Review of Literature Chapter 2

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REVIEW OF LITERATURE

In this chapter the literature with respect to the historical aspect of Genus

Berberis was first reviewed followed by three subsequent sections dealing with

Berberis systematics, medicinal aspects and molecular approaches. Medicinal plants

are the most important source of life saving drugs for the majority of the world’s

population. The analysis of genetic diversity and relatedness between or within

different populations, species, and individuals is a central task for many disciplines of

biological science. Classical strategies for the evaluation of genetic variability, such

as comparative anatomy, morphology, embryology, and physiology, have increasingly

been complemented by molecular techniques. These include the analysis of chemical

constituents, but most importantly relate to the development of molecular markers.

Marker technology based on polymorphisms in proteins or DNA has catalysed

research in a variety of disciplines such as phylogeny, taxonomy, ecology, genetics

and plant and animal breeding (Weising et al., 2005).

2.1 Determining Berberis Systematics:

Berberis is the largest genus in Berberidaceae (including Mahonia) (Ahrendt,

1961). There are conflicting views on generic delimitation in Berberis and Mahonia.

Ahrendt (1961) postulated that simple leaved Berberis are derived from compound

leaved Mahonia and that described Mahonia as distinct genus. Studies examining

chromosome number and floral anatomy (Terabayashi, 1978) found that there were no

significant differences between Berberis and Mahonia which suggests a close

phylogenetic relationship.

The family Berberidaceae was first established by A.L. de Jussieu (1789) as

‘Berberides’, was considered as one of the most primitive groups of Angiosperms. A

revision of the genus Berberis was done by Schneider (1905 and 1908), who recorded

13 new species and one variety from Indian region. Fedde (1902) published first

monographic work on Genus Mahonia. This was followed by a detailed work genus

Mahonia by Takeda (1917), who reported 44 species from of old world and Ahrendt

(1942-45) had published a monograph of section Wallichianae in which he

recognized 71 species in eight subsections, Ahrendt (1945) surveyed the Berberis

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species from Bhutan, Assam, South Tibet, Upper Burma and North West Yunnan, and

later Ahrendt (1961) published a detailed world revision of species of Berberis and

Mahonia.

Chamberlain and Hu (1975) revised section Wallichianae and treated 11

species, including one new species Berberis victoriana from Indian region. Jafari

(1975), in Flora of West Pakistan included only one species of Mahonia and 15

species of Berberis from Kashmir region. Landrum (1999) while doing work on

‘Revision of Berberis (Berberidaceae) in Chile and Adjacent Southern Argentina, had

reported 22 species of Berberis were identified from continental Chile, the Juan

Fernandez islands, and adjacent southern Argentina and none of the species are

common with Asian species.

Hooker (1875) for the first time compiled the taxonomic information available

until then for Berberidaceae in India. He had vast field knowledge and deep

understanding of Indian Berberidaceae as he spent several years in North-eastern

India. He included six genera and seventeen species under this family in his

monumental work ‘Flora of British India’. However, this treatment considered too

general and subsequent workers split up these species into 17 taxa and added many

more taxa which were new.

Chatterjee (1953) included 68 species of Berberis, 11 species of Mahonia, one

species of Epimedium and 2 species of Podophyllum in this family from I ndia. While

Ahrendt (1961) reported 52 species of Berberis and 11 species of Mahonia from

India. Several other floristic done work in different parts of India, Collett (1902)

while doing 'Flora of Simla district’ had reported five species of Berberis and one

species of Podophyllum. Rao and Hajra (1993) while treating the family for the ‘Flora

of India’ included 54 species of Berberis, one species of Epimedium and 13 species of

Mahonia from the Indian region. Aswal and Mehrotra (1994) recorded four species of

Berberis from Lahulspiti district of Himachal Pradesh. A work carried out by Rao et

al. (1998 a and b) reported 55 species of Berberis in India. Among all the Himalayan

states, Uttarakhand has the highest number of species (29 including sub-species). The

number of Berberis species (including sub-species) in Pakistan, Jammu and Kashmir,

Himachal Pradesh and Sikkim are 24, 25, 23 and 16 respectively (Jafari, 1975; Rao et

al., 1998 a and b).

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General collection of plants including Berberidaceae, mentioned in various

treatises. Collection of Strachey and Winterbottom that was revised by Duthie (1903-

1929) recorded two species of Holboellia and 11 species of Berberis, Osmaston

(1927) reported 13 species of Berberis, one species of Mahonia and two species of

Holboellia in his ‘Forest flora of Kumaon’. Naithani (1984) had reported 11 species

of Berberis and one species of Mahonia from Chamoli district of Uttarakhand. While

enumerating the plant wealth of Nanda Devi Biosphere Reserve (Hajara 1983, Hajra

and Balodi 1995 and Samant 1999) had reported 7 species of Berberis from the

vicinity. In ‘Flora of Garhwal’ by Gaur (1999) had reported 6 species of Berberis and

one species of Mahonia from the Pauri district in state. Rana et al. (2003) while

dealing with ‘Flora of Tons valley’ had reported 8 species of Berberis and one species

of Mahonia. 29 taxa of Berberis and 4 species of Mahonia are reported from

Uttarakhand by Uniyal et al. (2007). Tripathi et al. (2009) investigated different

gymnosperms in Nainital and also located 3 species of Berberis (B. aristata, B.

asiatica and B. chitria) in Banj oak forest of Nainital. Recently, Berberis rawatii sp.

nov. has been described by Tiwari and Adhikari (2011) from Uttarakhand.

2.2 Endangered status

Populations of many plant species, particularly threatened endemic taxa, have

sharply declined in many parts of the world during recent decades as a result of

habitat degradation, fragmentation and overexploitation (Colling and Matthies, 2006;

Dar et al., 2006 a, b and 2008). Small populations face the risks of extinction due to

demographic, environmental and genetic stochasticity (Bruna and Kress, 2002). Such

effects are further exacerbated by spatial isolation of the populations that limits the

replenishment of genetic variation and gene flow. Degree of out breeding in small

isolated populations may be severely constrained because such populations

experience reduced pollinator visitation and altered foraging behaviour of pollinators

(Byers, 1995). Massey and Whitson (1980) emphasized on the need for detailed

information on the life cycle of rare plant species in order to preserve them. Further,

several authors (e.g., Kruckeberg and Rabinowitz, 1985; Kunin and Gaston, 1993;

Linhart and Premoli, 1993) have encouraged research on the biology of rare plant

species to understand the causes of their rarity. However, the significance of

information for a single species, and in particular a rare one, is hard to evaluate unless

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comparisons are made with geographically-widespread, closely-related species

(Bradshaw, 1987). Information on the autecology of widespread species, based on

experimental research (Walck et al., 1997) or by literature review (Baskin et al.,

1997) serves as reference point for comparing with related rare endemic species for

which fewer ecological parameters may have been studied. Balodi (1995) studied on

the rare and endangered species of Pithoragarh and talk about their conservation.

Aswal and Goel (1985) had mention rare and lesser known plants of Garhwal and

Kumaon. Atkinson (1982) had given reference about ‘Flora of Garhwal and

Kumaon’, and Khanna (2001) has talked about endemic plants of Uttar Pradesh.

Autecology of narrow endemic species becomes all the more crucial for

understanding the phytogeography, adaptive evolution and associations and

dispersion (Huston, 1994). The genus Berberis exhibits an interesting pattern of

distribution in India. Of the 55 species recorded from the country, 16 species and 8

sub-species are endemic. Of these, about 19 taxa (11 species and 7sub-species and

varieties) are endemic to the Indian Himalayan region (Rao et al., 1998; Tiwari and

Adhikari, 2011). Western Himalaya and the state of Uttarakhand have 11 and six

endemic taxa of Berberis respectively. It is governed largely by climatic factors and

can be studied at varying scales from individual to community levels (Pau et al.,

2011). Hence, variation among species in their phenology is an important mechanism

for maintaining species coexistence in diverse plant communities, by reducing

competition for pollinators and other resources (Rathcke and Lacey, 1985). Likewise,

the timing of growth onset and senescence also determine length of growing season

driving annual carbon uptake in terrestrial ecosystems (Gu et al., 2003). Ecologists,

nevertheless, have only recently paid attention to the relationships between

phenological patterns, vegetative growth strategies and reproductive characteristics

(Arroyo et al., 1981, Chen et al., 2005). In highly harsh conditions at higher altitudes

of Himalayan region, flowering of plants must be completed during limited period, to

permit fruit maturation and seed dispersal. It has been predicted that autogamy and

apomixis, which reduce dependence on external pollinators, might be more frequently

encountered among species inhabiting at higher mountains (Arroyo et al., 1981).

Spatial and temporal displacement by competition for light and water (Terborgh,

1973), and temporal competitive adjustment for the most efficient utilization of

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pollinators (Levin and Anderson, 1970) and seed-dispersal agents, are important

factors governing niche differentiation in plant species.

2.3 The molecular approach

Molecular tools provide valuable data on genetic diversity through their ability

to detect variation at the DNA level. For evaluation of species diversity, it is essential

that individuals can be classified accurately. The identification of taxonomic units of

endangered species, whose genetic constitution is distinct from their more abundant

relatives, is important in the development of appropriate conservation strategies. In

population studies, molecular tools are being used to identify whether two individuals

are from the mating of specific parents and estimating the degree of relatedness

among individuals. DNA markers are useful in both basic (eg. phylogenetic analysis

and search for useful genes) and applied research (eg. marker assisted selection,

paternity testing and food traceability). A number of markers are now available to

detect polymorphisms in nuclear DNA (Weising et al., 2005).

2.4 Internal Transcribed Spacer region (ITS)

The internal transcribed spacer (ITS) region of the 18S, 5.8S and 28S nuclear

ribosomal DNA (nrDNA) genes are highly conserved among angiosperm families and

have proven to be a useful source of characters for phylogenetic studies. They show

little sequence divergence between closely related species and are useful for

phylogenetic studies among distantly related organisms (Baldwani et al., 1995).

Within each repeat unit, the conserved regions are separated by two internal

transcribed spacers, ITS 1 and ITS 2 (each <300bp), can be readily amplified by PCR

and show higher rates of divergence (Moncalvo et al., 1995c; Perlin and Park, 2001).

ITS regions were the most widely sequenced DNA regions. The widespread use of

the nrITS region at the species level makes it a good candidate for a potential plant

molecular clock.

There are specific segments in the ITS regions which have greater variability

than other segments. Moncalvo et al. (1995a) observed that the frequency of

nucleotide substitutions was similar in both ITS regions but found that variations were

mostly located in the central region of ITS 1 and close to the termini in ITS 2. They

also reported that nucleotide divergence between recently diverged taxa was usually

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in the ITS 2 region. This was also observed by Gottlieb et al., (2000), who reported

that a lower level of resolution of internal phylogenetic branches was obtained from

the ITS 1 data set.

In the last decade, the nuclear ribosomal internal transcribed spacer region

(nrITS) has revolutionized species level plant phylogenetics. Because concerted

evolution has generally homogenized sequence variation among the numerous

ribosomal DNA copies within an individual, direct sequencing of this region is

possible for most systems. This coupled with the availability of universal primers and

elevated substitution rates compared to most chloroplast regions makes it especially

accessible and appropriate for resolving interspecific phylogenetic relationships

(Small et al., 1998, Baldwin et al., 1995). Although reliance on nrITS as the sole

source of phylogenetic evidence has come under criticism because of certain features

of its evolution (Alvarez et al., 2003), it remains the most efficient locus for

generating species-level phylogenetic inferences in most plant groups.

Baldwin et al. (1995) reported that the ITS region of nrDNA is a valuable

source of evidence on angiosperm phylogeny. The two spacers of this region, ITS-1

and ITS 2 can be readily amplified by PCR and sequenced using universal primers

that attributed to rapid concerted evolution and allows direct sequencing of pooled

PCR products in many species. According to them, in reported studies, variation

between ITS sequences is mostly attributed to point mutation. Phylogenetic analysis

of combined data sets from both spacers yielded trees with greater resolution.

Therefore, the need for phylogenetic markers from the nuclear genome to compliment

the rapidly growing body of cpDNA data, makes the ITS region a particularly

valuable resource for plant systematics.

Liu et al. (2002) used ITS gene sequences to study molecular evidence for the

sister relationship of the eastern Asia-North America intercontinental species pair in

the Podophyllum group belonging to Berberidaceae family. A heuristic parsimony

analysis based on the trnL-F data identified the basal clade but provided poor

resolution of their inter-relationships. High sequence divergence was found in the ITS

data. The study confirmed the different patterns of species relationship between

Asian-North American disjuncts.

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One of the most popular sequences for phylogenetic inference at the generic

and infra-generic levels in plants is the ITS region. Homoplasy is shown to be higher

in ITS than in other DNA sequence data sets, most likely because of

orthology/paralogy conflation, compensatory base changes, problems in alignment

due to indel accumulation, sequencing errors, or some combination of these

phenomena. . In 2003, Alvarez et al. analayzed ribosomal ITS sequences and plant

phylogenetic inference. Despite the near-universal usage of ITS sequence data in plant

phylogenetic studies, its complex and unpredictable evolutionary behaviour reduce its

utility for phylogenetic analysis.

Kim et al. (2004) reported taxonomic and phytogeographic implications from

ITS phylogeny in Berberis. A phylogeny based on the internal transcribed spacer

(ITS) sequences from 79 taxa representing much of the diversity of Berberis (four

major groups and 22 sections) was constructed. The phylogeny was basically

congruent with the previous classification schemes at higher taxonomic levels, such as

groups and subgroups. The ITS phylogeny also suggested that a possible connection

between the Asian and South American groups through the North American species

(B. canadensis or B. fendleri) is highly unlikely. They reported that phylogenetic trees

based on the NJ and Bayesian method were almost identical in the relationships of

major lineages.

Kathleen et al. (2006) conducted a survey of nrITS substitution rates across

angiosperms. They found that herbaceous lineages have substitution rates almost

twice as high as woody plants. Angiosperm nrITS substitution rates vary by

approximately an order of magnitude, and some of this variation can be attributed to

life history categories. They also suggested that for lineages with independent

calibrations, much of the variation in nrITS substitution rates may come from

uncertainty in calibration date estimates, highlighting the importance of accurate

and/or multiple calibration dates.

Bottini et al. (2007) studied the relationships in Patagonian species of Berberis

(Berberidaceae) based on the characterization of rDNA internal transcribed spacer

sequences. Sequence analysis of the internal transcribed spacer (ITS) of the

18S(ITS1)-5.8S-26S(ITS2) rDNA region was performed in order to analyse the

phylogenetic relationships between 13 Patagonian species of the genus Berberis. ITS

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sequences, together with data obtained from morphological, biochemical, amplified

fragment length polymorphism, and cytological characterization, support the

existence of diploid and polyploid hybrid speciation in the genus.

Gilani et al. (2010) studied the genus Prunus to find out the phylogenetic

relationship among the 23 species of Prunus, selected from different regions of

Pakistan and GenBank using maximum parsimony analysis of sequence

polymorphism in nuclear ITS 9 and ITS 6 spacer DNA.

Balasubramani et al. (2011) developed ITS sequence based markers to

distinguish B. aristata DC. from B. lycium Royle and B. asiatica Roxb. which can be

used as a molecular pharmacognostic tool for quality control of herbal raw drugs.

They suggested that conventional macro-morphology and microscopic examination

does not aid in critically distinguishing the three species. Therefore, DNA markers

were developed by amplifying and sequencing the complete internal transcribed

spacer region (ITS1, 5.8S rRNA and ITS2) from the genomic DNA using universal

primers. The markers developed were efficient and reliable in authenticating B.

aristata, B. asiatica and B. lycium.

2.5 Random Amplified Polymorphic DNA (RAPD)

Studies on genetic diversity within populations have been simplified by the

introduction of molecular analysis techniques, such as randomly amplified

polymorphic DNA (RAPD) analysis (Williams et al., 1990). RAPD analysis is a

multilocus arbitrary fingerprinting technique that can be used for determining genetic

relationships of various species as well as in determining the components of herbal

medicinal mixtures (Shinde et al., 2007). In addition, RAPD analyses are efficient,

economical and tend to produce genetic markers suited to the assessment of

population, race and species-specific genetic variation (Aagard et al. 1998).

In 2003, Kumar et al. extracted high molecular weight DNA from dry root

tissue of Berberis lycium which was suitable for carrying out RAPD reaction. The

amplified DNA was digested with Taq I, Hind III and EcoR I and was examined on

agarose gels. Torres et al. (2003) studied genetic structure of an endangered plant,

Antirrhinum microphyllum (Scrophulariaceae) through allozyme and RAPD analysis.

13 allozyme loci and 68 RAPD markers were analyzed to assess the genetic diversity

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and population structure. According to allozyme data, species genetic diversity as

well as within-population genetic diversity was high when compared to average

estimates for other narrowly distributed plant species. Nei's genetic distances

estimated both from allozymes and RAPDs indicated low differentiation among

populations.

Abdel-Mawgood et al. (2005) reported application of RAPD technique for the

conservation of an isolated population of Capparis decidua, a rangeland plant species

growing in isolated regions in Saudi Arabia. The population was noticed to suffer

from lack of new regeneration. RAPD markers were used to study genetic diversity.

Cluster analysis showed that coefficient of similarity within population is high as

compared to control population. In addition, the percentage of polymorphic alleles in

population was within the range of other endangered plant species. Thus, indicated

that the population is iso1ated and suffering from narrow genetic base and of a

particular conservation concern. Similarly, in 2005, Qiu et al. studied genetic

variation in the endangered Chinese endemic species Dysosma versipellis

(Berberidaceae). 5 populations of D. versipellis and 1 population of D. pleiantha were

analyzed using starch gel electrophoresis of 9 enzymes that corresponded to 9

interpretable loci. A level of genetic polymorphism within populations was much

smaller than values for seed plants, as well as values for other endemic species. Mean

values for the FST across all D. versipellis populations tended to be high. An indirect

estimate of the number of migrants per generation indicated that gene flow is low

among populations. Hence, concluded that in situ conservation will be an important

and practical measure for maintaining this species.

Padmalatha et al. (2006) reported RAPD analysis of selected medicinal and

aromatic plants of conservation concern from Peninsular India. Genetic analysis of

plants relies on high yield of pure DNA samples. The technique is ideal for isolation

of DNA from different plant species and the DNA isolated was used for RAPD

analysis. RAPD protocol was optimized, reproducible amplifiable products were

observed and the results indicated that the optimized protocol for DNA isolation and

PCR was amenable to plant species belonging to different genera which is suitable for

further work on diversity analysis.

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Harisaranraj et al. (2008) studied analysis of inter-species relationships of

Ocimum species using RAPD markers. Genetic inter-relationship of 7 Ocimum

species was estimated using RAPD markers. The 15 selected RAPD primers out of

which 2 primers showed amplification in all Ocimum species. O. basilicum has very

close similarity (89%) with O. tenuiflorum and another two species of O. gratissimum

and O. micranthum. Results suggested that genetic relationships in Ocimum species

using RAPD banding data may be useful for plant improvement and an efficient way

to conserve genetic resources of Ocimum species, in addition to their effective

medicinal uses. In 2008, Kim et al. reported conservation genetics of endangered

Brasenia schreberi based on RAPD and AFLP markers. Genetic diversity was

examined within and among 6 populations using RAPD and AFLP. Polymorphisms

were more frequently detected per loci with AFLP (69.3%) than RAPD (36.8%). High

genetic diversity was recognized within populations. Great genetic differentiation was

detected among the 6 populations RAPD (0.670) and AFLP (0.196) and also a low

rate of gene flow RAPD (0.116) and AFLP (0.977). Furthermore, a Mantel test

revealed that no correlation existed between genetic distances and geographical

distances among the six local populations based on RAPD or AFLP markers.

Das et al. (2009) optimized DNA isolation protocol and PCR conditions for

RAPD analysis of banana / plantain (Musa spp.). The technique was ideal for

isolation of DNA from different plant species/cultivars and the isolated DNA were

used for RAPD analysis. The optimization of RAPD protocol was based on the use of

50 ng of template DNA. In all PCR reactions reproducible amplifiable products were

observed. Thus the results indicated optimized protocol for DNA isolation and PCR

was applicable to plant species belonging to different genera and this process is

suitable for further work on diversity analysis. In 2009, Kelley worked on genetic

variability in Hydrastis canadensis L., an endangered perennial wildflower species

native to eastern North America using RAPD analysis. Several populations (both

cultivated and wild type) were analyzed for genetic variability. RAPD analysis

technique was used to generate DNA profiles from individual plants and to estimate

genetic variability between groups, among populations within groups and within

populations using analysis of molecular variance (AMOVA) and a UPGMA

clustering phenogram. Results demonstrated that the bulk of genetic diversity may be

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within and among populations, but not between groups. Thus, indicated the need for

preservation and conservation efforts at the population level.

Al-Qurainy et al. (2011) reported RAPD profile for the assessment of

genotoxicity on a medicinal plant, Eruca sativa (Brassicaceae). The genotoxicity of

three heavy metals, viz., Zn, Pb and Cd was studied. RAPD technique was used for

detection of genotoxicity produced by these metals. 20 decamer primers were used, of

which 16 primers showed amplified products as monomorphic, whereas 3 primers

showed unique band from seedlings treated with medium and high concentrations of

Cd, Pb and Zn respectively. Genetic divergence among the seedlings was evaluated

with dendrogram and similarity matrix value was obtained from 47.83 - 95.83%. In

2010, Naik et al. reported assessment of genetic diversity through RAPD, ISSR and

AFLP markers in Podophyllum hexandrum, a medicinal herb from the North-western

Himalayan region. Genetic analysis of 28 populations was done with 19 RAPD

primers, 11 ISSR primers and 13 AFLP primer pairs. A total of 92.37% (RAPD),

83.82% (ISSR) and 84.40% (AFLP) genetic polymorphism among the

populations were detected. Similarly the mean coefficient of gene differentiation

(GST) were 0.69 (RAPD), 0.63 (ISSR) and 0.51 (AFLP) indicating that 33.77%

(RAPD), 29.44 % (ISSR) and 26% (AFLP) of the genetic diversity resided within the

population. Analysis of molecular variance (AMOVA) indicated that 53% (RAPD),

62% (ISSR) and 64% (AFLP) of the genetic diversity among the studied populations

was attributed to geographical location while 47% using (RAPD), 38% (ISSR) and

36% (AFLP) was attributed to differences in their habitats and markers. An overall

value of mean estimated number of gene flow (Nm) were 0.110 (RAPD), 0.147

(ISSR) and 0.24 (AFLP) markers indicating that there was limited gene flow among

the sampled populations. Goswami et al. (2005) reported the use of ISSR and RAPD

markers for detecting DNA polymorphism, genotype identification and genetic

diversity among Trichosanthes dioica Roxb. (Cucurbitaceae). 22 cultivars of male and

female of T. dioica from various agro-climatic regions of India have been

fingerprinted by RAPD and ISSR markers utilizing 15 primers respectively. To

understand genetic relationships among these cultivars, Jaccard’s similarity

coefficient and UPGMA clustering algorithm were applied to the two marker data

sets.

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Domyati et al. (2011) studied molecular markers associated with genetic

diversity of some medicinal plants in Sinai. The main objective of this work was to

fingerprint some selected plant germplasm which were economically important on the

medicinal and pharmaceutical levels. Molecular markers such as RAPD, ISSR and

AFLP technologies were used to detect genetic diversity of the selected medicinal

plants. The study showed that taxonomical locations can be distinguished for each

subspecies (with as low as 0 to 1% polymorphism using AMOVA analysis) using

their molecular fingerprint but it cannot be recognized as a different subspecies.

Sodagar et al. (2012) studied biosystematic study of the genus Berberis L.

(Berberidaceae) in Khorassan, Northeast Iran. 4 species of the genus Berberis L. have

been reported from different regions, including Khorassan provinces. Morphological,

palynological, chromosomal and molecular studies were conducted on specimens

collected. Morphological study resulted in recognition of three species of Berberis

and four unknown new taxa with new morphological characters were also identified.

Molecular studies were accomplished by RAPD and sequencing of the ITS region to

construct a framework of relationships between the taxa. Molecular studies

emphasized the difference in the four unknown taxa from others. Thus evidence

indicated that Berberis L. shows a high percentage of polyploidy and hybridization. In

2012, Uysal et al. studied genetic diversity in threatened populations of the endemic

species Centaurea lycaonica Boiss. and Heldr. (Asteraceae). The study aimed to

describe the genetic diversity of the populations of the threatened Centaurea

lycaonica species. A total of 160 unique bands were identified from a random

sampling of 62 individuals in the populations using 6 RAPD primers and 4 ISSR

primers. Genetic differences among individuals and polymorphisms in two natural

populations were measured. Of the bands scored, 91% (145 bands) were polymorphic

and 9% (15 bands) were monomorphic. Thus, indicating that these populations have

high genetic diversity to survive in this area.

Iqbal et al. (2013) reported genetic characterization of Berberis species

collected from kunhar river catchment using morphological and molecular markers.

The assessment of genus Berberis based upon 19 morphological characters. The data

obtained from numerical analysis was computed for getting dendrogram, which

classified 24 collections into two major groups. Molecular characterization was done

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with the help of 12 RAPD primes to elaborate genetic polymorphism in Berberis

collections. The primers based amplification in the collections revealed high level of

genetic polymorphism, i.e. 6-86%. The genetic diversity estimated as genetic

distances computed into dendrogram, separated the collections into 5 distinct groups.

2.6 Inter Simple Sequence Repeats (ISSR)

The first studies employing ISSR markers were published in 1994

(Zietkiewicz et al., 1994; Gupta et al., 1994). The initial studies focused on cultivated

species and demonstrated the hypervariable nature of ISSR markers. To test the utility

of the method in natural population a known hybrid complex of four species of

Penstemon was re-examined. Thus clearly demonstrated the utility of ISSR markers

for addressing questions of hybridization and diploid a hybrid speciation.

Joshi et al. (2004) studied role of molecular markers in herbal drug

technology. Herbal drug is used for technology converting botanical materials into

medicines, where standardization and quality control with proper integration of

modern scientific techniques and traditional knowledge is important. DNA-based

techniques have been widely used for authentication of plant species of medicinal

importance. In 2004, Dangi et al. optimized the assessment of genetic diversity of

Trigonella foenum-graenum and Trigonella Caerulea using ISSR markers. In this

report, they described the use of ISSR markers to study of genetic diversity in both

species of Trigonella. Seventeen accessions of Trigonella foenum-graenum and nine

accessions of Trigonella Caerulea representing various countries were analysed using

ISSR markers. Genetic diversity parameters (average number of alleles per

polymorphic locus, percentage polymorphism, average heterozygosity and marker

index) were calculated for ISSR marker in both the species and showed that plants

from different geographical regions were distributed in different groups in both the

species.

Balasaravanan et al. (2005) used ISSR markers to evaluate genetic

relationships within and between individuals of six Eucalyptus species. A total of 583

loci (256 to 1535bp) were amplified from 149 individuals belonging to six Eucalyptus

using seven primers. The ISSR fragments indicated significant polymorphism and

genetic diversity among the individuals. Cluster analysis and principal component

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analysis revealed the occurrence of wide genetic diversity among populations. In

2005, Pharmawati et al. studied the molecular variation and fingerprinting of

Leucadendron cultivars (proteaceae) using ISSR markers. Currently 100 cultivars

were used by industry and many of them were interspecific hybrids. The origins of

most cultivars are nuclear and their genetic diversity and relationship had not been

studied. This investigation evaluated the genetic variation and the relationship among

30 Leucadendron cultivars.

Wang et al. (2007) determined the genetic diversity in Paris polyphylla var.

yunnanensis, a traditional Chinese medicinal herb, detected by ISSR markers.

According to this report, it is an important Chinese herb. because of overharvesting,

the wild population of this herb have greatly declined and become fragmentized. ISSR

markers were used to determine the genetic diversity and genetic structure of this

variety represented by a total of 153 individuals from three natural populations and

three cultivated populations.

Barakat et al. (2007) compared the application and utility of ISSR marker

techniques for analysis of genetic diversity among Saudi wheat genotype under heat

stress and also developed the heat tolerance-associated DNA markers. 12 wheat

genotypes were used. The ISSR marker assayed to determine the genetic diversity of

the twelve wheat genotypes. In 2009, Mohamed et al. studied the molecular genetics

characterisation of some promising sugarcane varieties under smut disease. They

genetically characterized some identified promising sugarcane cultivars resistant to

smut fungus (Ustilago scitaminea) at an early stage of the breeding program. Ten

cultivars were used, including seven promising cultivars, one susceptible cultivar and

two were commercially cultivars. Resistant cultivars were detected using ISSR

markers. Mueller et al. (1999) reported that ISSR marker was used for medicinal

image retrieval information in research and diagnosis. Accordingly, researchers can

benefit from such a system through the use of visual image query. In diagnosis, find

image with the same anatomic region of a specific disease.

Petolescu et al. (2009) analyzed the genetic diversity of the in-vitro

regenerated alfalfa using ISSR markers. They highlighted that the genetic diversity is

one of the most important factors for crop improvement. On the other hand, for micro

propagation or genetic transformation the most crucial aspect is to retain genetic

Review of Literature

Phylogeny of genus Berberis and genetic diversity of its two threatened species across the

altitudinal zone in Uttarakhand

22

integrity with respect to the mother plants. In order to select genotypes with in vitro

stability or instability they evaluated the regeneration ability of the 30 Romanian

genotypes (lines and cultivars) and genetic diversity of the regenerates using ISSR

markers.

Vinayagam et al. (2008) reported that molecular markers can be effectively

utilized to diagnose and select a genotype. The molecular characterization was done

for genetic diversity analysis of Borassus flabellifer, 20 Palmyrah accessions using 20

ISSR primers were analyzed. In 2009, Wang et al. identified DNA markers through

fingerprinting technique and evaluated genetic diversity in C.goeringii cultivars. 25

primers were selected to produce a total of 224 ISSR loci for evaluation of genetic

diversity. A wide genetic variation was found. They suggested that the ISSR

technique provides a powerful tool for cultivars identification and establishment of

genetic relationships.

Xu-mei Wang (2010) used ISSR marker for authenticating the genuine species

of Rhubarb. They reviewed that Rhubarb is prescribed as the roots and rhizomes of

Rheum officinale Baill, Rheum palmatum L, and Rheum tanguticum in Chinese

pharmacopoeia. These three species are difficult to discriminate due to the

morphological and anatomical similarity of the aerial parts and herbal medicines.

Therefore, ISSR molecular fingerprinting markers were employed to authenticate

three genuine species of Rhubarb using 15 primers to discriminate R. officinale, R.

palmatum, and R. tanguticum. Four specific authentication markers were found to

authenticate three species of rhubarb. To enhance the efficiency of authentication,

ISSR fingerprinting codes were constructed using four polymorphic bands for

authenticating three genuine species of Rhubarb.

Datta et al. (2010) reported efficiency of three PCR based system for detecting

polymorphism in Cicer arietinum (L) and Cajanus Cajan (L) Millspaugh. They

studied that insufficient quality of molecular markers regarding their predictive and

diagnostic values has delayed the benefits of marker-assisted selection. To evaluate

the robustness of marker in the detection of DNA polymorphism, ISSR markers were

used to genetically differentiate two major pulse crops processing different important

traits.

Review of Literature

Phylogeny of genus Berberis and genetic diversity of its two threatened species across the

altitudinal zone in Uttarakhand

23

Other work at molecular level

Lafferriere (1997) described the transfer of specific and infra-specific taxa

from Mahonia to Berberis (Berberidaceae). Seventeen specific and infra-specific

names are formally transferred from Mahonia to Berberis. New combinations include:

Berberis aquifolium var. lyallii, B. aquifolium var. nutkana, B. x convoluta, B. x

herveyi, B. longipes, B. x moseri, B. paxii, B. pinnata var. hortensis, B. quinquefolia,

B. racemosa, B. repens var. macrocarpa, B. repens var. rotundifolia, B. russellii, B.

undulata, B. volcania, and B. zimapana. Berberis standleyi is a new name replacing

Mahonia glauca.

Lubell et al. (2008) reported AFLP identification of Berberis thunbergii

cultivars, inter-specific hybrids and their parental species. Using AFLP markers

differentiation between 43 B. thunbergii cultivars and genetic similarity of 62 B.

thunbergii genotypes, B. julianae, B. koreana, B. vulgaris and B. vulgaris

‘Atropurpurea’ were analysed. Result indicated variety of identical AFLP profiles

signifying only one genotype in cultivation and not a collection of similar genotypes.

Berberis aristata DC. is critically endangered species of Indian Himalaya due

to its extensively collection of roots for its Berberine alkaloid. In 2008, Majid et al.

studied vegetative propagation of Berberis aristata DC., an endangered Himalayan

shrub. They explored the possibility of propagating the species vegetatively to

maintain its genetic identity and population. The experiment was conducted by taking

different cutting portions viz., apical, sub-apical and basal which were treated with

various IBA concentrations viz., control, 2500, 5000 and 7500 ppm. Results revealed

that apical cuttings when treated with 5000 ppm IBA concentration performed

significantly better in sprouting (85%) and rooting percentage (50%) in comparison to

other treatments.

Tripathi et al. (2010) reported isolation and expression analysis of Berberis

chitria Lidl. specific transcripts using subtractive hybridization technique. The

method of suppressive subtractive hybridization was used to detect and clone

differentially expressed genes during the development of Berberis chitria Lidl. in cold

stress condition. cDNA library with insert size range from 100 to 600bp from B.

chitria roots at developmental stage was constructed. The cDNA after subtraction

Review of Literature

Phylogeny of genus Berberis and genetic diversity of its two threatened species across the

altitudinal zone in Uttarakhand

24

were cloned and a total 28 clones were identified, which was unregulated in B. chitria

root tissues, among these 8 were novel, while 20 showed homology with different

genes in database. They reported that the study of the genes identified may enhance

understanding of the genetic circuit involved in the development of Berberis.

In 2010, Rounsaville et al. studied cytogenetics, micropropagation, and

reproductive biology of Berberis, Mahonia, and Miscanthus to determine the genome

sizes and ploidy levels for a diverse collection of Berberis L. and Mahonia Nutt.

genotypes. Mean genome sizes varied between the two Mahonia subgenera

(Occidentales = 1.17±0.02, Orientales = 1.27±0.01), while those of Berberis

subgenera were similar (Australes =1.45±0.03, Septentrionales =1.47±0.02), and each

significantly larger than those of Mahonia. Polyploidy among both wild and

cultivated taxa was found to be rare. While the majority of species were determined to

be diploid with 2n=2x=28, artificially-induced autopolyploid Berberis thunbergii

seedlings were confirmed to be tetraploid and an accession of Mahonia nervosa was

confirmed to be hexaploid.

Thus, from the above literature review it was concluded that RAPD, ISSR and

ITS regions are the most preferred and reliable tools used across the world for

population genetics study and for phylogenetic analysis in Berberis as well as in other

plant species and were therefore employed in the present investigation.