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Analyses of Genetic Diversity and Population Structure of Anchote (Coccinia Abyssinica (Lam.) Cogn.) Using Newly Developed EST-SSR Markers

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Analyses of Genetic Diversity and Population Structure of Anchote (Coccinia Abyssinica (Lam.) Cogn.) Using Newly Developed EST-SSR Markers Genet Resour Crop Evol https://doi.org/10.1007/s10722-021-01132-5 (0123456789().,-volV)( 0123456789().,-volV) RESEARCH ARTICLE Analyses of genetic diversity and population structure of anchote (Coccinia abyssinica (Lam.) Cogn.) using newly developed EST-SSR markers Bekele Serbessa Tolera . Kifle Dagne Woldegebriel . Abel Teshome Gari . Mulatu Geleta Dida . Kassahun Tesfaye Geletu Received: 30 April 2020 / Accepted: 29 January 2021 Ó The Author(s) 2021 Abstract Anchote (Coccinia abyssinica (Lam.) Matsum. & Nakai] ESTs in the GenBank database. Cogn.) is a perennial root crop belonging to Cucur- Among those novel markers, eight were polymorphic bitaceae family. It is endemic to Ethiopia and and subsequently used for genetic diversity and distributed over wide range of agro-ecologies. For population structure analyses of 30 anchote accessions further improvement and efficient conservation of this collected from western Ethiopia. A total of 24 alleles crop, characterization of its genetic diversity and its were obtained across the eight polymorphic loci and pattern of distribution is a vitally important step. 30 accessions that revealed moderate level of genetic Expressed sequence tags-simple sequence repeats diversity in this minor crop. Among the eight loci, (EST-SSRs) markers were developed from publicly locus CA_06 was the most informative with six alleles available watermelon [Citrullus lanatus (Thunb.) and polymorphic information content (PIC) of 0.76. The accessions showed about threefold variation in terms of genetic diversity, with expected heterozy- Supplementary information The online version of this gosity (He) ranging from 0.15 (accession An) to 0.44 article (https://doi.org/10.1007/s10722-021-01132-5) contains supplementary material, which is available to authorized users. & B. Serbessa Tolera ( ) M. Geleta Dida Department of Biology, Wollega University, Department of Plant Breeding, Swedish University of P. O. Box 395, Nekemte, Ethiopia Agricultural Sciences, Box 101, 23053 Alnarp, Sweden e-mail: [email protected] e-mail: [email protected] K. Dagne Woldegebriel Á K. Tesfaye Geletu K. Tesfaye Geletu Department of Microbial, Cellular and Molecular Ethiopian Biotechnology Institute, Addis Ababa, Ethiopia Biology, Addis Ababa University, P. O. Box 1176, Addis Ababa, Ethiopia e-mail: [email protected]; kifl[email protected] K. Tesfaye Geletu e-mail: [email protected] A. Teshome Gari The John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK e-mail: [email protected] 123 Genet Resour Crop Evol (accession Dg). Other accessions with higher genetic Anchote tuber is rich in protein and calcium with diversity include Ar and Gu (He = 0.43 and 0.41, low content of anti-nutritional factors, and hence respectively). Analysis of molecular variance highly recommended as human food (Hora et al. (AMOVA) revealed that the variation within acces- 1995). It has also been used as traditional medicine, in sions and among accessions accounted for 84.7% and Ethiopia, to treat various illnesses such as Bone 15.3% of the total variation, respectively. The study fracture, backache, displaced joints and other diseases revealed low but significant population differentiation such as gonorrhea, tuberculosis, and cancer (Dawit in this crop with no clear pattern of population and Estifanos 1991; Gelmesa 2010). Even though structure. The EST-SSR markers developed in this anchote is economically, nutritionally, and medici- study are the first of their kind for anchote and can be nally valuable crop plant, there is limited information used for characterization of its wider genetic resources regarding its genetics, breeding, best agronomic for conservation and breeding purposes. practices and phylogeography to date. Recent studies on its nutritional contents (Desta 2011), morpholog- Keywords Accession Á Anchote Á Coccinia ical traits (Wondimu et al. 2014) and genetic diversity abyssinica Á EST-SSR Á Genetic diversity Á Population using ISSR markers (Bekele et al. 2014) revealed key structure information that can be used for its improvement. However, these studies targeted only limited geo- graphical areas where this crop is currently grown in Abbreviations Ethiopia, and hence the sample size and geographic AMOVA Analysis of molecular variance coverage in previous study did not fully represent the AR Allelic richness wider gene pool of anchote in the country. EST Expressed sequence tags In order to harness the nutritional values and other FMA Frequency of major allele benefits of this crop, quantifying its genetic diversity is SNP Single nucleotide polymorphism a primary step. At present, single nucleotide polymor- SSR Simple sequence repeat phism (SNP) markers are the marker of choice as they have genome-wide coverage and abundance in genic regions of most studied crops. However, such molec- Introduction ular markers require significant investment in terms of resources and time and hence are not an immediate Anchote (Coccinia abyssinica (Lam.) Cogn.) is a option for orphan crop like anchote. Alternatively, perennial root crop that belongs to Cucurbitaceae expressed sequence tags-simple sequence repeats family (Edwards et al. 1995; Hora et al. 1995). It is (EST-SSRs) are the second best option, as they are endemic to Ethiopia and distributed over a wide range quick and cheaper to develop and use (Ellis et al. of agro-ecologies adapting to altitudes as low as 2006). EST-SSRs markers have attributes, such as 550 m above sea level (masl) and as high as 2800 masl ease to develop, multi-allelic, transferable across (Getahun 1973). The plant grows well in areas where genera and are from expressed section of the genome. annual rainfall is between 950 and 2000 mm (Getahun They are also among markers of choice for QTL 1973; Westphal 1974). This species can reproduce analysis to identify genes driving traits of agronomic both sexually and asexually through their root tubers. interest (Ellis and Burke 2007). Therefore, developing Although anchote is monoecious, the main mode of such markers for anchote, would lead to enhanced sexual reproduction is through outcrossing due to understanding of its genome in general. In particular, protandry (Edwards et al. 1995). Asexually, it under- such molecular tool can guide in-situ and ex-situ goes an annual cycle in which its herbaceous shoots conservation efforts in Ethiopia and also play a role in die out and then new shoots emerge at the onset of breeding new varieties. Therefore, the present study rainy season from its ‘‘everlasting’’ rootstock. Sowing was aimed at developing new EST-SSR markers for seeds is a preferred means of anchote cultivation as the anchote and uses them for assessing the genetic crop produces hundreds of seeds per fruit (Hora et al. diversity and population structure of anchote acces- 1995; Wondimu et al. 2014). sions grown in Western Ethiopia where this crop is staple food. 123 Genet Resour Crop Evol Materials and methods containing sufficient silica gel for efficient desiccation and stored at room temperature until DNA extraction Plant material was conducted. Seed samples were collected from anchote accessions DNA extraction in 2013 from areas where the plant exists in nature and where it is widely cultivated by small-scale farmers Each silica gel dried leaf sample was transferred to a (Fig. 1). Thirty accessions, each represented by five 2 ml eppendorf tube containing two glass beads and individual plants, were selected based on their geo- was ground to fine powder using Mixer Mill MM 400 graphic distribution in the sampling area and their (Retsch GmbH, Germany). The DNA extraction from seeds were planted at Holeta Agricultural Research the ground samples was done following protocol Center, Ethiopian Institute of Agricultural Research, described in Geleta et al. (2012). The quality and located 41.7 km west of Addis Ababa during the year quantity of extracted DNA was assessed by conduct- 2013/14 crop growing season. Three weeks after ing agarose gel electrophoresis (1.5% (w/v) and planting, young and healthy leaves were separately NanoDrop measurement. The genomic DNA sampled from each seedling representing the 30 extracted from four of the samples was of poor quality accessions. Leaf samples were kept in a plastic zipper and consequently discarded. Hence, genomic DNA Fig. 1 Map of Ethiopia showing sample collecting sites (districts) of the 30 accessions used in this study 123 Genet Resour Crop Evol Table 1 List of 30 anchote No Accessions code Sampling district* Coordinates Altitude (m asl) accessions used in this study, their sampling 1 Ay Ayira 9°05050.1600 N35°23046.86 00E 1625.803 districts, and coordinates 2 La Lalo Asabi 9°10014.890 N35°28021.77 00E 1574.597 and altitude of the sampling 0 00 0 00 sites 3 Ds Dale Sadi 8°53 59.12 N35°13 28.30 E 1516.685 4 Dw Dale Wobera 8°55046.0100 N35°03009.30 00E 1606.296 5 Sy Sayo 7°36005.6800 N36°43031.35 00E 1843.43 6 An Anfilo 8°28019.6400 N34°35026.55 00E 1496.568 7 Gm Gimbi 9°10015.1500 N35°50008.88 00E 1967.789 8 Nj Nejo 9°30008.2600 N35°30010.22 00E 1870.862 9 Mt Metu 8°18000.0100 N35°35000.65 00E 1703.222 10 Al Alle 8°08057.0400 N35°32012.79 00E 1950.415 11 Hr Hurumu 8°20051.3100 N35°41049.24 00E 1815.084 12 Yy Yayo 8°20009.4200 N35°49020.82 00E 1598.981 13 Ch Chora 8°21039.8600 N36°07014.56 00E 2021.129 14 De Dega 8°34006.6900 N36°06036.03 00E 2266.798 15 Gc Gechi 8°20051.1100 N35°51053.91 00E 1642.872 16 Dh Dhidhesa 8°04053.5500 N36°27032.67 00E 1902.257 17 Gu Gumay 8°01000.0000 N36°31000.00 00E 1621.536 18 Go Goma 7°51026.250
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