distinguished by the frequency of different alleles in each subpopulation, and also by genetic separation between subpopulations (Chakraborty 1993). In addition, this second study analyzes the population structure of F. assafoetida. These ndings are further con rmed by the study of the Beysian structure. Pritchard et al. (2000) reported that the clustering method is based on the Bayesian statistical index for interpreting population structures.
Although this method can involve a small number of discontinuous markers, it is possible to analyze the population structure, accurately classify individuals into appropriate populations and recognize different individuals. Based on the results of this method, it can be stated that sometimes the classi cation of genotypes is independent of their geographical origin. In this research, the populations structure analysis was evaluated by structure software and studied populationsfor SCoT primers into 5 categories. URP, SCoT + URP were divided into 6 categories. Accordingly, most of these divisions were based on the geographical areas from which these populations originated, although they were not completely In addition, this nding shows that these regions may be a valuable source of heterogeneity in the discovery of new alleles and candidate genes. Such a rich germplasm makes breeding programs worthwhile, although some populations were not classi ed on the basis of geographical areas. For instance, the permanent isolation of samples could in uence a marker's nature, even as ahigh level of polymorphism of SCoTand URP markers could describe variations inaccurately (Tajbakht 2018). Similar ndings were reported in the case of other Apiaceae species, such as Nigella sativa Golkar and Nourbakhsh (2019) and Foeniculum vulgare (Maghsudi Kelardashti et al. 2018). These characteristics of F. assafoetida germplasm may have contributed to an enhanced adaptability, migration and gene ow across regions (Hamrick and Godt 1996). Our ndings revealed a high variation in the populations and this was con rmed by AMOVA. In addition, URP markers can be integrated with SCoTmarkers to yield more consistent results on genetic diversity.

Conclusion
Crop improvement is driven by knowledge of the extent and distribution of genetic variation, as well as relationships between breeding materials. This study investigates the population structure and genetic diversity of F.assafoetida germplasm. This study discovered a signi cant differential within populations. Furthermore, the ndings showed that URP and SCoT molecular markers were reasonably effective at evaluating genetic variation among F.assafoetida genotypes and separating various individuals from different F.assafoetida populations. F.assafoetida species had comparatively high admixture and could be categorized into distinct categories depending on their geographical distribution, according to the results of the study. The relatively high degree of gene transfer between species may be due to F.assafoetida pollination, seed dispersal, nonnuclear inheritance, and post transcription effects. The ndings of this study have indicated that the F.assafoetida species has a high potential for use in the pharmaceutical industry. Based on this knowledge, it is possible to infer that this plant is a natural source of useful phenolic compounds that can be used in breeding programs, industrial processes, and pharmaceuticals. Finally, more population selection for genetic diversity assessment will provide more informative data.    Population structure of 90 F.assafoetida accessions using SCoT (A), URP (B) and integrated data (C).