Evaluation of molecular diversity analysis and relation with environmental factors in accessions of Cumin (Cuminum cyminum L.) in Iran, revealed by inter-simple sequence repeat (ISSR) markers and start codon targeted (SCoT) markers Mohammad Samer Mohammadizadeh ( [email protected] ) Islamic Azad University Farzaneh Bahadori Agricultural Research Education and Extension Organization Leila Hakimi Agricultural Research Education and Extension Organization Abbas Dehshiri Agricultural Research Education and Extension Organization Research Keywords: Cuminum cyminum, Genetic diversity, Canonical correspondence analysis, ISSR, SCoT, Iran Posted Date: June 22nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-35133/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/14 Abstract Cuminum cyminum (as an aromatic herb) is the sources of cumin, which has been commonly used in traditional medicine for the treatment of various diseases especially in tropical Asia. Thus, the current experiment was performed to accesses molecular genetic diversity among 22 collected accessions of cumin from different area of Iran, based on inter simple sequence repeat (ISSR) markers and start codon targeted (SCoT) markers. Analysis of molecular variance revealed that a signicant genetic difference between the studied accessions reach to 43%. Mantel test between genetic diversity and geographical distance indicated that an increase in geographical distance of collected accessions did not inuence on genetic differentiation. Results about all agro- morphological traits showed a signicant difference between the ecotypes except the number of branches. Canonical correspondence analysis ballot of genetic features and environmental factors included 5 geographic and climatic factors for seed origin’s habitat, which have been show that the mentioned parameters had no strong effect on the plant genotypes. However, difference in elevation and latitude of accession origins had effect on genetic clustering in two major groups. The consensus tree of both molecular and morphological data relieved that the accessions Chah-Dashi, Damghan, Ferdows and Nehbandan formed discreet clusters based on genetic and agro-morphological features. In conclusion, the results conrmed that both ISSR and SCoT markers were reliable and useful tools for analyzing the genetic diversity of cumin in Iran. Introduction Cumin (Cuminum cyminum L.), the second most popular spice in the world, is belong to Apiaceous or Umbelliferae family, which has been regard as a medicinal herb, economical crop plant, and part of food culture in in wide geographical areas (KAFIE et al., 2002; LODHA and MAWAR, 2014) especially in tropical Asia (HASHEMIAN et al., 2013). There are numerous usages of cumin seed due to its phyto-chemicals components, minerals (like Fe, Cu, Ca, K, Mg, Se, Zn and Mn), vitamins, and many avonoid phenolic anti-oxidants (PARASHAR et al., 2014). Geographical distribution of cumin in Iran conned mainly to arid and semi-arid regions in eastern, south-eastern, and central provinces (HASHEMIAN et al., 2013). Estimation of genetic diversity of related plant sample is a requirement towards the helpful utilization and conservation of plant genetic resources (WEISING et al., 1995; SHAHLAEI et al., 2014). On the other hand, morphological and molecular surveys are used for genetic diversity, genotypes, and ecotypes of crop plants for upgrading the cultivars or varieties in purpose of breeding programs and conservation (AFSHAR et al., 2015). Molecular markers have been widely and effectively used for assessment of genetic diversity, phylogenetics, ngerprinting, variation, and differentiation in biology (XIONG et al., 2011; AMIRMORADI et al., 2012; MULPURI et al., 2013). Molecular markers, as a reliable method in detecting the polymorphism at DNA level, could be helpful in genetic relationships within and between species and ecotypes (MARSJAN and OLDENBROEK, 2007). DNA ngerprinting of all the genetic stocks is imperative in order to preparing a powerful molecular database of medicinal plants (WJITKUS et al., 1994). The most important characteristics of molecular markers are neutrality, not inuenced by age, environment, economical beneciary and more informative than morphological traits (SAMANTARAY et al., 2010; RAHIMI et al., 2018). Inter simple sequence repeat (ISSR) marker is preferred by many researchers due to many advantages such as simplicity, quickly, less costly and high reproducibility (PRADEEP REDDY et al., 2002; XU et al., 2018). ISSR markers rapidly reveal high polymorphic ngerprints and have been used frequently to determine the genetic diversity among date palm cultivars (AYESH, 2017). Also, SCoT (start codon targeted), as a novel marker, can be evaluating as the strong polymorphic markers which indicate differences between individuals extremely well (COLLARD and MACKILL, 2009). As yet, genetic relationships between cumin ecotypes were investigated in terms of agro-morphological traits (BAHRAMINEJAD et al., 2011) and molecular polymorphism by markers like RAPD, SCoT, CCMP and SSR (BAHRAMINEJAD et al. 2012; BAHRAMINEJAD and MOHAMMADINEJAD, 2013; ROSTAMI- AHMADVANDI et al., 2013; PARSSHAR et al., 2014). Considering the worth of medicinal plants in industry and commerce, the present study was survey to undertaken the agro-morphological traits and genetic differentiation (by ISSR and SCoT markers) of 22 cumin accessions collected from different regions of Iran. This study would be helpful in selecting parents in the cumin breeding programs and conservation of this medicinal plant. Furthermore, relationships between ecological factors and genetic differentiation will be discussed due to poor information about environmental inuence on different accessions of cumin. Materials And Methods Plant materials At current study 22 accessions of cumin from 9 Iran province were investigated (Figure 1, Supplementary 1). The accession number, origin and ecological condition of these accessions are presented in Table 1. The seeds were cultivated using randomized complete block design at the Research Field of Agriculture (Semnan, Iran; Table 2). And, the information about environmental factors of the study area was given from State Meteorological Organization of Iran (http://reports.irimo.ir/jasperserver/login.html). The measured agro-morphological traits were including: plant height, number of branches, number of umbel, mini-umbel and seed, 1000-seed weight, seed weight per plant and grain yield. These traits were measured through morphometric method. The photos of accession were given as Supplementary data 1. Table 1. Accession number, origin information of 22 accessions of cumin and their ecological factors Page 2/14 F Province Locality Accession .No Longitude Latitude Elevation (m) AMT (° C) AP (mm) Code. 1 Razavi-Khorasan Khaf 100 60° 8' 10.509'' 34° 34' 17.431'' 972 16.7 200 2 Razavi-Khorasan Khoshab 101 57° 59' 26.287'' 36° 25' 30.785'' 1187 13.8 207 3 South-Khorasan Chah-Dashi 102 59° 43' 20.238'' 31° 28' 14.567'' 1065 20.05 86 4 South-Khorasan Ayesk 103 58° 22' 58.742'' 33° 53' 14.868'' 1368 16.7 135 5 East-Azarbayjan Tabriz- Ilkhchi 104 45° 58' 35.354'' 37° 56' 19.467'' 1308 11.6 318 6 East-Azarbayjan Shabistar-Shendabad 105 45° 37' 44.730'' 38° 8' 39.281'' 1309 10.9 320 7 Isfahan Khur and Biabanak 106 55° 5' 6.655'' 33° 46' 25.764'' 831 20.2 79 8 Isfahan Mobarakeh-Talkhuncheh 107 51° 33' 36.569'' 32° 15' 44.604'' 1731 15.1 154 9 Kerman Rig-Mahan 108 57° 17' 29.504'' 30° 3' 40.064'' 1901 15.4 156 10 Kerman Kuhbanan 109 56° 16' 58.527'' 31° 24' 37.072'' 1990 14.1 146.1 11 Yazd Yazd 110 54° 21' 20.827'' 31° 53' 15.208'' 1222 18.9 55 12 Semnan Dlạzyạn 111 53° 24' 23.810'' 35° 29' 53.300'' 1042 17.2 130 13 Semnan Damghan 112 54° 20' 27.193'' 36° 9' 49.639'' 1154 15.4 213 14 Semnan Sorkheh-Biabanak 113 53° 16' 6.488'' 35° 24' 42.841'' 1034 17.4 129 15 South-Khorasan Birjand1 2001 59° 13' 0.940'' 32° 52' 24.009'' 1454 17 129 16 South-Khorasan Birjand3 2003 59° 13' 0.940'' 32° 52' 24.009'' 1454 17 129 17 South-Khorasan Boshruyah 2004 57° 25' 41.322'' 33° 52' 6.185'' 879 19.7 96 18 South-Khorasan Ferdows 2005 58° 10' 0.473'' 34° 0' 20.273'' 1269 17.2 130 19 South-Khorasan Nehbandan 2006 60° 3' 1.431'' 31° 32' 26.144'' 1185 18.4 93 20 South-Khorasan Faizabad 2007 58° 47' 33.697'' 35° 1' 10.119'' 942 26 220 21 Tehran Tehran 2008 51° 22' 50.599'' 35° 42' 2.604'' 1214 16.4 220 22 North-Khorasan Bojnord 2014 57° 19' 44.809'' 37° 28' 20.124'' 1071 13.2 257 F (Features); AMT (mean year temperature); AP (mean year rainfall). Table 2. Geographical condition of seeds were cultivated Parameters Longitude Latitude Altitude (m) Temperature (mean year, ° C) Rainfall (mean year, mm) Semnan 53°23' 20.077'' 35° 35' 0.752'' 1164 17.2 130-145 DNA Extraction and ISSR and SCoT PCR amplication Total genomic DNA was extracted from young leaves using cetyl trimethylammonium bromide method described by Doyle and Doyle (1990) with a little modication (Ghasemzadeh-Barkaki et al., 2018) that included combination of β-mercaptoethanol, polyvinyl pyrrolidone, sodium N-lauroyl sarcosine and sodium dodecyl sulfate. To determine the quality of DNA, 0.8% agarose gel electrophoresis were used. In this study, 5 SCoT primers commercialized by UBC (the University of British Columbia) using for amplication (COLLARD and MACKILL, 2009). Specic primers (Table 3) were provided by Invitrogen, USA. Polymerase chain reaction was performed in 25 μL reaction volume containing 10 mM Tris-HCl, pH 8.3, 2.5 mM Mg Cl2, 1 mM dNTPs mix, 0.2 μM of each primer, 1U of Taq DNA polymerase, and 25 ng of template DNA. Thermocycling conditions (BIORAD.T100) were as follows: after initial denaturation at 94oC for 300s; 36 amplication cycles were performed at 94oC for 60s, 60 oC for 60s; Finally 60 oC for 60s, 72 oC for 90s, and 72 oC for 10 min.