Research article
Computational analysis of atpB gene promoter from different Pakistani apple varieties

https://doi.org/10.1016/j.compbiolchem.2016.05.002Get rights and content

Highlights

  • atpB gene promoter region was amplified and sequenced from six apple varieties.

  • We analyzed atpB promoter region using various bioinformatics tools.

  • Phylogenetic analyses have revealed monophyletic origin of studied apple varieties.

  • Common as well as unique cis-regulatory elements along their functions were identified.

Abstract

Apple is the fourth most important fruit crop grown in temperate areas of the world belongs to the family Rosaceae. In the present study, the promoter (∼1000 bp) region of atpB gene was used to evaluate the genetic diversity and phylogeny of six local apple varieties. atpB gene is one of the large chloroplastic region which encodes β-subunit of ATP synthase and previously it had been used largely in phylogenetic studies. During the present study, atpB promoter was amplified, sequenced and analyzed using various bioinformatics tools including Place Signal Scan, MEGA6 and BLASTn. During the phylogenetic analysis, obtained phylogram divided the studied varieties into two clusters revealing the monophyletic origin of studied apple varieties. Pairwise distance revealed moderate genetic diversity that ranges from 0.047–0.170 with an average of 0.101. While identifying different cis-acting elements present in the atpB promoter region, results exhibited the occurrence of 56 common and 20 unique cis-regulatory elements among studied varieties. The identified cis-acting regulatory elements were mapped as well. It was observed that Kala Kulu has the highest unique features with reference to the availability of cis-acting elements. Moreover, the possible functions of all regulatory elements present on the promoter sequence of atpB gene were predicted based on already reported information regarding their in vivo role.

Introduction

Apple (Malus domestica), which belongs to the family Rosaceae, is one of the significant cultivated fruit crops of the world temperate regions (Janick and Moore, 1996). The total number of species in the genus Malus varies between different studies as Robinson et al. (2001) recognized seventy eight while Harris et al. (2002) reported 55 species. Global apple production exceeds 75.4 million tonne in 2013 (Sonmezuglu and Kutuk, 2014). Apple is the fourth major fruit crop of Pakistan and mainly cultivated in northern areas of Khyber Pukhtunkhwa, Punjab and Baluchistan. However, Baluchistan province is the major contributor of apple production. Various varieties of apple being grown in Pakistan includes Kala Kulu, Golden Delicious, Super, Gaja, Red Delicious, Mashadi, etc. (Mukhtar et al., 2010, Muhammad et al., 2011). Jasra et al. (2001) reported severe decline in apple yield in Baluchistan (Pakistan) due to selection of variety as pollinizer as well as attack of insects. So, accurate identification and assessment of genetic polymorphism of the existing cultivar is of utmost importance for successful apple breeding programs.

Malus domestica is a hybrid species from a number of progenitor species, including M. sylvestris, M. prunifolia, M. dasyphyllus, M. orientalis and M. praecox (Korban and Skirvin, 1984, Cornille et al., 2013, Nikiforova et al., 2013). The domestication of apple orchards started in Central Asia along with secondary contribution from the European crabapple dated back in near four thousand years after the first use of grafting practices (Cornille et al., 2014). The road named Old Silk that connects Eastern China with Black Sea has been considered the mainstream way in the evolution of Malus (Juniper et al., 1999).

Complete genome sequence of Malus domestica Borkh has been published (Velasco et al., 2010). All members in genus Malus contain equal number of x = 17 chromosomes (Evans and Campbell, 2002). Genetic markers collectively selected from nuclear DNA (nrDNA), mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) have been widely used to trace the origin of domesticated apples. Moreover, extensive studies have been reported on apple to examine its genetic variability using technique such as random amplified polymorphic DNA (RAPD) (Dantas et al., 2000; Goulao and Cristina, 2001), amplified fragment length polymorphism (AFLP) (Goulao et al., 2001, Hokanson et al., 2001), restriction fragment length polymorphism (RFLP) (Nybom and Schaal, 1990, Gardiner et al., 1996, Khadivi-Khub et al., 2014), trnH-psbA intergenic spacer (Volk et al., 2015), inter-simple sequence repeat (ISSR) (Kashyap et al., 2010) and simple sequence repeat (SSR) (Gharghani et al., 2009, Zhang et al., 2012, Gross et al., 2014).

cpDNA sequences are traditionally used as staple data source for studying the molecular phylogeny of plants (Patwardhan et al., 2014). This is because cpDNA is relatively conserved as well as smallest as compared to nuclear and mitochondrial genome along with the ease of PCR amplification and sequencing of chloroplast genes (Dong et al., 2012). cpDNA as a molecular marker can be used efficiently to determine genetic diversity as well as phlyogenetic lineage (Coart et al., 2003). Several cpDNA genes including matK, rbcL, atpB, rpl16, rps16, rps11 and ndhF have been used for inferring phylogeny in plants at different taxonomic levels (Zhang, 2000; Gao et al., 2008, Li, 2008). Previously phylogenetic analyses based on different regions of the chloroplast genome have been conducted in the family Rosaceae (Lo and Donoghue, 2012).The atpB gene, located on the cpDNA, encodes the β (beta) subunit of ATP synthase. In many studies scientist used atpB gene to elucidate phylogenetic relationship for different plant species because it has a relatively slow rate of nucleotide substitution compared to most genes encoded by the cpDNA (Savolainen et al., 1995, Magee et al., 2010). On the other hand no elaborative studies have been undertaken for atpB gene characterization in any single aspects for apple chloroplast. Therefore, atpB based analysis can be a good candidate to study the phylogeny of Malus species. The present study has therefore been conducted with an aim of evaluating the genetic diversity and phylogeny of six apple varieties based on atpB gene promoter sequences and to analyze the functional importance and identification of its regulatory elements.

Section snippets

Plant material and extraction of genomic DNA

Fresh leaves samples of the selected apple varieties were collected from Pishin, Baluchistan (Pakistan) and stored at 4 °C for further processing. Total genomic DNA was extracted using CTAB-based method (Richard, 1997) with slight modification by incubating the homogenized mixture at 70 °C for 30 min.

Primer designing

Primers was designed for atpB gene promoter using Primer 3 (version 4.0) (http//primer3.sourceforage.net/) based on cpDNA sequences of tobacco available at NCBI GenBank (www.ncbi.nih.gov). The

Results and discussion

The obtained sequence data of atpB gene promoter was analyzed. Sequences were aligned and compared with nucleotide sequences in the GenBank using ClustalW and BLASTn to determine conserved regions and similarities respectively. Blast results showed higher than 90% sequence identity with Malus cpDNA.

Conclusion

Our results showed the occurrence of significant novel cis-regulatory elements in the atpB gene promoter from studied apple varieties that may regulate atpB gene expression. These regulatory elements can be used for experimental verification, selection and conservation of genetic resources and for future breeding programs. However, to determine the exact role of these cis-regulatory elements, in vivo studies are required.

Acknowledgement

We are thankful to Higher Education Commission, Islamabad, Pakistan for providing financial assistance.

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