Indian J. Genet., 71(4): (2011) The sugarcane varieties under cultivation are man made complex interspecific hybrid derivatives, primarily between Saccharum officinarum L., the ‘noble’ cane and the wild S. spontaneum L., with the contributions also from S. barberi Jesw., S. sinense Roxb. S. robustum Brandes and Jesweit ex Grassl and the related genera such as Miscanthus Anderss. and Erianthus Michx. [1]. The cytoplasmic diversity in the cultivated sugarcane varieties are very limited, because only a few S. officinarum clones such as Black Cheribon, Vellai, Ashy Mauritius, Bandjermasin Hitam, and Badila were involved as female parents in their ancestry [2]. The enhancement of genetic base of sugarcane is being done by gene introgression from wild related species S. spontaneum and Erianthus arundinaceus (Retz.) Jesweit, which are cross compatible with sugarcane. Attempts are also being made to diversify the cytoplasm base also of sugarcane with the related species [3]. The homoplasmy of the organellar genomes facilitate the determination of the cytoplasm background of different species and hybrids by organellar DNA polymorphism. The conserved nature of the intergenic segments of the chloroplast and mitochondrial DNA can be PCR amplified using the universal primers identified which are common to most of the angiosperms [4, 5]. The cytoplasm of the genera Saccharum and Erianthus could be differentiated by the PCR-RFLP of the chloroplast DNA segments psbC-trnS and trnL intron [6]. There are no previous reports on the PCR primers which could differentiate the cytoplasm of S. spontaneum from that of S. officinarum. The present report is on the mitochondrial gene nad4/3-4, which differentiates cytoplasm of S. officinarum, S. spontaneum and Erianthus species. The materials used in the study were Saccharum officinarum clone 28 NG 210; sugarcane cultivars Co 775 and CoC 671; S. spontaneum clones Iritty-2, Coimbatore, SES 106 A, SES 168 and SES 515/7; Erianthus arundinaceus clones IK 76-62 and IK 76-99, and E. bengalense clone SES 240. These varieties are clonally maintained at Sugarcane Breeding Institute, Coimbatore. The genomic DNA was isolated from freshly collected young leaves using the CTAB method [7]. The nad 4/3-4 region of mitochondrial genome was amplified by a set of primer pairs: 5’-GGA GCT TTC CAA AGA AAT AG-3’and 5’-GCC ATG TTG CAC TAA GTT AC-3’ [8]. Genomic DNA was amplified in 25 μL reaction mixture containing 100 ng of template DNA, 10mM Tris-HCl (pH 9.0), 1.5mM MgCl 2 , 50mM KCl and 0.01% gelatin, 100μM of dNTPs, 0.5μM of each primer and 1 unit of taq DNA polymerase. The PCR conditions were 1 cycle of 4 min at 94°C, 35 cycles of 45 sec at 94°C, 45 sec at 57°C and 3 min at 72°C and one cycle at 72°C. The amplified products along with 1 kb DNA marker were separated by agarose-gel (1%) electrophoresis and stained with ethidium bromide [8]. For RFLP studies, the restriction endonuclease enzymes MspI (C ? CGG) and AluI (AG ? CT) were used and the PCR products were digested according to the manufacturer’s instructions (GeNei TM , Bangalore) and restriction pattern was studied after agarose-gel (1%) electrophoresis. Amplification of nad4/3-4 yielded products of different band sizes of approximately 2 kb for S. officinarum and the commercial sugarcane varieties, and 1.8 kb for S. spontaneum and Erianthus clones (Fig. 1A). The restriction of the PCR product with MspI *Corresponding author’s e-mail: premsbi@rediffmail.com Published by Indian Society of Genetics & Plant Breeding, F2, First Floor, NASC Complex, PB#11312, IARI, New Delhi 110 012 Online management by indianjournals.com Differentiation of cytoplasm of Saccharum and Erianthus species by mitochondrial DNA polymorphism V. Raffee Viola, Maya Lekshmi and M. N. Premachandran* Crop Improvement Division, Sugarcane Breeding Institute, Coimbatore 641 007 (Received: Revised: Accepted: )