Selecting among putative DNA barcodes and universal primers

Selectinga DNA barcode should be done with universally (Kocher et al.

, 1989) and consistently amplified with standardized primers,which means the higher rates of recovery of a barcode region by PCR where thesame locus was analyzed in a broad range of taxa from various phyla (Lobo et al., 2013). Instead 100% recovery itmeans the highest relative rate of recovery among putative DNA barcodes anduniversal primers may anneal in highly conserved genes (Erickson et al., 2008). According to Hajibabaei et al.

(2005) the standard recoveryshould be 95%. However, as described in Erickson et al. (2008) it should be minimum 90% for multiple geneticmarkers. As described in Ekrem et al.,(2007), the power of DNA barcode is directly proportional to the database ofbarcodes and it was increased by a complete DNA barcode database.1.1.1.

1.   Discrimination(Species differentiation)Selecting a DNA barcode should bedone with universally (Kocher et al.,1989) and consistently amplified with standardized primers, which means thehighest rates of recovery of a barcode region by PCR where the same locus wasanalysed in a broad range of taxa from various phyla (Lobo et al., 2013). Instead 100% recovery it means the highest relativerate of recovery among putative DNA barcodes and universal primers may annealin highly conserved genes (Erickson etal., 2008). According to Hajibabaei etal. (2005) the standard recovery should be 95%.

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However as described inErickson et al. (2008) it should beminimum 90% for multiple genetic markers. As described in Ekrem et al.

, (2007), the power of DNA barcodeis directly proportional to the database of barcodes and it was increased by acomplete DNA barcode databaseCowan and Fay, 2012).1.1.1.2.   LocicomplementationIndividualloci were unresolved, due to less informative phylogenetic sites (Chase et al., 2007).

A sufficient sequencevariation should be required in a useful DNA barcode to identify species. Landplants have lower intrinsic rates of sequence evolution than animals so it hashigh sequence divergence at species level. Kress and Erickson, 2007 recommendedtwo-locus global land plant barcode for species discrimination (Kress et al., 2005; Kress and Erickson, 2007;Chase et al.

, 2007; Rubinoff et al., 2005).1.1.1.      Potentialbarcode lociFormallymatK located in Large Sub Unit (LSU)with approximately 1550 bp.

Phylogenetically intergenic or inter-specificrelationships were resolved by molecular information about matK as it is the closest one to the mitochondrial gene cytochromeoxidase 1(COI) used as the animal barcode (Hollingsworth et al., 2011) with highest rate evolution out of the other plastidregions. (Barthet and Hilu, 2007; Hilu etal., 2003; Wicke and Quandt, 2009). According to the codon position of matK (Soltis and Soltis, 2004) forangiosperms it could be a universal barcode (Lahayeet al., 2008) .On other hand, there are many drawbacks of matK which might lead to unsuccessfulresults (CBOL, 2009; Hidalgo et al.

,2004) such as low PCR amplification due to unavailability of universal primersets, rapid rate of substitution with frame shift indels and presence ofpremature stop codons.Typically,rbcL has 1428 bp in length with an extremelyrare indels that encodes the large subunit of ribulose-1, 5-bisphosphatecarboxylase (RUBISCO) which is highly conserved and widely used in many planttaxa (Bousquet et al., 1992; Gaut et al., 1992; Morgan and Soltis, 1993).

This gene was initially used for comparison of higher level inter families andrecently used for intra family. Usually, the phylogenetic relationship below thefamily level is poor (Doebley et al.,1990).Chloroplastregion rpoC1 has a very high amplification rate (Sass et al. 2007) while the rate of evolution is lower than thenon-coding and some plastid genes like matK(Newmaster et al.

2008)trnH-psbAchloroplast intergenic spacer consist of psbA 3′ UnTranscribed Region andhighly variable psbA-trnH intergenic spacer (IGS) which could use for landplant determination (CBOL, 2009; Kressetal., 2005; Liu et al., 2012) withapproximately 450 bp and easily used for low taxonomic levels too (Pang et al., 2012).But, it is difficult touse trnH-psbA complex as an individual barcode (Hao et al., 2010; Storchova and Olson, 2007).ITSregion located on two sides of 5.8S genes with ITS 1 and ITS 2 in the nuclearribosomal DNA.

This is the most widely used nuclear DNA sequences to determine thebarcode gap between inter and intra-specific variations. It has been used fordiscrimination of both plants and animals (Baldwin et al., 1995; Chen et al.,2010).Li et al. (2011) studies aboutnuclear internal transcribed spacer (ITS) region as the standard barcode. ITSregions can amplify easily as the presence of highly universal primers withhigh evolutionary divergence rate (Kress etal.

2005) suggest that its use as a barcode should perhaps not bediscredited entirely and there’s a congruence between chloroplast DNA andnuclear DNA. Despite several successful attempts concerted evolution,availability of pseudogenes, Secondary structure and compensatory base changes anddifficulties in PCR are some limitations in ITS region. Incorrectidentification of species may occur due to divergent copies within theindividuals (Bailey et al., 2003;Zheng et al. 2008).

CBOL(2009)and Fazekas (2008)proposed the combination of matK and rbcL as twolocus approach. According to Hollingsworth etal. (2011) further improvement should be done due to the lack of the discriminatorypower of rbcL and primer universalityfor matK. rbcL and trnH–psbA also evaluated as multilocus approach(Ferriet al., 2008; Kress and Erickson, 2007;Kress et al., 2005; Panget al., 2012; Tripathi et al., 2013).Numerous loci fromdifferent genomic regions, including rbcL,matK, ITS, ITS2 and psbA-trnH havebeen tested with different degrees of success tabulated by Mishra (2016) shownin Table 2.