Life Sciences Leaflets FREE DOWNLOAD ISSN 2277-4297(Print) 0976–1098(Online)

ENDEMIC LILIUM MACKLINIAE SEALY (LILIACEAE): A

RECOLLECTION AND FIRST TIME DNA BARCODE

REPORT FROM NATIVE MANIPUR, AFTER PLANTING

IN POTS OUTSIDE ENDEMIC HABITAT

AYAM VICTOR SINGH DEPARTMENT OF BOTANY, RAJIV GANDHI UNIVERSITY (RGU), RONO HILLS, DOIMUKH-791112,

ARUNACHAL PRADESH.

Received on: Corresponding author’s e-mail: [email protected] 11th June 2013 ABSTRACT: Accepted on: 14th June 2013 Lilium mackliniae Sealy (Liliaceae), endemic to Siroi Hills, Ukhrul, Manipur is collected and has been enumerated with DNA sequence of taxonomic Revised on: 20th August 2013 marker chloroplast DNA segments (ribulose-1-5-bis phosphate carboxylase/rbcL and maturaseK/matK), and nuclear ribosomal DNA segment Published on: 1st Oct 2013 (Internal transcribe spacer/ITS) for identification and sequence conservation. Chloroplast rbcL gene has been found to be more reliable for L. mackliniae Volume No. Online & Print identification and sequence has been reported to NCBI. This is the first time 10(2013) report from the Manipur and India. The adaptability of the plant outside

Page No. natural habita has also been enumerated here. 01 to 08 KEY WORD: Lilium mackliniae, Imphal Valley, Genbank, Accession

nNumber, RBCL, MATK ,ITS2 Primers.

Life Sciences Leaflets is an international INTRODUCTION: open access print & Out of the estimated tens of millions of species on earth only about 1.7 million e journal, peer species could be described taxonomically [1]. Tropics which harbour the major reviewed, worldwide abstract listed, diversity of Earth’s species is under potential thread by global climate change published every month [2]. Global climate change is majorly due to human activities and possible with ISSN, RNI Free- future changes in the global climate is predicted by climatologists. With the membership, downloads and access. predicted climate change and no immediate change in the behaviour, there will be a drastic impact on the biosphere within just a few decades, with the number of species facing extinction estimated to be around one million by

http://lifesciencesleaflets.ning.com/ PEER-REVIEWED Page | 1

Life Sciences Leaflets FREE DOWNLOAD ISSN 2277-4297(Print) 0976–1098(Online)

2050 [3]. Therefore, it requires the use of tools like DNA bar coding for quicker identification of species, in biological conservation such as biodiversity survey or when traditional methods are unrevealing i.e. identification of specimen from the remains of the specimens such as egg and larvae, analysis of stomach contents or excreta to determine the food webs [4]. A DNA barcode is a short, highly variable, standardized, orthologous DNA sequence use to identify species through comparison to a DNA sequence database [5]. DNA Bar coding construct a public reference library of species identifiers which could be used to assign unknown specimens to known species [6]. One such important plant under thread, whose number is declining, is Lilium mackliniae, the state flower of Manipur [Fig.no.1]. The plant is endemic to the state. The main threats to the species and habitats are frequent forest fire and rapid colonization by a dwarf bamboo after forest fire [7]. Uncontrolled visits by tourists and collection of the Lily has also led to its severe decline [8]. The word “Lilium” (latin) derives from leirion (Greek) used by Theophrastus for Madonna lily. Lilies are horticulturally classified in Division 9 under eight sub-divisions and Lilium mackliniae comes under subdivision “H” [9]. There are approximately 2000 lily species under 200 genera of the family Liliaceae. The genus Lilium comprises of 110 to 120 species [10]. The species of the genus Lilium have the largest genomes in plant kingdom with a haploid DNA content (1C) ranging from 32.75 to 47.90 pg, which makes it a difficult crop for molecular marker techniques [11]. After 60 years of the original discovery of Lilium mackliniae by Frank kingdom Ward in 1949 from Mount Siroi (2,700m), two more Lilium species have been claimed to be discovered and spread to two other mountains, viz. Mount Saramati (3,700m), Japvo (about 3,000m) with the three peaks approximately 100km apart [12]. There are 34 recognized biodiversity hotspots in the world which cover 2.3 percent of the Earth's land surface. These hot spots have been declined by 70 percent from their original vegetation coverage. But despite the decreasing size of these hotspots they are still the home to 50 percent of the world's plant species [13]. Observing the fast changing nature in the biodiversity, continuous thread in the Lilium population and unavailability of the taxonomic characteristic genomic sequence of the endemic lily in the GeneBank library (no research report published of the plant from Manipur, India, the native to the endemic L. mackiliniae), the present study of DNA bar coding of the endemic plant was carried out.

METHODOLOGY: i) Plant material Lillium mackliniae was collected during June, 2007 from Siroi hill which is 1730m–2590m above sea level in Ukhrul district, located under 25° 7' 0N latitude and 94° 22' 0E longitude and an altitude of 1661m [14] of Manipur, India [Figure No.2], which experiences, subtropical hill and mid-temperate climate with wet summer and extreme cold, occasionally snowfall and dry winter. Plant siblings collected were planted under natural conditions without fertilizer in earthen pots at home at Imphal West, Manipur, located at 24.82°N, 93.95°E, average

http://lifesciencesleaflets.ning.com/ PEER-REVIEWED Page | 2

Life Sciences Leaflets FREE DOWNLOAD ISSN 2277-4297(Print) 0976–1098(Online)

elevation of 786metres [15] which is comparatively warmer or hotter and dryer than Siroi. The adaptability and survivality under warmer/hotter and dryer sub-tropical climate was observed. Herbarium voucher specimen is deposited at Manipur University, Kanchipur, Imphal, Manipur [MUID No.000817, 2012]. Plant material under fresh condition are performed DNA barcoding using chloroplast rbcL and matK genes with nuclear ribosomal ITS gene as markers. The DNA Sequences generated in the lab were blasted to NCBI BLAST with those available in GeneBank for checking their similar base sequences availability. The sequence of matK was not on report hence it is submitted to NCBI, USA [GenBank accession number KC539824], later it is release on May 5, 2013 and made it available to EMBL [Europe] and DNA Data Bank [Japan]. ii) DNA extraction, amplification and sequencing Genomic DNA was extracted from fresh leaves of Lillium mackliniae following the protocol associated with the Plant Genomic DNA Kit (Sigma GeneElute TM Plant Genomic DNA Miniprep kit, Sigma-Aldrich, Inc., USA). Polymerase chain reaction (PCR) of the chloroplast rbcL, matK and nuclear ribosomal ITS regions were carried out in a Mastercycler personal 120V (Eeppendorf, USA) using approximately 30ng of genomic DNA as a template in a 20 µl

reaction mixture (2µl of 10 x PCR buffer without MgCl2, 2µl of 25mM MgCl2, 2µl of 2mM dNTPs, 0.5µl each of forward and reverse primers, 0.2 µl of (5u/ µl) Tag DNA polymerase (AmpliTaq Gold® 360 DNA Polymerase, 1000U, Biosystems, USA), and 9.8µl of HPLC grade distilled water. Universal primers such as rbcL primers, matK primers and ITS2 primers were used (CCDB, Canada). The PCR products were run on a 1.0% agarose gel in 0.5XTBE buffer and purified with ExoSAP-IT treatment by incubating to PCR at 37C for 1hr then at 85C for 15 mins. The purified PCR product was sequenced by Sanger dideoxy sequencing Technology under sequencing PCR conditions in GeneAmp® PCR System 9700 (Applied Biosystems Inc.) using 1µl (~20ng) of cleaned PCR product in a 10µl reaction volume (1.86µl of 5X sequencing buffer, 0.28 µl of Sequencing Mix (Big Dye Terminator, Applied Biosystems), 0.32µl of primer (forward/reverse-10 µM), 6.45µl of distilled water (HPLC grade)). The post sequence cleaning is done using in master mix of 5 µl distilled water, 0.1 µl 0.5M EDTA and 1 µl of 3M sodium acetate and 44 µl of ethanol per 10 µl sequence PCR reaction. The cleaned up sequence is air dried and sequenced in ABI 3730DNA Analyzer (Applied Biosystems). Gene scan is done in 1.0 µl PCR product using ABI 3730 in the sample volume of 20 µl obtained from the mixture of 20 µl Hi-Di Formamide and 0.5 µl of GeneScan 600 Liz-Size standard.

http://lifesciencesleaflets.ning.com/ PEER-REVIEWED Page | 3

Life Sciences Leaflets FREE DOWNLOAD ISSN 2277-4297(Print) 0976–1098(Online) iii) Sequence alignment and data analysis: Separate sequences forward (F) and reverse (R) were obtained for each of the chloroplast rbcL and chloroplast matK and nuclear ribosomal ITS marker genes of Lillium mackliniae and extracted the sequences using Sequence Scanner (Applied Biosystems) and by alignment following Assembly Method using Geneious Pro 5.0.3, the consensus sequences for each of the markers were obtained. The consensus sequences of the each of the markers were perform nucleotide BLAST to confirm whether marker sequence of the plant is already in the GeneBank and so as to make a new submission or if already submitted confirming for the correct identification and checking of the best BLAST hit for the query sequence.

RESULT: Measurement of DNA Sequences alignment for Lillium mackliniae for matK and ITS2 markers in nucleotide BLAST shows 100% similarity score with the one reported in NCBI, GeneBank from Japan. There was no sequence report for rbcL genes and was submitted as a new sequence submission by direct submission to NCBI and was assigned with the accession number KC539824. The sequence results for matK and ITS are comparatively inferior than rbcL as both contains wrong and mismatch pair with a few stop codons within the length of the sequences. No phylogenetic tree is constructed as the study focuses on the submission of the new rbcL marker sequence to GeneBank for open access and public use and not on their phylogeny. The nucleotide and a respective protein sequence of the marker gene are given bellow:

GenBank flat file: LOCUS KC539824 749 bp DNA linear PLN 30-APR-2013 DEFINITION Lilium mackliniae voucher 000817 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene, partial cds; plastid. ACCESSION KC539824 VERSION KC539824 KEYWORDS . SOURCE plastid Lilium mackliniae ORGANISM Lilium mackliniae Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida;

Liliales; Liliaceae; Lilium.

REFERENCE 1 (bases 1 to 749) AUTHORS Singh,A.V.

http://lifesciencesleaflets.ning.com/ PEER-REVIEWED Page | 4

Life Sciences Leaflets FREE DOWNLOAD ISSN 2277-4297(Print) 0976–1098(Online)

TITLE DNA sequence of Lilium mackliniae Sealy using rbcL primer JOURNAL Unpublished REFERENCE 2 (bases 1 to 749) AUTHORS Singh,A.V. TITLE Direct Submission JOURNAL Submitted (28-JAN-2013) Botany, Rajiv Gandhi University, Rono Hills, Doimukh, Arunachal Pradesh 791112, India COMMENT ##Assembly-Data-START## Assembly Method :: Geneious Pro v. Geneious Pro 5.0.3 Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##

FEATURES Location/Qualifiers source 1..749 /organism="Lilium mackliniae" /organelle="plastid" /mol_type="genomic DNA" /specimen_voucher="000817" /db_xref="taxon:82321" /country="India: Siroi Hills, Ukhrul District,

Manipur" gene <1..>749 /gene="rbcL" CDS <1..>749 /gene="rbcL" /codon_start=3 /transl_table=11 /product="ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit" /protein_id="AGK45626"

Protein sequence

/translation="PQTETKASAGFKAGVKDYKLTYYTPDYETKDTDILAAFRVTPQP GVPPEEAGAAVAAESSTGTWTTVWTDGLTSLDRYKGRCYHIESVVGEENQYIAYVAYP LDLFEEGSVTNMFTSIVGNVFGFKALRALRLEDLRIPTSYSKTFQGPPHGIQVERDKL NKYGRPLLGCTIKPKLGLSAKNYGRAVYECLRGGLDFTKDDENVNSQPFMRWRDRFLF CAEALYKAQAETGEIKGHYLNATAGTCEKKK"

http://lifesciencesleaflets.ning.com/ PEER-REVIEWED Page | 5

Life Sciences Leaflets FREE DOWNLOAD ISSN 2277-4297(Print) 0976–1098(Online)

ORIGIN (nucleotide sequence) 1 caccacaaac agagactaaa gcaagtgctg gattcaaagc tggtgttaaa gattacaaat 61 tgacttatta tactcctgac tatgaaacca aagatactga tattttggca gcattccgag 121 taactcctca acccggagtt ccacccgaag aggcaggggc agcggtagcc gccgaatctt 181 ccactggtac atggacaact gtgtggactg atggacttac cagtcttgat cgttacaaag 241 ggcgatgcta ccacatcgag agcgttgttg gggaggaaaa tcaatatatt gcttatgtag 301 cttatccttt agaccttttt gaagaaggtt ctgttactaa catgttcact tccattgtgg 361 gtaatgtatt tggtttcaaa gccctacgag ctctacgtct ggaggatctg cgaattccta 421 cttcttattc caaaactttt caaggcccgc ctcatggcat ccaagttgaa agggataaat 481 tgaacaagta tggtcgtccc ctattaggat gtaccattaa accaaaattg ggattatctg 541 caaagaacta tggtagagcg gtttatgaat gtctgcgcgg tggacttgat tttaccaagg 601 atgatgaaaa cgtgaactca caaccattta tgcgttggag agaccgtttc ttattttgtg 661 ccgaagcact ttataaagcg caagccgaaa cgggtgaaat caaaggacat tacttgaatg 721 ctactgcagg tacatgcgaa aaaaaaaaa

Growing of Lillium outside endemic habitat

Lilium mackliniae was once claimed as endemic to moist, rainy, cold, high altitude mountain of Siroy hills of Ukhrul district of Manipur, India. The habitat of Lillium has been reported to be deteriorated due to frequent forest firing, trace passers, tourists, visitors and climate change. The plant is deminising from the natural habitat, due to climate change and various other stress factors. When plants are under stress they develop more amount of secondary metabolites or a change in the secondary metabolite occurs while trying to adapt to the changing environment. Secondary metabolites increase in response to elevated temperatures [16], but reported decreased[17]. The responses of secondary chemicals to increased temperature are less well understood, although, an increase in volatile organic compounds has been generally detected [18], which at any case means there is a change in the secondary metabolite due to climate change. The warming temperature and rising CO2 level could alter growth cycles of alpine plants and active constitutes of the plants may change due to physiological changes [19]. An attampt was made by collecting the L. mackliniae and planting in pots with the available soil without treatments and special nutrient supplimentation just like other potted plants, surprisingly the plant grows and flowers as in their natural habitat. This shows that the plant have undergone the needed changes to aclamatize the existing changed environment which outside their natural endemic habitat. Though facing stress condition in their natural habitat, it acquires more resiliant characters to adapt to new habitats, thus from endemism it may become cosmopolitant in the near future.

DISCUSSION: From the nucleotide BLAST result of rbcL and ITS markers the sequences under BLAST shows highest similarity to Lilium mackliniae, it was noticed that the sequences contain internal stop codons and/or

http://lifesciencesleaflets.ning.com/ PEER-REVIEWED Page | 6

Life Sciences Leaflets FREE DOWNLOAD ISSN 2277-4297(Print) 0976–1098(Online) reading frame shifts (insertions/deletions based on BLAST similarity search results and/or an alignment. This indicates errors in the nucleotide sequence, which is of low quality, in rbcL and ITS markers. There could be problems with feature annotation which may be due to: translation in the wrong reading frame or on the wrong strand translation and will not have the correct exon spans translation includes noncoding sequence, such as introns or UTR's. On the contrary matK marker shows better sequence result without stop codons and the matK sequence of L. mackliniae is not yet reported by any researcher to any GeneBank from elsewhere, and it’s the first report made of Lilium mackliniae for matK marker to be made available for public domain.

ACKNOWLEDGEMENT: Special thanks to the local people for helping in plant collection, thanks to MU, herbarium repository, Department of Botany, Kanchipur, for facilitating the submission of the collection. And Thanks to RGCB, Thiruvananthpuram, Kerala for the technical support and RGU, Rono Hills, Arunachal Pradesh, for the partial financial support during the study.

REFRENCES: 1. Vernooy R, Haribabu E, Muller mr, Vogel Jh, Hebert PDN, et al. (2010). Barcoding life to conserve biological diversity: beyond the taxonomic imperative. Plos Bio 8: e1000417. Doi: 10.1371/Journal.pbio.1000417.

2. Bradshaw CJA, Sodhi NS, Brook BW (2009). Tropical turmoil: a biodiversity tragedy in progress. Front Ecol Environ 7: 79-87. Doi: 10.1890/070193.

3. Savolainen V, Reeves G (2004). A plea for DNA banking. Science 304: 1445-1445. Doi: 10.1126/science. 304.5676.1445b.

4. Stoeckle M (2003). Taxonomy, DNA,and the Bar Code of Life. BioScience: 53:9 2-3.

5. Maia VH, Mata CSd, Franco LO, Cardoso SRS, et al. (2012). DNA Barcoding Bromeliaceae: Achievements and Pitfalls. PloS ONE 7(1): e29877. Doi: 10.1371/journal.pone.0029877.

6. www. Barcoding.si.edu/whatis.html

7. Mao A. A., & R. Gogoi. (2013). Lilium Mackliniae Sealy an Endemic Lily of North East India, its Distribution and Status in the Wild. The Indian Forester. 139(2), Abstract.

8. Important Bird Areas in India – Manipur. C739M739Y739K739.

9. www.pecificbulbsociety.org/pbslist/2003-june/n2818putmap2bm33dhve367680.html

10. www.bdlilies.com/research.html

11. Tuyl J.M.V., Arens P., Ramanna M.S., Shahin A., Khan N, Xie S., Marasek-Ciolakowska A., Lim K.B. and R. Barba-Gonzalez (2011). Wild Crop Relatives: Genomic and Breeding Resources. Springer pub. pp 161-183.

12. http://www.rhs.org.uk/Plants/RHS-Publications/Journals/The-Plantsman/2010- http://lifesciencesleaflets.ning.com/ PEER-REVIEWED Page | 7

Life Sciences Leaflets FREE DOWNLOAD ISSN 2277-4297(Print) 0976–1098(Online)

issues/March/Variation-in-Lilium-mackliniae.

13. http://edition.cnn.com/2010/TECH/science/05/07/biodiversity.hotspots/index.html

14. http://www.fallingrain.com/world/IN/17/Ukhrul.html.

15. http://en.wikipedia.org/wiki/Imphal.

16. Litvak ME, Constable JVH, Monson RK (2002). Supply and demand processes as controls over needle mononterpene synthesis and concentration in Douglas fir [Pseudotsuga menziesii (Mirb.) Franco]. Oecologia. 132: 382–391.

17. Snow MD, Bard RR, Olszyk DM, Minster LM, Hager AN, Tingey DT (2003). Monoterpene levels in needles of Douglas fir exposed to elevated CO2 and temperature. Physiol. Plantarum., 117: 352–358.

18. Loreto F, Barta C, Brilli F, Nogues I (2006). On the induction of volatile organic compound emissions by plants as consequence of wounding or fluctuations of light and temperature. Plant Cell Environ., 29: 1820–1828.

19. Chaturvedi AK, Vashistha RK, Prasad P, Nautiyal MC (2007). Need of innovative approach for climate change studies in alpine region of India. Curr. Sci., 93(12): 1648-1649.

http://lifesciencesleaflets.ning.com/ PEER-REVIEWED Page | 8