Tag-derived Simple Sequence Repeat Markers of Olive

Tag-derived Simple Sequence recite Markers of Olive naming and operative annotation of expressed era Tag-Derived Simple Sequence Repeat Markers of chromatic (genus Olea europaea)Olive tree (Olea europaea L.) is one of the approximately important oil producing crops in world, the genetic appellation of several(prenominal) genotypes by using molecular markings is the offshoot step in life programmes. A heavy(p) number of Olea europaea expressed while pits (ESTs) 11,215 were done from the NCBI database and designd to search for microsatellites. Our result Explained that 8295 SSRs were correspond and its fate of occurrence which somewhat 77.6%,11.84%,8.62%,0.84%,0.77% and 0.29% for Mononucleotide, trinucleotide, dinucleotide, hexanucleotide, pentanucleotide and tetranucleotide respectively. The air of the AAG/CTT recapitulate was highly parcel in trinucleotide and percentage of AG/CT was highly in dinucleotide reiterates. By using flanking region of SSRs repeat we des igned 1,801 EST-SSR f charactere drive pairs. The result obtained from useful annotation of chromatic EST orders restraining SSRs indicated that 81% of these sequence having homology with know proteins, while 1.55% was homologous to hypothetical or un cognize proteins and the 17.37% sequences did not possess homology with some(prenominal) know proteins. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that EST being SSRs were implicated in diverse biologic process include cellular and metabolic process, while in molecular lean includes catalytic activity, binding and enzyme regulator activity. A total of 93 different pathways were significant matches in the KEGG database, which divided onto Carbohydrate metabolism much(prenominal)(prenominal) as glycolysis/gluconeogenesis pathway and the Energy metabolism such as carbon paper fixation in photosynthetic organism pathway, also this included 11 different pathways from lipide metabolism su ch as Fatty demigod biosynthesis pathway. We isolate a genomic desoxyribonucleic sulphurous from 9 olive cultivars and time- tried with 25 random selected solid ground pairs for amplification and pleomorphism detection. All tested dry lands, exhibited successfully amplified and detected polymorphism.Olive tree (Olea europaea L.) is one of the near overaged and important long lived fruit species in Mediterranean 1, its diploid species (2n = 2x = 46), and the genome size of it range amongst 2.90 pg/2C and 3.07 pg/2C, with 1C = 1,400 1,500 Mbp 2. Olea europaea is one of the first domesticated crops from family Oleaceae family, and it cultivated for table olives and edible oil 3, a long register of olive cultivation in the Middle East was descriptions by archeology and botanists 4. The olive cultivars are high of number that more than 1200 cultivars 5, also the accessions are functional in a large numbers in olive producing countries, that occurrence a problems for germplas m preservation and it management 6.The genetic identification and characterizing of several genotypes by using molecular markers is the first step in breeding programs 7, and by increased rate of mutation in microsatellites repeats that show a highly level of length polymorphism 8. With the improvement and increasing of deoxyribonucleic acid sequencing technology, sequencing of expressed gene are employ to obtain a large collection of EST which are separated from a specific tissue and pointedness on organism 3. Recent EST-SSR studies have describe that the EST is uses a seed of SSRs and that reveal highly polymorphism 9. EST sequences Available in public database and by using bioinformatics tools can gibe and stimulatement of SSR markers in that EST sequences 10. In olive that can be allow to development of new working(a) marker and use it in molecular breeding 11. Also it can use as useful tools for gene and marker discovery, gene mapping and functional proportional studi es. EST-SSRs recently reported in several plant species, such as genus Musa 12, Finger Millet 13, Jatropha Curcas 14, Pineapple 15, Celery 16, Lettuce 17, barley 18, Radish 19, Citrus 20, Watermelon 21, Sugarcane 22, grape vines 23, Cereal species 24 and scraping shuck 25. A large number of EST sequences in olive are acquirable on database it can be a useful resource to develop gene based markers. The aim of this study was to use bioinformatics tools to develop and get a line a new genic marker EST-SSR in Olive, to compare the frequence and distribution of different repeat types in genic sequences. Also determine the localization of these primers in different pathways in plant, to use it as tools to differences between the olive cultivars.The Source of Sequence, screening and primer designing of microsatellites.EST database apply a source of olive EST sequences from NCBI (http//www.ncbi.nlm.nih.gov). A total of 11,215 ESTs sequences of Olea europaea are available and used in this study. Identification of SSRs by using the perl script MISA (MIcroSAtellite identification tool http//pgrc.ipk-gatersleben.de/misa/).The criteria used to determine SSR repeat were mononucleotide 10, dinucleotide 6, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide 5, and the maximal number of bases interrupting deuce SSRs in a compound microsatellite are 100 bp. The flanking region of SSR papers used to design SSR primers by using primer3_core 26. The parameters used were best length of primer 20 nucleotides, optimum annealing temperature (Tm) of 58C, expected amplified products size of 100-500 bp and optimum G/C content 50 %.Validation of designed primerFor primer validation, we designed 25 EST-SSR primers and test these primers on 9 olive cultivars. Total genomic DNA was extracted from olive leaves using engraft Genomic DNA equip (QiGen). PCR amplification was conducted in 25 l reactions containing 50 ng of guide DNA, 2.5 mM MgCl2, 5 l 5X PCR buffe r, 0.5 mM from each one primer, 0.5 U Taq DNA polymerase, and 2.5 mM dNTPs. The PCR cycling profile was 94C for 5 min, 35 cycles at 94C for 45 s, the optimum annealing temperature for each primer pair shown on (Table S1) for 50 s, 72C for 45 s, and a final extension at 72C for 10 min. The quality of the PCR product was checked by mixing it with an equal heap of loading buffer and then visualizing the band on a 1.5% agarose gelatine in TBE buffer at 100 W for 120 min.Identification of EST-SSRs Putative Function annotationThe putative function annotation of EST sequences contains SSR performed by used Blast2go program 27 to BLAST against a reference database. Also blast2go program are containing many features such as Gene Ontology (GO), Enzyme Commission (EC), and KEGG annotation. diffusion of various repeat type in oliveOur result referred to 4,088 EST sequences about 36.45% from 11,215 of Olea europaea EST sequences as containing 8,295 various motif SSRs that Due to the EST seque nces maybe contain more than one SSR motif (Table 1), and this number based on the criteria we used it to identify SSR motifs in the EST sequence.The investigation of different types of SSR repeats in our result showed that the highest percentage of appearance mononucleotide repeats were 77.64%, followed by trinucleotide 11.84%, dinucleotide 8.62%, hexanucleotide 0.84%, pentanucleotide 0.77% and tetranucleotide 0.29% (Fig. 1). The high abundant of trinucleotide in label regions were arranged with the previous studies in eukaryotic genomes 28, 31.In mononucleotide A/T repeats 88.8% were high than the G/C 11.2% motifs, and these results were proportionate with SSRs analytic thinking of chloroplast SSRs on Olea species 29 and with SSRs abbreviation of major cereal organelle genome 28. GA motifs were representing 55% from dinucleotide motifs in olive EST sequences. According to previously studies from foxtail millet 31, barley, maize, sieve, sorghum and wheat 30, GA motifs were the most abundant motifs in these crops. AG/CT and GA/TC motifs were the most frequent respectively and CG motifs the lowest frequencies were put in olive, this case reported in the distribution of microsatellites on three different plant families that Brassicaceae, family Solanaceae and Poaceae 32. Dinucleotide motif can represent to multiple codons that depending on the denotation frame and can restate into different amino acids such as, AG/CT motif can represent AGA, GAG, CUC and UCU codons in mRNA and translate into the amino acids Glu, Arg, Leu and Ala respectively, Ala and Leu were present in protein at higher frequencies, hence the higher incidence of GA, CT motifs in the EST sequences 33. This could be one of the reasons why GA, CT motifs are present at such highly appearance in EST collections 34, dinucleotide repeats that located on coding regions are more sensitive to any change such as any addition or ablation because that causes a frame shifts and will give differ ent amino acids 35. As for trinucleotide TCT, TTC were the most common repeat motif in olive EST (Table 2), sequence AAG/CTT motifs were the most common in chloroplast of Olea species SSRs 29, however, in other crops such as barley, maize, rice, sorghum and wheat CCG or AAC were the most common trinucleotide repeat 30.Our results revealed that tetranucleotide motifs AATC, CTTT are the most common however the most common in Olea species SSRs chloroplast were AAAG, CTTT 29. Pentanucleotide and hexanucleotide AAAAT and GAAAAA respectively are the most common repeat motif in our results while 29 found AATCC was the most common on pentanucleotide in Olea species SSRs chloroplast and hexanucleotide was not found.Design and vali betrothal of EST-SSRIn this study, we designed 1,801 PCR primer pairs from the 8295 SSR motifs of Olea europaea EST, The designed primers were referred as Oe-ESSR_xxxx, where Oe-ESSR referred to Olea europaea EST SSR, xxxx was referred the number of EST-SSR from 1 -1801 and that different of the previously SSR primers designed from chloroplast sequences of Olea species 29. This primers were listed in the (Table S1), and provide with all information cogitate it such as Primer name, GenInfo Identifier gi number of EST sequence, Repeat type, Repeat Sequence, Length of Repeat, Repeat start on sequence, Repeat end on sequence, Forward and Reveres Primer, Tm (C), Length of Primer (bp), product Length (bp), sequence of EST, Sequence Description, gene ontology, Enzyme code and Enzyme Name.We use a sample of 25 primers randomly from these 1,801 EST-SSR primers to validate it by using a genomic DNA isolated from 9 olive cultivars. All tested primers, exhibited successfully amplified and detected polymorphism (Fig. 2).Putative Function annotation of EST-SSRsThe putative function annotation of the EST sequences containing SSR performed by used Blast2go program 27. According to the Blast2go result, 81% from EST sequence as homology with known proteins, w hile hypothetical or unknown proteins were 1.55%, and 17.37% of this EST sequences did not homology with any known proteins. The gene ontology of olive EST sequences containing SSRs using Blast2GO revealed that in the biologic processes, the highly appearance of SSR were involved in cellular processes, metabolic, response to stimulus, biologic regulation and developmental process, while Signaling, rhythmic processes and growth had the lowest SSR contents among these EST. The molecular function category includes catalytic activity and binding, while cell membrane and organelle were assigned in the cellular component category (Fig. 3). The Similar results were found on functional annotation of the citrus and date palm EST sequences containing SSRs 20, 36. Our results organization with the similar results obtained in 20 which suggested that genes were involved in protein metabolism and biosynthesis were easily conserved in plants.Functional classification by KEGG pathway analysesTh e KEGG tract analysis is useful tool to understand the molecular interaction and biologic functions 37. Our study exhibited a total of 93 different pathways include 253 enzymes rear by 381 EST-SSR primers were significant matches in the KEGG database (Table S2), this data can Visualization by using circos software 38 (fig. 4).The higher occurrence of SSR on pathways indicated a vertical potential for using these molecular markers to targeting the enzyme related to the trait subjected in our study. This EST sequences contain SSR were categorized into metabolism, as well as its subcategories, including lipid metabolism (Table 3), kale metabolism, energy metabolism, amino acid metabolism, nucleotide metabolism and metabolism of cofactors and vitamins.In details, the mapping result can further investigated against the glycolysis/gluconeogenesis (Fig. 5), Oxidative phosphorylation (Fig. 6) and Fatty acid degradation (Fig. 7) pathways as an example of Carbohydrate metabolism, Energy metabolism and Lipid metabolism respectively.ConclusionSSR markers are very important because it is co-dominant, highly polymorphous and can render from functional regions of the genome. EST-SSR technique have the potential to generate phenotypically linked functional markers and a useful tool can use on genetic diversity, marker assisted selection and genome mapping in olive. This study exhibited the functional categorization of olive EST sequences containing SSR revealed that these ESTs representing in genes with cellular component, biological process and molecular function. This EST-SSR primers also providing with useful information to understand the biological functions and genes interactions according to the localization of this primers in different pathways related to possible phenotypic differences between the olive cultivars.ReferencesGaby E, Mbanjo N, Tchoumbougnang F, Mouelle AS, Oben JE, Nyine M, et al. Development of expressed sequence tags-simple sequence repeats ( E ST-SSRs ) for Musa and their applicability in authentication of a Musa breeding population. Afr J Biotechnol. 201211(71)1354659.Naga BLRI, Mangamoori LN, Subramanyam S. Identification and characterization of EST-SSRs in finger millet (Eleusine coracana (L.) Gaertn.). J Crop Sci Biotechnol. 201215(10)916.Wen M, Wang H, Xia Z, Zou M, Lu C, Wang W. 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