Organellar Genome Diversity in Saccharum and Erianthus spp. revealed by PCR-RFLP  

Nerkar G. , Farsangi F. , Devarumath R.
Molecular Biology and Genetic Engineering Division, Vasantadada Sugar Institute, Manjari (Bk.), Pune- 412307, Maharashtra, INDIA
Author    Correspondence author
Molecular Plant Breeding, 2015, Vol. 6, No. 11   doi: 10.5376/mpb.2015.06.0011
Received: 24 Apr., 2015    Accepted: 03 Jun., 2015    Published: 19 Jun., 2015
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Preferred citation for this article:

Nerkar et al., Organellar Genome Diversity in Saccharum and Erianthus spp. revealed by PCR-RFLP, Molecular Plant Breeding, 2015, Vol.6, No. 11 1-11 (doi: 10.5376/mpb.2015.06.0011)

Abstract

The organellar genome diversity in Saccharum and Erianthus species was analysed by chloroplast deoxyribonucleic acid (cpDNA) and mitochondrial deoxyribonucleic acid (mtDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Six different chloroplast primers (psbC-trnS, trnL, psaA, clpP, matK and ccsA); and ten mitochondrial primers (nad1, nad4/1-2, nad4/2-3, nad5/1-2, nad5/4-5, 18S-5SrRNA, coxI, matR, cob and mttb) were used to amplify the genes/intergenic spacers of 8 different members of Saccharum complex namely S. officinarum, S. robustum, S. spontaneum, S. barberi, E. arundinaceus, E. ciliaris, E. elegans and CoC 671 (S. officinarum hybrid). The amplified PCR-products were digested with ten different restriction enzymes namely AluI, BamHI, BglII, DraI, EcoRI, HaeIII, HindIII, HinfI and PstI, TaqI. Our results suggest that although monomorphic bands were observed with the PCR using chloroplast and mitochondrial primers; there exists restriction fragment length polymorphism in these genes/intergenic spacers. Out of the sixty primer-enzyme combinations studied, thirty primer-enzyme combinations revealed cpPCR-RFLP while out of hundred primer-enzyme combinations studied fifty-seven primer-enzyme combinations revealed mtPCR-RFLP in sugarcane. Differentiation in Saccharum and Erianthus species was found in the psbC-trnS region of the chloroplast digested using enzyme HaeIII and also in the trnL region digested using enzyme TaqI. One new finding in our work was the mtPCR-RFLP revealed by nad4/2-3region restriction digested using enzyme AluI which was able to differentiate Saccharum species and Erianthus species. Our results may add to the knowledge about organellar genome diversity in sugarcane and may be useful for identification of sugarcane hybrids.

Keywords
PCR-RFLP; Chloroplast genome diversity; Mitochondria genome diversity; Sugarcane; nad4/2-3

Sugarcane is a tall growing monocotyledonous C4 plant with high capability for carbon fixation and sucrose accumulation. It is an important industrial crop cultivated in the tropical and sub-tropical regions of the world primarily for its ability to store high concentrations of sucrose (99.50%) or sugar in the internodes of the stem. It ranks among the world’s top 10 crops and accounts for nearly 80% of the sugar production worldwide. Traits for tolerance to biotic and abiotic stresses are particularly important for selection of cultivars in the breeding programme towards the development of elite sugarcane varieties. Besides the nuclear genome, organellar genomes also contribute to these important traits especially drought, salinity, water use efficiency, carbon assimilation and resistance to diseases. Understanding the cytoplasmic diversity of Saccharum species based on chloroplast and mitochondrial genome should facilitate an understanding of the genetic relationships of Saccharum species, which have complex nuclear genome structures and are polyploids with significant levels of chromosomal mosaicism (Burner and Legendre, 1993; D’Hont et al., (1993); Takahashi et al., (2005). The markers that target specific loci from chloroplast and mitochondrial genomes of sugarcane germplasm would be useful to evaluate the variation in these genomes within the genus Saccharum, its wild relatives and hybrids. The use of the information revealed by diversity studies of organellar genomes will facilitate to modulate the organellar genomes by genetic engineering towards transgenic development (Daniell, 2002).

As a result of the extensive research conducted in the past two decades, cpDNA analysis brought about fundamental changes to the systematics of plants. Chloroplast DNA (cpDNA) sequence variations are now widely used to investigate interspecific relationships among angiosperms and other plants (Palmer et al., 1988; Clegg et al., 1991; Liu et al., 2011a, Agrawal et al., 2014). The chloroplast genome is ideal for phylogenetic analyses of plants for several reasons. First, it occurs abundantly in plant cells and is taxonomically ubiquitous. And since it is well researched, it can be easily tested in the laboratory conditions and analyzed in comparative programs. Moreover, it often contains marker structural features cladistically useful, and, above all, it exhibits moderate or low rate of nucleotide substitution (Cleeg and Zurawski, 1992). Specific chloroplast genes and/or intergenic spacers can be amplified (Taberlet et al., 1991; Demesure et al., 1995; Tsumura et al., 1995, 1996; Dhingra 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, 1996; Lakshmi et al., 2000; Parani et al., 2000, 2001; Komatsu et al., 2001; Kishimoto et al., 2003; Nwakanma et al., 2003; Zhu et al., 2003; Asadi Abkenar et al., 2004, 2008; Van Droogenbroeck et al., 2004; Ibrahim et al., 2007; Sehgal et al., 2008; Jena et al., 2009; Poczai et al., 2011).
The cytoplasmic diversity in the cultivated sugarcane varieties is very limited, because only a few S. officinarum L. clones such as Black Cheribon, Vellai, Ashy Mauritius, Bandjermasin Hitam, and Badila were involved as female parents in their ancestry (Tew, 1987). The enhancement of genetic base of sugarcane is being done by gene introgression from wild related species S. spontaneum L. and Erianthus arundinaceus (Retz.) Jesweit, which are cross compatible with sugarcane. Attempts are also being made to diversify the cytoplasm base of sugarcane with the related species (Bakshi et al., 2007). 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). Virupakshi and Naik (2008) used lSSR markers to characterize chloroplast and mitochondrial DNA for red-rot disease in sugarcane, thereby providing a powerful complementary method for studying the genetic relationship and diversity of sugarcane species. Viola et al. (2011) reported differentiation of S. officinarum L., S. spontaneum L. and Erianthus species based on PCR-RFLP approach based on the mitochondrial gene nad4/3-4 restriction digested using enzyme AluI.
The present work aims at studying the organellar diversity in Saccharum complex using PCR-RFLP based on universal and gene-specific chloroplast and mitochondrial primers which will allow the differentiation of