Species identification in seven small millet species using polymerase chain reaction - restriction fragment length polymorphism of trnS-psbC gene region

Abstract

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCR-RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.

Organellar Genome Diversity in <i>Saccharum</i> and <i>Erianthus</i> spp. revealed by PCR-RFLP

“…Also, we included two more species from the genus Saccharum namely: Saccharum robustum and Saccharum edule and two other species from Erianthus namely: Erianthus ciliaris and Erianthus elegans in addition to the Saccharum and Erianthus species used by Premachandran et al (2006) which further confirm that the sets of primer-enzyme combinations used by Premachandran et al (2006) are able to distinguish Saccharum and Erianthus but there is not difference at this locus of psbC-trnS at interspecific level. These results differ from Parani et al (2001), Sehgal et al (2008) and Agrawal et al (2014) who could successfully differentiate Panicum, Carthamus and Eleusine species respectively at interspecific level using PCR-RFLP for psbC-trnS region using the restriction enzyme HaeIII. Unlike the reports on Carthamus (Sehgal et al, 2008) and Eleusine (Agrawal et al, 2014) the cpPCR-RFLP patterns for psaA/TaqI and psaA/AluI could not yield distinguishable patterns for Saccharum and Erianthus.…”

“…The chloroplast DNA segments psbC-trnS and trnL introns, which are non-coding sequences, were found to have interspecific polymorphism in many plant taxa (Demesure et al, 1995;Taberlet et al, 1991;Parani et al, 2001) including sugarcane (Premachandran et al, 2006). Restriction site mutations have also been reported using the chloroplast psaA region (Sehgal et al, 2008).…”

“…The PCR-RFLP approach to study organellar genome diversity has been reported in many plants (Tsumura et al, 1995(Tsumura et al, , 1996Lakshmi et al, 2000;Parani et al, 2000Parani et al, , 2001Komatsu et al, 2001;Kishimoto et al, 2003;Nwakanma et al, 2003;Zhu et al, 2003;Asadi Abkenar et al, 2004Van Droogenbroeck et al, 2004;Ibrahim et al, 2007;Sehgal et al, 2008;Jena et al, 2009;Poczai et al, 2011). This work aimed at deciphering the polymorphism in the chloroplast and mitochondrial genomes in the Saccharum spp.…”

“…Specific chloroplast genes and/or intergenic spacers can be amplified (Taberlet et al, 1991;Demesure et al, 1995;Tsumura et al, 1995Tsumura et al, , 1996Dhingra and Folta, 2005;Heinze, 2007). The amplicons can be directly sequenced or restriction endonuclease digested (PCR-RFLP) or subjected to cleaved amplified polymorphic sequence (Tsumura et al, 1995(Tsumura et al, , 1996Lakshmi et al, 2000;Parani et al, 2000Parani et al, , 2001Komatsu et al, 2001;Kishimoto et al, 2003;Nwakanma et al, 2003;Zhu et al, 2003;Asadi Abkenar et al, 2004Van Droogenbroeck et al, 2004;Ibrahim et al, 2007;Sehgal et al, 2008;Jena et al, 2009;Poczai et al, 2011).…”

“…Since the early 1990s, there have been several attempts to study the cytoplasmic genome diversity in sugarcane. The chloroplast DNA segments psbC-trnS and trnL introns, which are non-coding sequences, were found to have interspecific polymorphism in many plant taxa (Demesure et al, 1995;Taberlet et al, 1991;Parani et al, 2001;Sehgal et al, 2008;Agrawal et al, 2014). The polymorphism in the chloroplast DNA segments psbC-trnS and trnL intron amplified by polymerase chain reaction and fragmented with restriction enzymes HaeIII and TaqI, respectively could differentiate the Saccharum L. and Erianthus cytoplasm (Premachandran et al, 2006).…”