Loading...

Evaluation of genetic diversity of jackfruit (Artocapus heterophyllus Lam) varieties based on sequence analysis of the rbcL gene

by Dr. Prem Jose Vazhacharickal (Author) Jiby John Mathew (Author) Ajesh C Kuriakose (Author) Sajeshkumar N.K (Author)

Scientific Study 2016 69 Pages

Biology - Genetics / Gene Technology

Excerpt

Table of contents

Table of figures

Table of tables

List of abbreviations

Evaluation of genetic diversity of jackfruit (Artocapus heterophyllus Lam) varieties based on sequence analysis of rbcL gene

Abstract

1. Introduction
1.1Taxonomical classification

2. Review of literature

3. Hypothesis

4. Materials and Methods
4.1 Botanical description and varieties
4.2 Nutritional and vitamin composition
4.3 Study area
4.4 Sample collection
4.5 Morphological descriptors for jackfruit tree classification
4.6 Isolation of DNA
4.7 Quantification of DNA
4.8 PCR amplification
4.9 Data sequencing
4.10 Data analysis
4.11 Statistical analysis

5. Results and discussion
5.1 General characteristics
5.2 Leaf characteristics
5.3 Fruit
5.4 DNA isolation
5.5 Polymerase chain reaction
5.6 Molecular phylogentic analysis using ‘rbcL’ gene
5.7 Restriction fragment length polymorphism
5.8 Polymorphic sites
5.9 Analysis

6. Conclusions

Acknowledgements

References

ACKNOWLEDGEMENTS

Firstly we thank God Almighty whose blessing were always with us and helped us to complete this project work successfully.

We wish to thank our beloved Manager Rev. Fr. Dr. George Njarakunnel, Respected Principal Dr. V.J.Joseph, Vice Principal Fr. Joseph Allencheril, Bursar Shaji Augustine and the Management for providing all the necessary facilities in carrying out the study. We express our sincere thanks to Mr. Binoy A Mulanthra (lab in charge, Department of Biotechnology) for the support. This research work will not be possible with the co-operation of many farmers.

We are gratefully indebted to our teachers, parents, siblings and friends who were there always for helping us in this project.

Prem Jose Vazhacharickal*, Sajeshkumar N.K, Jiby John Mathew and Ajesh C Kuriakose

*Address for correspondence

Assistant Professor

Department of Biotechnology

Mar Augusthinose College

Ramapuram-686576

Kerala, India

premjosev@gmail.com

Table of figures

Figure 1. Mean monthly rainfall (mm), maximum and minimum temperatures (°C) in Kerala, India (1871-2005; Krishnakumar et al., 2009).

Figure 2. Introduced and indigenous regions of Artocarpus heterophyllus around the world (Modified after; Haq, 2006).

Figure 3. Map of Kerala showing the various sample collection points; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20).

Figure 4. Jackfruit trees a) jackfruit with varying sizes; b) different stages of fruiting; c) tree bearing fruits; d) fruits plucked; e) small type of jack fruit; f) jackfruit cut opened; g) jackfruit seeds; h) opened jackfruit flakes; i) flakes unopened.

Figure 5. Sample Ku (A1) description a) tree bearing jackfruits, b) jackfruit with exposed pith, c) longitudinal section of jackfruit, d) flake, e) fruit stalk leaf, branch leaf, flake, seeds and spine, f) pith, g) seed.

Figure 6. Sample Tv (A3) description a) jackfruit, b) cross section of jackfruit, c) reddish flake, d) fruit stalk leaf, branch leaf, flake, seeds and spine.

Figure 7. Sample Uc (A7) description a) tree bearing jackfruits, b) bunch of round jackfruit, c) spherical shaped jackfruit, d) longitudinal section of jackfruit, e) fruit stalk leaf, branch leaf, flake, seeds and spine, f) spines, g) depression in stalk attachment to fruit.

Figure 8. Sample Va (A9) description a) tree bearing jackfruits, male and female flower, b) fruit stalk leaf, branch leaf, flake, seeds and spine, c) flake and seed, d) leaf apex (retuse).

Figure 9. Sample Pv (A16) description a) jackfruit, b) leaf apex shape, c) fruit stalk leaf, branch leaf, flake, seeds, d) flake and seed.

Figure 10. Sample Sv (A20) description a) jackfruit, b) spine, c) longitudinal section of jackfruit, d) flake, e) fruit stalk leaf, branch leaf, flake, seeds and spine, f) inflated stock attachment to fruit.

Figure 11. Description and morphology of samples Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20).

Figure 12. DNA on agarose gel a) agarose gel electrophorosis of the DNA of samples; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20), b) amplified DNA samples.

Figure 13. rbcL gene sequences of six different Artocarpus heterophyllus varities; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20).

Figure 14. Pairwise genetic distance between six Artocarpus varities; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20) based on rbcL gene sequence (Overall distance is 0.001).

Figure 15. Phylogenetic tree based on rbcL sequence of six Artocarpus varities; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20) by a) Maximum likelihood (ML) method b) Neoghbour joining (NJ) method c) Unweighted pair group method with arithmetic mean (UPGMA) method. 49

Table of tables

Table 1. Phenolic, flavinoid content and antioxidant activity of araticum, papaya and jackfruit in undigested and digested extracts (Modified after; Pavan et al., 2011).

Table 2. Biochemical difference in various jackfruit varieties in South India (Chrips et al., 2008).

Table 3. Uses of different jackfruit parts (Chrips et al., 2008).

Table 4. Uses of different lectins from jackfruit parts (Chrips et al., 2008).

Table 5. Different vernacular names of Artocarpus heterophyllus in India (Modified after; Baliga et al., 2011).

Table 6. Common names, uses and distribution of major Artocarpus species (Modified after; Jagtap and Bapat, 2010).

Table 7. Chemical composition of jackfruit (Modified after; Jagtap and Bapat, 2010).

Table 8. Description and geographic features of the jackfruit varieties; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20).

Table 9. Branch leaf features and properties of jackfruit varieties; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20) in Kerala.

Table 10. Fruit stalk leaf composition and properties of jackfruit varieties; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20) in Kerala.

Table 11. Fruit features and properties of jackfruit varieties; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20) in Kerala.

Table 12. General features and properties of jackfruit varieties; Ku (A1), Va (A9), Uc (A7), Pv (A16), Tv (A3) and Sv (A20) in Kerala.

Table 13. Maximum composite likelihood estimate of the pattern of nucleotide substitution. 51

List of abbreviations

Abbildung in dieser Leseprobe nicht enthalten

Evaluation of genetic diversity of jackfruit (Artocapus heterophyllus Lam) varieties based on sequence analysis of rbcL gene

Prem Jose Vazhacharickal1*, Sajeshkumar N.K1, Jiby John Mathew1 and Ajesh C Kuriakose1

* premjosev@gmailcom

1 Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kerala, India-686576

Abstract

Jackfruit (Artocarpus heterophyllus) is commonly grown in home gardens of tropical and sub-tropical countries. The fruit which contain high levels of carbohydrates, protein, starch, calcium and vitamins. Jack fruit has diverse medicinal uses especially anti-oxidant, anti-inflammatory, antimicrobial, anti-cancer and anti-fungal activity. Jackfruit is considered to be an underutilized fruit where most of the fruits get wasted due to ignorance, lack of post harvest technology and gaps in supply chain systems. Jackfruit contains more protein, calcium, iron, vitamins and other essential nutrients when compared to the common fruits. To find out the morphological variation among jack fruits among Kerala we conducted an elaborative survey and find out verities then the parameters such as tree characteristics, leaf characterises, fruit characteristics of selected samples are measured. Both qualitative and quantitative data are measured using field visit and standard methods and morphological indicators, after analysis of data there is a morphological variation among the jack fruit samples are identified. Advances in the genetic markers such as RFLP and PCR based methods are more reliable for identification of genetic diversity than morphological markers although each technique has advantages and limitations. The objective of this research work was to estimate the level of genetic diversity and to assess genetic relationships among six varieties of jackfruit using ‘rbcL gene’ based on PCR technique and RFLP markers. The partial sequence of ‘rbcL’ gene of six different Artocarpus varities was used in the analysis. The size of amplified products was approximately 700 bp. After sequencing and sequence editing, sequence information on a 651 bp region was finally obtained for analysis. The alignment of sequences revealed two haplotypes out of 651 sites. The nucleotide frequencies are 27.96% (A), 29.47% (T/U), 19.69% (C), and 22.89% (G). Being one of the underutilized fruits in India, Artocarpus heterophyllus Lam. has promising leads to further scientific researches and livelihood strategies. The study of rbcL gene using PCR and RFLP seems to a promising tool in establishing genetic diversity among jackfruit varities.

Keywords: Anti-oxidant; Jackfruit; rbcL; Monecious; RFLP; Underutilized fruit.

1. Introduction

Arocarpus heterophyllus (Jackfruit tree) is an evergreen tree from Moraceae family (Bose, 1985., Bhattacherjee, 1986). It is believed to have originated in Western Ghats in India (Reddy et al, 2004) and bear the largest tree born fruit (Alagaipillai et al, 1996). Jackfruit is used in different ways; unripe fruits used in local cuisine and ripe fruit can be eaten raw. Jackfruit consist mainly of 3 parts; a fruit axis that is the core of jackfruit and has several laticiferous cells, the true fruit which develops over the ovary (Sharma, 1964), and a perianth. The perianth has three parts a fleshy edible region called bulb, a middle fused region called rind and an upper spiny region (Jagadeesh et al., 2007a; Jagadeesh et al., 2007b; Baliga et al., 2011; Saxena et al., 2009b; Hameed, 2009; Swami et al., 2012).

Normal Jackfruits can weigh up to 10-25 kg when mature (Rahman et al,1995) while large varieties can weigh up to 50 kg (Selvaraj and Pal,1989). An average jackfruit has 29% bulb, 12% seeds and 59% rind (Bhatia et al, 1955). The tree is medium sized that can grow up to 10-25 m height and a trunk diameter of 30-60 cm.

Longer seed dormancy, large variation in fruit quality (in different as well as same plant) makes jackfruit unsuitable for being a commercial crop (Samaddar, 1985). Its functional properties include foaming, emulsifying and gelation properties, water and oil absorption capacities and protein solubility (Lawhon et al., 1972; Beuchat et al., 1975; Coffman and Garcia, 1977; Narayana and Rao, 1982).

Several classifications were done on jackfruits, but none of them was given a universal name, since same variety can have different phenotype in two different regions. Several factors influence this including temperature, climate, soil quality, nearby vegetation, etc. Common classification in India is one having small fibrous soft and spongy flakes with sweet carpels, this is known as koozha, this is of high quality compared to other. The other variety is crunchy but not as sweet as koozha, this is called varikka. Several other varieties have been identified but they all have some common characters to these two varieties (Baliga et al., 2011; Haq, 2006; Prakash et al., 2009). Other than them an intermediate variety is also common in Kerala known as paathivarikka. It has flakes with lower half of koozha and upper half of varikka. Unda chakka is a variety that got the name from its small size and round shape; it can exhibit property of either koozha or varikka depending on the parent tree.

Cetyl Trimethyl Ammonium Bromide (CTAB) method (Doyle and Doyle, 1990) and its modifications are the most widely used method for plant genome isolation. A modified CTAB method was used for the DNA isolation which was standardised through repeated experimentation to be suitable for the experiment. DNA was isolated from plant leaf since it has higher concentration of DNA than flakes or other parts. Components in CTAB method include;

a) The extraction buffer (This includes a detergent such as CTAB or sodium dodecyl sulphate (SDS) which disrupts the membranes, a reducing agent such as β- mercaptoethanol which helps in denaturing proteins by breaking the disulfide bonds between the cysteine residues and for removing the tanins and polyphenols present in the crude extract, a chelating agent such as ethylene diamine terta acetic acid (EDTA) which chelates the magnesium ions required for DNase activity , a buffer which is almost always Tris at pH 8 and a salt such as sodium chloride (NaCl) which aids in precipitation by neutralizing the negative charges on the DNA so that the molecules can come together)

b) Phenol chloroform extraction (Nucleic acid solutions commonly contain undesirable contaminants that are chiefly made of proteins. A classic method of purifying is phenol –chloroform extraction by which the nucleic acid solution is extracted by successively washing with a volume of phenol (pH 8.0); a volume of phenol: chloroform: isoamyl alcohol (25: 24: 21) and chloroform: isoamyl alcohol (24:1). Centrifugation is performed intermittently and the upper aqueous phase is transferred to a new tube while avoiding the interphase. The contaminants are denatured and accumulate in the organic phase or in the marginal layer between the two phases and the nucleic acids are preserved in the aqueous phase. Another way of removing proteins is by using the enzyme proteinase K which however again is denatured by phenol via phenol chloroform extraction),

c) Precipitation of nucleic acids (Alcohol precipitation is the most commonly used method for nucleic acid precipitation. This requires diluting the nucleic acid with a monovalent salt, adding alcohol to it and mixing gently. The nucleic acid precipitated spontaneously and can be pelleted by centrifugation. The salts and alcohol remnants are removed by washing with 70% alcohol. The most commonly used salts include CH3COONa (sodium acetate) pH 5.2 (final volume 0.3 M), NaCl (final concentration 0.2 M), NH4CH3CO2 (ammonium acetate) (2- 2.5 M), lithium chloride (0.8 M) and potassium chloride. Ethanol (twice the volume) or isopropanol (two thirds volume) are the standard alcohols used for nucleic acid precipitation) and

d) Resuspending DNA (The nucleic acid pellet can be resuspended in either sterile distilled water or TE buffer (10 mM Tris:1mM EDTA)).

DNA was analysed using electrophoresis which separate nucleic acid based on their size (Muyzer et al., 1993; Smith et al., 1981; Botstein et al., 1980). Electrophoresis uses the negative charge of DNA to make it migrate through an electric field created by the positive and negative electrode (Cohen et al., 1987; Heng et al., 2004; Westermeier 2016). The movement of DNA was based on pre size of gel and size of DNA (Southern et al., 1987; Kaji et al., 2004; Lee et al., 2012; Lalande et al., 1987). Pore size of gel can be varied in anyway by changing the quantity of agarose used. It also allows purification of DNA by eluting the desired molecular weight portion from gel (Cohen et al., 1987; Heng et al., 2004; Westermeier 2016; Southern et al., 1987; Kaji et al., 2004; Lee et al., 2012; Lalande et al., 1987). Agarose gel electrophoresis is widely employed to estimate the size of DNA fragments after digesting with restriction enzyme and used in molecular diagnosis via analysis of polymerase chain reaction (PCR).

Polymerase chain reaction is a molecular based technique used widely in research, detection and characterisation of infectious organisms and other medical application. Polymerase Chain Reaction is a technique for DNA amplification In virology, PCR technique is reliably used since it has high sensitivity and reproducibility in viral genomic detection and strain characterisation. For a PCR based technique purity and quantity of DNA is of great importance (Powledge, 2004; Hill and Wachsmuth, 1996; Gilbride et al., 2006).

Study was conducted on ribulose-bisphosphate carboxylase gene (rbcL) and a phylogenetic relation was drawn between 6 varieties of jackfruit. They are varikka, Singapore varikka, paathivarikka, theenvarikka, undachakka and koozha. These samples were selected since they were the most common variety seen in Kerala and known in different local names. A phylogenetic analysis was conducted based on PCR amplification and sequence analysis.

Rubisco (EC 1.1.1.39) is a key enzyme in photosynthetic carbon assimilation and catalyzes the first committed step of CO2 fixation in the Calvin cycle. It has been estimated that nearly 70% of soluble leaf protein is in the form of Rubisco (Miller and Huffaker, 1982). Higher plant Rubisco is an oligomer composed of eight large subunits (Mr 52,000-55,000) and eight small subunits (Mr 12,000-15,000) (Enyedi and Pell, 1992). The large subunit contains the active site for carboxylation and oxygenation reactions, and the small subunit protein is hypothesized to maintain the apoenzyme in a stable conformation capable ofactivation to a catalytically active holoenzyme (Miziorko and Lorimer, 1983). The small subunit protein of Rubisco is nuclear encoded by the rbcS gene which exists as a multigene family (Dean et al., 1989). The gene for the large subunit of the rbcL gene is an appropriate choice for inference of phylogenetic relationships at higher taxonomic levels (Chase et al., 1993; Duvall et al., 1993; Les et al., 1991). Because of its slow synonymous nucleotide substitution rate in comparison with nuclear genes and its functional constraint that reduces the evolutionary rate of non synonymous substitutions (Wolfe et al., 1987), rbcL is considered to be more useful than the isozymes. The primary reduction of CO2 into organic matter (CO2 fixation) via the Calvin cycle represents a pathway essentially conserved throughout evolution (Tabita, 1988) and serves as the mechanism of primary production in nearly all ecosystems. Comprised of a large and small subunit, the enzyme is highly conserved from cyanobacteria to higher plants in both subunit organization (L8S8) and primary structure of the large subunit (ca. 70% homology at the nucleotide level and 78 to 81% homology at the amino acid level (Shinozaki et al., 1983).

Jackfruit is considered as national fruit in Bangladesh and highly appreciated in India due to cheap and availability in summer seasons were food is scarce (Muralidharan et al., 1997; Morton, 1987; Schnell et al., 2001; Schnell et al., 1996). The fruit provide 2 MJ per kg/wet weight of ripe perianth and contain high levels of carbohydrates, protein, starch, calcium and vitamins (Swami et al., 2012; Ahmed et al., 1986; Burkill, 1997; Saxena et al., 2009a). Boiled and cooked jackfruit seeds are included in the diets which have 77% starch content, which is exploited as a potent source of starch (Bobbio et al., 1978; Tulyathan et al., 2002; Mukprasirt and Sajjaanantakul, 2004; Odoemelam, 2005). Jackfruit is widely used in culinary preparation, baking, candid jackfruit, baby food, jams, jellies, juice, chips, deserts and the advances in food processing technologies further expanded the possibilities (Burkill, 1997; Swami et al., 2012; Selvaraj and Pal, 1989; Narasimham, 1990; Roy and Joshi, 1995 Haq, 2006). Jackfruit is widely accepted by consumers, researchers and food industries due to the presence of bioactive compounds and diversity products made out of it (Swami et al., 2012; Saxena et al., 2009a; Dutta et al., 2011; Lin et al., 2009; Devalaraja et al., 2011). Various parts of jackfruit tree have been used for medicine and the hard wood used for construction (Roy and Joshi, 1995).

1.1Taxonomical classification

Kingdom: Plantae-- planta, plantes, plants, vegetal

Subkingdom: Tracheobionta -- vascular plants

Division: Magnoliophyta -- angiosperms, flowering plants, phanerogames

Class: Magnoliopsida -- dicots, dicotyledones, dicotyledons

Subclass: Hamamelidae

Order: Urticales

Family: Moraceae - mulberries

Genus: Artocarpus - breadfruit

Species: Artocarpus heterophyllus Lam.

2. Review of literature

The Artocarpus heterophyllus is an average tree and is commonly known as Jackfruit, belongs to the family Moraceae. Jackfruit is a native to Western Ghats of India, and the rain forest of Malaysia. It is also found in central and eastern Africa, south eastern Asia, the Caribean, Florida, Brazil, Australia, Puerto Rico, and many Pacific Islands (Rajasekhar et al., 2010; Theivasanthi and Alagar, 2008; Kanjilal et al., 1940; Talbot, 1911; Brandis, 1906; Gamble, 1902; Beddome, 1873; Wight, 1843). Jackfruit tree produce heavier yield compared to other tree and bear the largest known edible fruit weight up to 35 kg. It can reach a height of 10 to 15 m tall at the age of 5, with dark green oval shaped leaves. Jackfruit height increases 1.5 m/yr, when the tree reaches maturity slowing to 0.5 m. The tree has the life span of 60 to 70 years (Narkhede et al., 2011). The wood is strong, hard durable and easy to carve, machine, saw, or machine. It is used to make furniture, house construction such as, windows, doors, roof, oars, masts, rafters, implements and some musical instruments and tambooras (Haque, 1999; Gunasena, 1993). Artocarpus heterophyllus is a bisexual plant, both male and female flowers are found on the same plant. At the early stage the male flower which is green in colour at maturity in it become yellowish, the pollen grains are yellow in colour, then it fall off from the tree. The female flower which is an aggregate of small flowers (Brown and Crane, 2010).

Artocarpus heterophyllus Lam. is originated in Western Ghats India, and been cultivated for centuries over the lowlands of South East Asia (Acedo, 1992; Samaddar, 1985; Soepado, 1991). Several classifications were done based on the morphological characteristics (Hossain, 1996; Saha et al., 1996; Jagadeesh et al., 2007a). A minimum of 30 varieties have been studied and only two of them were recognized with maximum distinct characteristics (Odoemelam, 2005). The total components of a jackfruit has been determined: vitamin content (Bose, 1985; Ahmed et al., 1986; Selvaraj and Pal, 1989), starch (Bobbio et al., 1978; Hossain et al., 1990; Rahman et al., 1995; Rahman et al., 1999), water soluble sugars (wills et al., 1986; Selvaraj and Pal, 1989), free sugars and fatty acids (Choudhery et al., 1997; Choudhery et al., 1991) and flavour volatiles (Swords et al., 1978; Rasmussen, 1983; Maia et al., 2004; Chandrika et al., 2005; De Faria et al., 2009). Due to availability of superior germplasm, modern growing, and emerging ethnic and mainstream marketing opportunities jackfruit has gained popularity in United States (Campbell and El sawa, 1998; Campbell and McNaughton, 1994). A small collection of jackfruit cultivars has been established at Fairchid tropical Garden (FTG) which is providing germplasm throughout tropical America (Campbell and McNaughton, 1994). Some of the varieties were specially selected for production in South Florida and have contributed to a substantial increase in commercial production.

CTAB is a total genomic DNA (nuclear, chloroplast, and mitochondrial) isolation method used successfully on a wide variety of plant groups and some animals (Doyle, 1991; Olmstead and Palmer, 1994; Jansen et al., 2005; Weising et al., 2005; Lang and Burger, 2007). It is a rapid, inexpensive method that is suitable for use in conjunction with other protocols, such as DNA isolation of DNA enriched for cpDNA, it is also easy to scale down for use in population sampling, using 0.01g or less of fresh tissue (Cato and Richardson, 1996; Sass et al., 2007; Nemeth et al., 2004; Varma et al., 2007; Wang and Messing, 2011). Other applications include isolation of DNA from herbarium specimens (Doyle and Dickson, 1987), and isolation of RNA. A brief word on the history of the protocol is in order. This procedure was modified by us (Doyle and Doyle, 1987) for use with fresh plant tissue from a method of Saghai-Maroof et al. (1984) who used lyophilised tissue. They in turn had developed their procedure from earlier protocols.

Genetic diversity is a criterion inevitable in plant breeding which is measured by genetic distance or genetic similarity (Weir, 1990). Morphological and biochemical classification are restricted to few traits and low polymorphism level. It can also vary in its manifestation based on environmental factors (Melchinger et al., 1991). While molecular based studies have capability to define a clear diversity based genomic content.

A molecular marker is any measure character and molecular characteristic that is inherited in a simple mendalian fashion (Avise, 2012; Hadrys et al., 1992; Rieseberg and Brunsfeld, 1992). The discovery of molecular markers in recent years has greatly enhanced the scope for detailed genetic analysis and approaches to improve crop plants (Collard and Mackill, 2008; Egan et al., 2012; Snowdon and Friedt, 2004; Reynolds et al., 2009). The molecular technology has indirectly improved the efficiency of plant breeding programs (Collard and Mackill, 2008; Egan et al., 2012; Snowdon and Friedt, 2004). Molecular markers play two main roles in plant breeding programs, firstly as a source of genetic finger prints and as a selected marker linked to phenotyphic traits of interest to breeder (Koebner et al., 2001; Eagles et al., 2001; Skøt et al., 2005; Haussmann et al., 2004; Perez-de-Castro et al., 2012). Markers are broadly classified into morphological markers, protein based markers and DNA based markers (Eagles et al., 2001; Liu and Cordes, 2004; Avise, 2012; Fukami et al., 2004; Halward et al., 1991). DNA based markers like, random fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and amplified fragement length polymorphism (AFLP) can act as excellent tools to study the genetic diversity eliminate duplicate in germplasm to study the genetic relationships, gene tagging, genome mapping and to use in plant variety rights (PVR) (Karp, 1997; Meudt and Clarke, 2007; Hegarty and Hiscock, 2005; Sharma et al., 2008; Langridge et al., 2001). These markers measure, diversity at DNA level in all tissue at all of plant and are seldom influenced by environmental condition (Gopalsamy et al., 2012; Hegarty and Hiscock, 2005; Sharma et al., 2008).

A mutable fragment of chloroplast genome, described by ogihara et al. (1991) was amplified by polymerase chain reaction by Arnold et al. (1991), later analysed for taxonomic data by restriction analysis of this fragment (Liston, 1992; Rieseberg et al., 1992; Badenes and Parfitt, 1995).

Genetic diversity in Artocarpus heterophyllus Lam. based on AFLP was done by Shyamalamma et al. (2008). In their research, genetic diversity and genetic relatedness of 50 jackfruit accessions were acessed using AFLP markers. Since jackfruit is tetraploid, its somatic chromosome number is 4n (56). Therefore the basic chromosome number is 14 (Darlington and Wylie, 1956). Diversity and genetic relatedness of jackfruit was first studied by Schnell et al. (2001) where he studied 26 accessions from different parts of world using AFLP.

To date, very little attempts have made to estimate the genetic diversity of different jackfruit cultivars in India. A comprehensive understanding of genetic diversity and molecular characterization of jackfruit cultivars is needed for formulating appropriate sampling and management strategies. A detailed analysis of a large number of genetic markers will provide us with useful gene conservation strategies and help in popularizing this species as a commercial crop.

Rubisco (EC 1.1.1.39) is a key enzyme in photosynthetic carbon assimilation and catalyzes the first committed step of CO2 fixation in the Calvin cycle (Geiger and Servaites, 1994; Tcherkez and Farquhar, 2005; Balmer et al., 2003; Foyer and Noctor, 2000; Kleczkowski, 1994). It has been estimated that nearly 70% of soluble leaf protein is in the form of Rubisco (Miller and Huffaker, 1982). Higher plant Rubisco is an oligomer composed of eight large subunits (Mr 52,000-55,000) and eight small subunits (Mr 12,000-15,000). The large subunit contains the active site for carboxylation and oxygenation reactions, and the small subunit protein is hypothesized to maintain the apoenzyme in a stable conformation capable ofactivation to a catalytically active holoenzyme (Miziorko and Lorimer, 1983). The small subunit protein of Rubisco is nuclear encoded by the rbcS gene which exists as a multigene family (Dean et al., 1989; Coruzzi et al., 1984; Rowan et al., 1996; Manzara and Gruissem, 1988; Lamppa et al., 1985). The gene for the large subunit of the rbcL gene is an appropriate choice for inference of phylogenetic relationships at higher taxonomic levels (Chase et al., 1993; Duvall et al., 1993; Les et al., 1991). Because of its slow synonymous nucleotide substitution rate in comparison with nuclear genes and its functional constraint that reduces the evolutionary rate of non synonymous substitutions (Wolfe et al., 1987), rbcL is considered to be more useful than the isozymes. The primary reduction of CO2 into organic matter (CO2 fixation) via the Calvin cycle represents a pathway essentially conserved throughout evolution (Tabita, 1988) and serves as the mechanism of primary production in nearly all ecosystems. Comprised of a large and small subunit, the enzyme is highly conserved from cyanobacteria to higher plants in both subunit organization (L8S8) and primary structure of the large subunit (ca. 70% homology at the nucleotide level and 78 to 81% homology at the amino acid level (Shinozaki et al., 1983).

The morphological features of a variety may vary due many factors including variety feature, soil and climatic parameters, management practices. The nutritional quality may also differ in parts other than fruit especially leaves, seeds, flowers, stem and bark. Some varieties have a significant difference exist between them while may have slight morphological character difference. The objective of this research work was to estimate the level of genetic diversity and to assess genetic relationships among six varieties of jackfruit using ‘rbcL gene’ based on PCR technique and RFLP markers.

3. Hypothesis

The current research work is based on the following hypothesis

1) Jackfruit varieties in Kerala differ in genotypical features
2) Phylogenetical differentiation could be obtained using rbcL gene
3) Phlogentic tree building using rbcL gene could establish the evolutionary relationships exist among the selected jackfruit varieties.

4. Materials and Methods

4.1 Botanical description and varieties

Artocarpus species (15 edible fruits) are known to occupy various niches and habitats, comprise mainly bread fruit and jackfruit (Jagtap and Bapat, 2010; Wangchu et al., 2013). Jackfruit is monecious and pollinated flowers develop several months to develop into ripe fruit, depending on climatic and soil conditions (Morton, 1987; Baliga et al., 2011). According to Prakash et al. (2009) jackfruit consist of lower fleshy edible region (bulb), middle fused region (syncarp) and out spiney region (spike). When ripe the fruit get fleshy, outer spines widened and flesh get soft and yellow (Saxena et al., 2009b). Except the thorny outer bark and axis are not edible (Baliga et al., 2011).

The jackfruits were classified based on their phonotypical and organoleptic characteristics with variation in bulb colour as well as shape, size, odour, flake size, flake colour and period of maturity (Haq, 2006; Prakash et al., 2009; Jagadeesh et al., 2007b; Jagadeesh et al., 2007a). Two types of ecotypes are recognised flake characteristics, one with soft and spongy while other with firm carpals which called different in regional languages (Baliga et al., 2011; Amma et al., 2011; Shyamalamma et al., 2008; Muralidharan et al., 1997; Odoemelam, 2005).

4.2 Nutritional and vitamin composition

Studies have proved that the nutritional and photochemical composition among jackfruit varies depending on the cultivar as well as region (Baliga et al., 2011; Arkroyd et al., 1966; Azad, 2000; Haq, 2006; Narasimham, 1990). It is a good source of vitamins (A, C, thiamine, riboflavin, niacin) and minerals especially calcium (Ca), potassium (K), iron (Fe), sodium (Na) and zinc (Zn) (Swami et al., 2012; Haq, 2006; Narasimham, 1990; Arkroyd et al., 1966; Azad, 2000). Protein and carbohydrate concentration also varied in seeds across India were some varieties contain 6.8% of protein content in seeds (Baliga et al., 2011; Chrips et al., 2008).

Table 1. Phenolic, flavinoid content and antioxidant activity of araticum, papaya and jackfruit in undigested and digested extracts (Modified after; Pavan et al., 2011).

Abbildung in dieser Leseprobe nicht enthalten

Numbers represent means ± one standard deviation (SD) of the mean

Table 2. Biochemical difference in various jackfruit varieties in South India (Chrips et al., 2008).

Abbildung in dieser Leseprobe nicht enthalten

Numbers represent means ± one standard deviation (SD) of the mean.

4.3 Study area

The samples are collected from different places in Kerala, including high altitude areas. Kerala state covers an area of 38,863 km² and situated between Western Ghats to east and Arabian Sea to the west. There are more than 3 crore people lives in Kerala. The soil conditions in Kerala are mostly acidic ranging from pH 4.5 to 6.2.

4.4 Sample collection

Sampling locations were selected in Kerala based on an elaborative baseline survey conducted during February 2015 to March 2015. The samples were collected based on an elaborative iterative survey as well as traditional knowledge from local people. Six samples were collected from different parts of Kerala, locations of the sample collection areas were recorded using a Trimble Geoexplorer II (Trimble Navigation Ltd, Sunnyvale, California) and data were transferred using GPS pathfinder Office software (Trimble Navigation Ltd, Sunnyvale, California). Fresh leaves were collected and transferred to polyethylene zipper bags placed along with frozen ice packs; transported immediately to the laboratory. The leaves were washed with sterile distilled water and placed in deep freezers till further analysis. The six different varieties (koozha, varikka, undachakka, paathi varikka, thaen varikka, Singapore varikka) of jackfruit leaves were collected and abbreviated as koozha (Ku; A1),varikka (Va; A9), unda chakka (Uc; A7) paathi varikka (Pv; A16), theen varikka (Tv; A3) and singapore varikka (Sv; A20).

4.5 Morphological descriptors for jackfruit tree classification

Different verities of jackfruit are found in Kerala, specific characteristics of different verities can be studied. The instruments used to collect data are, measuring scale (30 cm), tape (160 cm) weighing machine, camera, field book, twine etc., first we conducted an elaborative survey, from this survey six samples and data such as type, local name GPS position were selected. The parameters taken about general characters were tree vigour, age of tree, tree height, trunk perimeter, canopy structure, branching density, branching type, trunk surface, branching pattern (Khan et al., 2010).

Branch leaf and fruit stock leaf were also collected and analysed for various parameters including length, breadth, tip length, petiole length, petiole width, longest distance between veins, shortest distance between veins, leaf colour, leaf texture, leaf blade shape, leaf apex shape, leaf base shape. From fruit the information collected are fruit bearing position, fruit shape, stalk attachment to fruit, fruit rind colour, fruit surface, shape of fruit spine, spine density, fruit attraction, flake colour, flake taste (FT), fruit stalk length (FSL), fruit stalk breadth (FSB), fruit length (FL), fruit diameter (FD), fruit perimeter (FP), fruit weight (FW), fruit pith length (FPL), pith width (PW), total flakes (TF), flake length (FL), flake with seed weight (FSW), flake weight (FW), seed length (Sl), seed width (SW), seed perimeter (SP), seed weight (SW), number of partially developed seeds (NPDS), fruit rind width (FRW), spine length (SL), number spines per inch (NSPI).

4.6 Isolation of DNA

The total genomic samples from jackfruit leaves were isolated using a modified CTAB method with the addition of β-mercaptoethanol (Simon et al., 2007; Shyamalamma et al., 2008). Three gram leaves samples were wiped with 70% alcohol and chopped into fine pieces and later homognized along with 10 ml extraction buffer using a pre-chilled pestile and motar. The extraction buffer contains 100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl, 3% (w/v) CTAB, 2% polyvinyl pyrrolidine (PVP) and 1% β-mercaptoethanol. The contents were slowly mixed and incubated in water bath at 65°C for 1 hr with slight shaking. The contents were brought to room temperature after incubation and 5 ml chloroform: isoamyl alcohol mixture (24:1) was added. The tubes were centrifuged at 8000 rpm for 20 min at 4°C till a clear supernatant was obtained. After the final spin, the DNA was precipitated using ice-cold isopropanol for overnight at 4°C. The tubes were further centrifuged at 5000 rpm and the pellet washed with 2-3 drops of 70% alcohol, air dried and redissolved in 50 µl Tris-EDTA (TE) buffer and stored at -20°C till further analysis (Simon et al., 2007; Shyamalamma et al., 2008; Xiaoming and Xiuxin, 2001).

4.7 Quantification of DNA

The purity of the isolated DNA was checked spectrophometrically in a UV-Vis spectrophotometer by checking the 260/280 values (Thompson and Dvorak, 1989; Muller et al., 2003). Concentration of DNA (ng/µl) were calculated using the formula

DNA (ng/µl) = OD @ A260 x 50 x 100 x 0.1

Where OD @ A260 is the optical density at absorbance 260 nm

50 is the calculation factor

100 is the dilution factor

0.1 is the total volume of DNA

4.8 PCR amplification

The polymerase enzymes, adaptors and primers were purhasec from Genie life science technology, Bangalore, India. The PCR reaction was perfomed with a Biorad MJPt100 thermocycler (Bio-Rad Laboratories, Bangalore, India). PCR amplifications were performed on two stages. The pre-selecctive amplification was performed with an amplification profile of 94°C for 30 s, anneling at 56°C for 1 min, extension at 72°C for 1 min, repeated for 20 cycles, and then cooling at 10°C for 30 min. Further amplification was performed with a cycling profile of 94°C for 30 s, 65°C for 30 s, 72°C for 24 cycles followed by a cooling of 10°C for 30 min. The primers used for the selective amplification ended with three nucleotide extensions at 3’ ends. The PCR products were stored at -20°C in a deepfreezer. Electrophoresosi of the samples was carried out on agarose gels, by loading 10 µl of each DNA samples at 50 V for 3 hrs tracking dye has properly moved across the gel. The gels were lated viewed on a gel docuing sataion () and photographed.

4.9 Data sequencing

The PCR products were cleaned up using GenEluteTM PCR Clean-Up Kit (Sigma-Aldrich). Purified PCR products were sequenced by dideoxy chain termination method (Sanger et al., 1977) using AB3730XL capillary sequencer for the six jackfruit samples.

4.10 Data analysis

Taxonomical identification of the Artocarpus varities collected from different locations were carried out using molecular techniques. The sequences were aligned using the ClustalW algorithm (Thompson et al., 1994) in Bioedit 7.0 (DNA Sequence Analysis Software 224 package). Molecular phylogeny of Artocarpus heterophyllus varieties were studied using the partial sequence of the rbcl gene. Phylogenetic trees were constructed by the maximum likelihood (ML), neighbour joining (NJ) and unweighted pair group method with arithemetic mean (UPGMA) analysis with the software MEGA version 6 (Tamura et al., 2007), and using partial sequence of rbcl gene of the six Artocarpus heterophyllus varieties with 1,000 times boots trapping. Pair wise genetic distances between the Artocarpus heterophyllus varieties were calculated based on Kimura 2 parameter model and was used to calculate estimates of nucleotide diversity (Tajima, 1989), singleton variation, parsimony informative sites, and haplotype diversity. Statistical significance of Artocarpus heterophyllus varieties within the inferred trees was evaluated using the bootstrap of 1,000 replications.

4.11 Statistical analysis

The survey results were analyzed and descriptive statistics were done using SPSS 12.0 (SPSS Inc., an IBM Company, Chicago, USA) and graphs were generated using Sigma Plot 7 (Systat Software Inc., Chicago, USA).

[...]

Details

Pages
69
Year
2016
ISBN (eBook)
9783668383289
ISBN (Book)
9783668383296
File size
2.6 MB
Language
English
Catalog Number
v351290
Institution / College
Mar Augusthinose College
Grade
Tags
evaluation artocapus

Authors

Share

Previous

Title: Evaluation of genetic diversity of jackfruit (Artocapus heterophyllus Lam) varieties based on sequence analysis of the rbcL gene