Genetic Diversity in Species of Abutilon Mill. from North-West India

Arneet Grewal and Ramneet Kaur*

Department of Botany, Punjabi University, Patiala 147002, Punjab, India

Received September 15, 2019; accepted October 24, 2019

Summary The course of male meiosis was studied in six species of Abutilon collected from different localities in North-West India. Abutilon bidentatum, A. indicum, A. muticum, A. pannosum and A. theophrasti showed gametic chromosome number n=21 and were found to be hexaploid (6x). A. ramosum was found to be diploid (2x) with chromosome number n=8. A. muticum (n=21) has been worked out for the ﬁrst time. The present study revealed that genus Abutilon is dibasic having basic chromosome numbers x=7 and 8. The genetic divergence among the selected accessions of six species of Abutilon was analyzed by using ITS of rDNA as a DNA marker. The sequences of ITS showed divergence at 126 nucleotide positions. Two major clades of Abutilon species were resolved in the phylogenetic tree with basic chromosome numbers x=7 and 8. A. ramosum (x=8) lies genetically between Abutilon species with x=7 and those species of Abutilon already shifted to genus Callianthe with x=8.

Keywords Abutilon, Chromosome number, ITS, Molecular phylogeny, Basic number.

Abutilon is a medicinally important genus of Malva- A. ramosum and A. theophrasti was evaluated by col- ceae consisting of about 160 species (Donnell 2012). It lecting the germplasm from the North-Western region includes annual or perennial herbs, shrubs or even small of India (Table 1). The voucher specimens were depos- trees, which are widely distributed in the tropical and ited in the Herbarium of Department of Botany, Punjabi subtropical regions of the world (Sivarajan and Pradeep University, Patiala and accession numbers (PUN) were 1996). The genus is represented by 18 species in India, obtained. of which 12 species found in North-West India (Bamber Gametophytic chromosome number was determined 1916, Babu 1977, Sharma and Tiagi 1979, Chowdhery by studying male meiosis. Young floral buds were fixed and Wadhwa 1984, Kumar 2001, Singh et al. 2002). in Carnoy’s fixative (ethanol : chloroform : glacial acetic The species of Abutilon are characterized by the pres- acid=6 : 3 : 1) for 24 h then transferred to 70% ethanol ence of pubescent or rarely glabrous stem, ovate leaves till further use in a refrigerator. Anthers were smeared with cordate base, pentamerous and actinomorphic flow- in 1% acetocarmine on a slide for observing pollen ers, monadelphous stamens with monothecous anthers, mother cells (PMCs). Several slides were prepared for carpels with capitate stigma, presence of echinate pol- each collection to determine the chromosome number. len, schizocarpic fruit-bearing five to numerous meri- Microphotographs of the preparations were taken using carps and absence of epicalyx. Abutilon is found grow- a Nikon Eclipse 80i microscope equipped with a digital ing under a wide temperature range of 5°C to 40°C. The camera. plants are distributed up to an altitude of 1500 m (Ra- Molecular studies were carried out on the DNA ex- jurkar et al. 2009). Abutilon plants mostly inhabit waste- tracted from young leaves of each species. Genomic lands and are found commonly along the roadsides in DNA was extracted and purified by using the protocol North-Western region of India. During the present study, as given in HiPurA Plant Genomic DNA Miniprep Pu- an attempt was made to determine the gametophytic rification Kit (Sigma-Aldrich). The extracted DNA was chromosome number and molecular phylogenetic rela- stored at -20°C until further use. DNA amplification tionship of the species of Abutilon from North-Western of ITS region of nuclear DNA was done by PCR using region of India. ITS-4 and ITS-5 primers (White et al. 1990). The PCR was performed using 50 µL reaction mixture under fol- Materials and methods lowing thermal cycling conditions: Initial denaturation at 94°C for 5 min, 35 repeats of denaturation at 94°C for The genetic diversity of six species of Abutilon viz. 1 min, annealing of primers at 55°C for 1 min, exten- A. bidentatum, A. indicum, A. muticum, A. pannosum, sion of primers at 72°C for 1 min and final extension at 72°C for 5 min using the Thermocycler (Bio-Rad). The * Corresponding author, e-mail: [email protected] sequence of amplified DNA was obtained by outsourcing DOI: 10.1508/cytologia.85.45 to Chromous Biotech Pvt. Ltd., Bengaluru and SciG- 46 A. Grewal and R. Kaur Cytologia 85(1)

Table 1. Localities, chromosome number, ploidy level of six Abutilon species examined.

Species Voucher Number (PUN) Locality Chromosome number (n) Ploidy level

A. bidentatum Hochst. ex A. Rich. 59683 Sangrur (PB) 21 6x A. indicum (L.) Sweet 59686 Patiala (PB) 21 6x A. muticum Del. ex DC. 59685 Bikaner (RJ) 21 6x A. pannosum (G. Forst.) Schltdl. 59994 Bikaner (RJ) 21 6x A. ramosum (Cav.) Guill. & Perr. 59801 Sangrur (PB) 8 2x A. theophrasti Medik. 59684 Patiala (PB) 21 6x

PB: Punjab; RJ: Rajasthan enom Labs Pvt. Ltd., Cochin, Kerala. The ITS sequences of studied species were analyzed with a sequence dataset consisting of 22 species of Abutilon retrieved from GenBank (NCBI) including those shifted to genus Callianthe. Sida spinosa and Gos- sypium robinsonii were also retrieved from GenBank as outgroup sequences. The alignment of nucleotide sequences was done by ClustalW (Thompson et al. 1994). The phylogenetic tree was generated by using the maximum likelihood method based on the Tamura-Nei model (Tamura and Nei 1993) using software of molecular evolutionary genetics analysis (MEGA) version 6.06 (Tamura et al. 2013).

Results

Cytological studies revealed that the accessions of A. bidentatum, A. indicum, A. muticum, A. pannosum and A. theophrasti were hexaploid with chromosome number n=21 based on basic chromosome number x=7, while A. ramosum was diploid with a meiotic chromosome count of n=8 based on basic chromosome number x=8 (Table 1, Fig. 1). The ampliﬁed nuclear DNA of the studied accessions was observed to be nearly 800 bp long comprising of partial sequences of 18S rRNA gene, the complete Fig. 1. (a) PMC of A. bidentatum showing 21 bivalents at metaphase sequence of ITS 1, 5.8S RNA gene and ITS 2 and I. (b) PMC of A. indicum showing 21 bivalents at metaphase partial sequence of 26S rRNA gene. Analysis of the I. (c) PMC of A. muticum showing 21 bivalents at Diakinesis. sequences revealed genetic variation among present ac- (d) PMC of A. pannosum showing 21 bivalents at metaphase I. (e) PMC of A. ramosum showing 8 bivalents at metaphase cessions of Abutilon. The species showed divergence at I. (f) PMC of A. theophrasti showing 21 bivalents at meta- 126 nucleotide positions (48 positions in ITS 1 region, phase I. Scale bars=10 µm. 4 in 5.8S and 74 in ITS 2 region) in the sequenced region. The complete ITS sequences of A. bidentatum, A. with A. pannosum (bootstrap value=70%). A. ramosum indicum, A. muticum, A. pannosum, A. ramosum and (x=8) formed a different clade (bootstrap value=42%) A. theophrasti were submitted to NCBI (see accession from other species of Abutilon having basic chromosome numbers in Fig. 2). The sequence data generated from number x=7. the molecular studies were used to prepare the phylogenetic tree to inter-relationship among the species Discussion studied at present along with the previously submitted sequences of species of Abutilon (Fig. 2). It was evident The chromosome numbers (n=21) for A. bidentatum, that species from Punjab and Rajasthan formed separate A. indicum, A. muticum, A. pannosum and A. theophrasti clades. A. indicum showed a close genetic relationship are in line with the previous records (Skovsted 1935, with A. fruticosum. Similarly, A. bidentatum showed a Ford 1938, Podlech and Dieterle 1969, Bir and Sidhu close relationship with A. theophrasti and both formed 1978, 1979, 1980, Uhrikova and Majovsky 1980, Krish- a sister clade with 82% bootstrap value as showed by nappa and Munirajappa 1983, Husain et al. 1988, Muni- the phylogenetic tree. A. muticum formed a sister clade rajappa and Krishnappa 1993, Cheng and Tsai 1999 and 2020 Genetic Diversity of Genus Abutilon Mill. 47

a need for analysis of more samples of other Abutilon species with base numbers x=7, 8 and 9 for establishing the tribasic nature of the genus and its relation with Cal- lianthe at the molecular level.

Acknowledgements

The authors acknowledge the ﬁnancial and material facilities provided by UGC and DST (New Delhi) under DRS, DSA and WOS-A programmes. Thanks are due to the Head, Department of Botany, Punjabi University, Patiala for providing the necessary infrastructural facilities. The authors also thankful to IPLS-DBT Project, Punjabi University, Patiala for providing instrumental facilities for molecular analyses.

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