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Utilizing Bioinformatics to Detect Genetic Similarities Between African Honey Bee Subspecies

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Utilizing Bioinformatics to Detect Genetic Similarities Between African Honey Bee Subspecies Journal of Genetics (2019)98:96 Ó Indian Academy of Sciences https://doi.org/10.1007/s12041-019-1145-7 (0123456789().,-volV)(0123456789().,-volV) RESEARCH ARTICLE Utilizing bioinformatics to detect genetic similarities between African honey bee subspecies HOSSAM F. ABOU-SHAARA* Department of Plant Protection, Faculty of Agriculture, Damanhour University, Damanhour 22516, Egypt *E-mail: [email protected]. Received 27 December 2018; revised 23 June 2019; accepted 30 July 2019 Abstract. Various honey bees, especially subspecies Apis mellifera, occur in Africa and are distribute across the continent. The genetic relationships and identical genetic characteristics between honey bee subspecies in Africa (African bee subspecies) have not been widely investigated using sequence analysis. On the other hand, bioinformatics are developed rapidly and have diverse applications. It is anticipated that bioinformatics can show the genetic relationships and similarities among subspecies. These points have high importance, especially subspecies with identical genetic characteristics can be infected with the same group of pathogens, which have implications on honey bee health. In this study, the mitochondrial DNA sequences of four African subspecies and Africanized bees were subjected to the analyses of base composition, phylogeny, shared gene clusters, enzymatic digestion, and open reading frames. High identical base composition was detected in the studied subspecies, and high identical results from all tests were found between A. m. scutellata and A. m. capensis followed by A. m. intermissa and A. m. monticola. The least genetic relationships were found between A. m. lamarckii and the other subspecies. This study presents insights into the genetic aspects of African bee subspecies and highlights similarity and dissimilarity aspects. Also, Africanized honey bees derived from A. m. scutellata showed high genetic similarities to other African bees, especially A. m. capensis. Additionally, specific primers to identify these subspecies were designed and tested. Keywords. primers; mitochondrial DNA sequences; African honey bee; phylogeny; Apis mellifera L. Introduction hybridization between A. m. lamarckii and Carniolan bees beside other subspecies from Europe (Page et al. 1981; Honey bees, Apis mellifera L., have many subspecies of Sheppard et al. 2001; Kamel et al. 2003). which some are distributed in Europe and Asia, and others in The phylogenetic analyses from previous studies support the Africa (Garnery et al. 1992; Arias and Sheppard 1996; presence of close relationships between African bees in one Oleksa and Tofilski 2015). Some of the studies support that branch (Garnery et al. 1992), and there are differences between Africa is the origin of honey bees while other studies do not African and European bees in random amplified polymorphic suppot this fact (Franck et al. 1998; Whitfield et al. 2006; DNA (Suazo et al. 1998). A few studies have been conducted in Han et al. 2012; Wallberg et al. 2014). The African honey Africa on the genetic characterization of the honey bees, e.g. bee subspecies include Apis mellifera lamarckii Cockerell, studying mtDNA polymorphisms in Moroccan and Spanish bee 1906 or commonly known as Egyptian honey bees in Egypt, populations (Garnery et al. 1995) and in subspecies from Kenya A. m. intermissa Maa, 1953 in other north African countries, (Meixner et al. 2000). Additionally, A. m. scutellata were A. m. monticola Smith, 1961 in east Africa, and A. m. introduced to South America giving rise to the problem of scutellata Lepeletier, 1836 and A. m. capensis Eschscholtz, Africanized honey bees or commonly known as killer bees 1822 in South Africa. Other subspecies exist in limited (Taylor 1977;Winston1992). Many genetic studies have con- regions, such as A. m. sudanese in Sudan (Woyke 1993) and centrated on Africanized bees (e.g. Sheppard et al. 1991, 1999). A. m. sahariensis in Morocco and south Algeria (Smith et al. Detecting the identical genetic similarities between honey bee 1991; Adjlane et al. 2016). Some of these subspecies have subspecies has high importance especially subspecies with exposed to extensive hybridization over years, e.g. hybrid identical genetic characteristics can share the same pathogens or bees are at present common in Egypt due to the be infected with the same group of pathogens, which have 96 Page 2 of 7 Hossam F. Abou-Shaara implications on honey bee health. In fact, comprehensive Biotechnology Information (https://www.ncbi.nlm.nih.gov/) studies to compare genetic characteristics and relationships (table 1). These subspecies occur in different parts of Africa, based on sequences between African honey bee subspecies are but the sequence of A. m. scutellata is to Africanized honey not common. Therefore, it is hypothesized that using bioin- bees occur in America (Gibson and Hunt 2016). formatics can greatly detect variations and similarity aspects in DNA sequence between African bee subspecies, and between Africanized bees and African bees. Sequence analysis Fortunately, the availability of many free genetic resources encourage researchers to extract more genetic information from The percentage of A, T, C and G, beside the AT-skew and target organisms. The full sequences of mitochondrial DNA of GC-skew (Perna and Kocher 1995) were calculated for each some subspecies in Africa are freely available (Hu et al. 2016; subspecies and 13 genes that was identified (table 2). Eimanifar et al. 2016, 2017a, 2017b). Therefore, this study aimed to utilize bioinformatics using various analytical tools to highlight the genetic relationships between some African honey bee subspecies and the Africanized honey bees derived from A. Phylogenetic tree m. scutellata. Also, specific primers to identify African and Africanized bee subspecies were designed. Two trees were constructed for the studied subspecies, the first one was based on the whole sequences while the second one was based on the sequences of the 13 genes. Sequences Methods were aligned using ClustalW and IUB as DNA weight matrix, then the maximum likelihood method as statistical Sequences of African honey bee subspecies method, the Jukes–Cantor model (Jukes and Cantor 1969), and bootstrap method to test of phylogeny were used to The mitochondrial DNA sequences of some African honey construct the two phylogenetic trees using MEGA7 (Kumar bee subspecies were downloaded from National Centre for et al. 2016). Table 1. The information of A. mellifera subspecies used in this study. Gene Occurrence Subspecies Size (bp) number Accession Reference North Africa (Libya, Tunisia Morocco and Algeria) A. m. intermissa 16336 13 KM458618 Hu et al. (2016) North Africa (Egypt) A. m. lamarckii 16589 13 KY464958 Eimanifar et al. (2017a) East Africa A. m. monticola 16343 13 MF678581 Eimanifar et al. (2017b) Africanized honey bees in America A. m. scutellata 16411 13 KJ601784 Gibson and Hunt (2016) South Africa A. m. capensis 16470 13 KX870183 Eimanifar et al. (2016) Table 2. Thirteen genes identified in sequences of the African honey bee subspecies. Sequences (from - to) Genes A. m. intermissa A. m. lamarckii A. m. monticola A. m. scutellata A. m. capensis 1 498–1499 513–1514 502–1503 507–1508 508–1509 2 1798–3358 1802–3362 1794–3354 1803–3368 1803–3363 3 3620–4297 3887–4564 3618–4295 3645–4320 3693–4368 4 4446–4604 4713–4871 4444–4602 4471–4629 4520–4678 5 4586–5266 4853–5533 4584–5264 4611–5291 4660–5340 6 5287–6066 5554–6333 5285–6064 5312–6091 5362–6141 7 6187–6540 6481–6834 6185–6538 6253–6606 6302–6655 8 Complement Complement Complement Complement Complement (6894–8558) (7196–8860) (6892–556) (6960–8624) (7009–8673) 9 Complement Complement Complement Complement Complement (8646–9983) (8949–10259) (8644–9987) (8712–10019) (8761–10071) 10 Complement Complement Complement Complement Complement (9989–10252) (10292–10555) (9991–10254) (10048–10311) (10104–10367) 11 10439–10942 10744–11247 10441–10944 10499–11002 10555–11058 12 11002–12153 11306–12457 11004–12155 11062–12213 11118–12269 13 Complement Complement Complement Complement Complement (12300–13217) (12606–13523) (12302–13219) (12364–13281) (12419–13336) Genetic similarities between African bee subspecies Page 3 of 7 96 Shared gene cluster Table 3. Components of sequences of the studied African bees. The OrthoVenn as a platform for comparison of orthologous AT- GC- gene clusters (http://www.bioinfogenome.net/OrthoVenn/) Subspecies A% T% G% C% skew skew utilizing proteins downloaded from Uniprot was used to A. m. 43.17 41.41 5.65 9.75 0.02 -0.26 identify the shared gene clusters between studied subspecies. intermissa A. m. lamarckii 43.33 41.55 5.56 9.53 0.02 -0.26 A. m. monticola 43.11 41.60 5.58 9.60 0.01 -0.26 A. m. scutellata 43.24 41.45 5.61 9.68 0.02 -0.26 Enzymatic digestion A. m. capensis 43.24 41.51 5.61 9.61 0.02 -0.26 A method by Abou-Shaara (2019) was used to compare the fragments resulted from the enzymatic digestion. The Table 4. Components of sequences of the 13 genes of studied sequences of studied subspecies were digested using African bees. available restriction enzymes, namely BamHi, BclI, BglI, AT- GC- BglII, EagI, EcoRI, EcoRV, HindIII, HpaI, NdeI, NheI, Subspecies A% T% G% C% skew skew NotI, PstI, SacI, SalI,SamI, SphI, XbaI and XhoI at Gen- ome Compiler 2.2.88 (http://www.genomecompiler.com) A. m. 42.04 40.95 6.40 10.59 0.01 -0.24 and then fragments were simulated on gel using NEB intermissa 100-bp ladder. A. m. lamarckii 42.07 41.08 6.41 10.42 0.01 -0.23 A. m. 42.05 41.24 6.31 10.38 0.009 -0.24 monticola A. m. 41.99 41.01 6.41 10.56 0.01 -0.24 Open reading frames (ORFs) scutellata A. m. capensis 41.98 41.07 6.42 10.51 0.01 -0.24 The default options of Genome Compiler 2.2.88 were used to identify ORFs for the studied African bees with start codon of ATG, minimum length of amino acids of 60, and (table 4).
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