1 RESEARCH ARTICLE Nucleotide diversity and phylogenetic relationships among Gladiolus cultivars and related taxa of family Iridaceae NIRAJ SINGH1, BALESHWAR MEENA1, ASHISH KUMAR PAL1, ROOP KUMAR ROY1, SRI KRISHNA TEWARI, SUSHMA TAMTA2 AND TIKAM SINGH RANA1* 1CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow -226001, India 2Department of Botany, D. S. B. Campus, Kumaun University, Nainital-263001, Uttarakhand, India Running Title: Nucleotide diversity in Gladiolus cultivars E-mail: [email protected]; [email protected] Keywords Gladiolus, Iridaceae, Plastid genome, Phylogenetic analyses Abstract The plastid genome regions of two intergenic spacers, psbA-trnH and trnL-trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA-trnH region was higher than trnL-trnF region of chloroplast. We employed Bayesian, maximum parsimony and Neighbor-Joining approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA-trnH and trnL-trnF intergenic spacers of Gladiolus and related taxa like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis, Asphodeline lutea) were used in the present investigation. Results showed that Sub-family Iridoideae have sister lineage with sub-family Ixioideae and Crocoideae. Hymenocallis littoralis and Asphodeline lutea were separately 2 attached at the base of tree as the diverging Iridaceae relative’s lineage. Present study revealed that psbA-trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL-trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop. Introduction The genus Gladilous L. is comprised of about 265 species in the world, and is one of the largest genera of family Iridaceae. The Cape of Good Hope (South Africa) is considered to be the centre of diversity for the genus Gladiolus. It is distributed throughout tropical Africa, Madagascar, Arabian Peninsula, the Mediterranean basin, Europe and Asia including Iran and Afghanistan (Goldblatt and Manning 1998; Goldblatt et al. 2001). The basic chromosome number is x=15, ranging from diploid (2n=30) to hypododecaploid (2n=180), (Bamford 1935; Ohri and Khoshoo 1983). Hybridization and polyploidy have been greatly responsible for the evolution of Gladiolus (Ohri and Khoshoo 1983). Gladiolus is out-breeding in nature, and exhibited diverse pollination mechanism (Ohri and Khoshoo 1983; Goldblatt and Manning 2002). Gladiolus cultivars possess enormous diversity in colours, sizes, textures, numbers and types of flowers and forms of inflorescence (Buch 1978; Ohri and Khoshoo 1985b; Anderson and Park 1989). In India, Gladiolus is primarily grown in the states of Uttar Pradesh, Uttarakhand, Himachal 3 Pradesh, Haryana, Delhi, Karnataka, Punjab, West Bengal, Assam, Sikkim and Meghalaya, having sub tropical climates. Artificial hybridization and selection for desired phenotypes have already produced large number of cultivars. Phenotypic characters have been widely used to characterize and identify the cultivars; however, it is often difficult and even misleading in case of specific cultivars. Therefore, molecular characterization of different cultivars of Gladiolus is very significant in understanding the genetic relationships among the cultivars and its close relatives in the family Iridaceae. Characterization of different cultivars/genotypes using molecular markers will not only help in establishing the relationship and affinities among various taxa, but will also pave the ways for selection of elite germplasm from large sets of parental genotypes, and could further be used in broadening the genetic base of Gladiolus through breeding (Pragya et al. 2010). Earlier, Gladiolus cultivars have been investigated using RAPD (Takatsu et al. 2001; Jingang et al. 2006; Pragya et al. 2010), ISSR (Jingang et al. 2008; Raycheva et al. 2011) and AFLP (Ranjan et al. 2010) to study the variability. It is well known that most of the angiosperm species, the nuclear genome is bi-parentally inherited and can spread by pollens and seeds, whereas chloroplast and mitochondrial genome is maternally inherited and spread only by seeds (Greiner et al. 2015). However, there is no comprehensive study available on the characterization of varied Gladiolus cultivars using chloroplast DNA regions like psbA-trnH and trnL-trnF intergenic spacers. However, plastid DNA regions, rbcL, rps4, trnL intron, trnL-F, matK, and rps16 have been used to infer phylogenetic analysis and molecular systematics of family Iridaceae (Souza-Chies et al. 1997; Reeves et al. 2001; Goldblatt 2011). The chloroplast genome has been extensively used for phylogenetic reconstructions in plant systematics, because of its relatively small size and conservative mode of evolution. In the present study, we therefore, aimed to estimate the nucleotide diversity and establish phylogenetic relationships among Gladiolus cultivars and related taxa of family Iridaceae 4 using two plastid genome markers. Material and methods Plant materials and isolation of genomic DNA In the present study 66 Gladiolus cultivars were procured from the Botanic Garden of CSIR- National Botanical Research Institute (CSIR-NBRI), Lucknow (India). Total genomic DNA was extracted from young fresh leaves of all the collected genotypes following CTAB method (Doyle and Doyle 1990). Quantitation of purified DNAs was carried out by using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., USA). PCR amplification and sequencing The primer sequences of psbA (5′-GTTATGCATGAACGTAATGCTC-3′), trnH (5′- CGCGCATGGTGGATTCACAAATC-3′), trnL (5’-AAAATCGTGAGGGTTCAAGTC-3’), trnF (5’-GATTTGAACTGGTGACACGAG-3’) available in the public domain (Shinozaki et al. 1986; Taberlet et al. 1991) were custom synthesized from Sigma Aldrich Chemicals Pvt. Ltd. India. The amplification of chloroplast region psbA-trnH and trnL-trnF intergenic spacers was performed in 20 μl reaction volume with the following components: 10 μl 2X phusion master mix, 1 μl (10 picomole) forward primer, 1 μl (10 picomole) reverse primer, 2 μl genomic DNA (40ng) and water 6 μl (protease, DNase, RNase free) using Proflex PCR System (Applied Biosystems, Life Technologies, USA). After initial denaturation at 98 °C for 30 s, each cycle consisted of 10 sec denaturation at 98 °C, 20 s of annealing at 54 °C, 30 s of extension at 72 °C and a final 5 min extension at 72 °C at the end of 35 cycles. The amplified products obtained were electrophoresed on 0.8% agarose gels in 1x TBE buffer at a constant voltage 5 V/cm. After electrophoresis gel was stained in ethidium 5 bromide, visualized and archived using gel documentation system (UV Technology, UK). A known DNA ladder of 100 base pair differences was also loaded on the first well of the gels. Since the size of psbA-trnH and trnL-trnF regions of Gladiolus cultivars were known (approximately 600 bp and 700 bp respectively), the bands of interest were identified with reference to DNA ladder loaded in the gels, were excised for purification of DNA. PCR products eluted from the agarose gel were purified using QIAquick Gel Extraction Kit (QIAGEN). Quantitation of purified PCR products was carried out by UV spectrophotometery using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., USA). Sequencing was conducted using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the ABI 3730 (Applied Biosystems). After the completion of capillary gel electrophoresis the fluorescence data were displayed as an electropherogram with the help of data collection software (ABI PRISM® DNA Sequencing Analysis Software v.5.0). Only, sequence data with reliable read lengths were considered in the present study. The sequences determined for the amplified psbA-trnH and trnL-trnF regions were uploaded to the EMBL/GenBank nucleotide database for storage and archiving. Phylogenetic analysis In the present phylogenetic analyses, a total of 86 accessions of psbA-trnH and trnL-trnF intergenic spacer sequences, comprising 66 Gladiolus cultivars, 18 accessions of other taxa representing family Iridaceae (Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis) and two out group species Hymenocallis littoralis and Asphodeline lutea were considered. The sequences of interest available in the NCBI database were downloaded to use as a supplement in the phylogenetic study of Gladiolus cultivars. The names of the taxa 6 downloaded from GenBank have been presented in table 1, along with their accession numbers. The chloroplast sequences were aligned with Clustal W (Thompson et al. 1994) using Bio Edit Sequence Alignment Editor (Hall 1999), MEGA6 software (Tamura et al. 2013) and MUSCLE (Edgar 2004). The alignment of sequences was further examined and edited manually as well. The boundaries