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Studies on genetic relationships among locally cultivated Musaceae varieties in Kerala employing rbcL and matK gene using PCR technique and RFLP markers

by Dr. Prem Jose Vazhacharickal (Author) Sajeshkumar N. K. (Author) Jiby John Mathew (Author) Mathew Sebastian (Author) Betty Sebastian (Author)

Scientific Study 2016 58 Pages

Biology - Micro- and Molecular Biology

Excerpt

Table of contents

Table of figures

Table of tables

List of abbreviations

Studies on genetic relationships among locally cultivated Musaceae varieties in Kerala employing rbcL and matK gene using PCR technique and RFLP markers

Abstract

1. Introduction
1.1 Objectives
1.2 Objectives of the study
1.3 Taxonomical classification

2. Review of literature
2.1 Uses and importance
2.2 Banana stem and flower
2.3 Banana Leaves:
2.4 Environmental status:
2.5 Molecular markers

3. Hypothesis

4. Materials and Methods
4.1 Study area
4.2 Sample collection
4.3 Description of the species
4.4 Species identification
4.3 Isolation of DNA
4.5 Quantification of DNA
4.6 PCR primers
4.7 PCR amplification
4.8 Data sequencing
4.9 Data analysis
4.11 Statistical analysis

5. Results and discussion
5.1 BLAST search for rbcL: sample A; Palayankodan
5.2 BLAST search for rbcL: sample B; Njalipoovan
5.3 BLAST search for rbcL: sample C; Robusta
5.4 BLAST search for matK: sample A; Palayankodan
5.5 BLAST search for matK : sample B; Njalipoovan
5.6 BLAST search for matK: sample C; Robusta
5.7 Multiple sequence alignment for partial rbcL gene for Musaceae
5.8 Multiple sequence alignment for partial matK gene for Musaceae
5.9 Phylogenetic tree based on partial sequence rbcL gene by UPGMA method
5.10 Phylogenetic tree based on partial sequence rbcL gene by Maximum likelihood method
5.11 Calculation of genetic distance using rbcL gene
5.12 Phylogenetic tree based on partial sequence matK gene by UPGMA method
5.13 Phylogenetic tree based on partial sequence matK gene by Maximum likelihood method
5.14 Calculation of genetic distance using matK gene

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. Joseph V. J, 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 Betty Sebastian

*Address for correspondence

Assistant Professor

Department of Biotechnology

Mar Augusthinsoe 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. Map of Kerala showing the various sample collection points. Authors own work

Figure 3. Musaceae description a) robusta banana, b) palayankodan banana, c) njalipoovan banana, d) nendravazha banana, e) banana for sale in shop, f) banana flower and leaves for sale. Photo courtesy (d, e, f): Wikipedia

Figure 4. Musaceae description a) different types of banana fruit, b) cavandish banana, c) cross section of wild type banana, d) and e) banana flower, f) banana fruit during Hindu workship cermony. Photo courtesy: Wikipedia

Figure 5. Musaceae description a) banana corm, b) banana leaf, c) fish cooked on banana, d) lunch served on plantain leaf, e) making of banana leaf plate. Photo courtesy: Wikipedia

Figure 6. Musaceae description a) prasadm (offerings from temple) on banana leaf, b) and d) banana chips, c) banana chips coated with jaggery, e) banana chips made from semi-ripe fruit, f) preparation of banana chips. Photo courtesy: Wikipedia

Figure 7. Gel electrophoresis of genomic DNA; A) Sample 1, B) Sample 2 and C) Sample

Figure 8. BLAST search for rbcL gene of locally collected Musaceae family species (Palayankodan), Sample A1. Authors own work

Figure 9. BLAST search for rbcL gene of locally collected Musaceae family species (Palayankodan), Sample A2. Authors own work

Figure 10. BLAST search for rbcL gene of locally collected Musaceae family species; (Palayankodan), Sample A3. Authors own work

Figure 11. BLAST search for rbcL gene of locally collected Musaceae family species; (Njalipoovan), Sample B1. Authors own work

Figure 12. BLAST search for rbcL gene of locally collected Musaceae family species; (Njalipoovan), Sample B2. Authors own work

Figure 13. BLAST search for rbcL gene of locally collected Musaceae family species; (Njalipoovan), Sample B3. Authors own work

Figure 14. BLAST search for rbcL gene of locally collected Musaceae family species; (Robusta), Sample C1. Authors own work

Figure 15. BLAST search for rbcL gene of locally collected Musaceae family species; (Robusta), Sample C2. Authors own work

Figure 16. BLAST search for rbcL gene of locally collected Musaceae family species; (Robusta), Sample C3. Authors own work

Figure 17. BLAST search for matK gene of locally collected Musaceae family species (Palayankodan), Sample A1. Authors own work

Figure 18. BLAST search for matK gene of locally collected Musaceae family species (Palayankodan), Sample A2. Authors own work

Figure 19. BLAST search for matK gene of locally collected Musaceae family species (Palayankodan), Sample A3. Authors own work

Figure 20. BLAST search for matK gene of locally collected Musaceae family species; (Njalipoovan), Sample B1. Authors own work

Figure 21. BLAST search for matK gene of locally collected Musaceae family species; (Njalipoovan), Sample B2. Authors own work

Figure 22. BLAST search for matK gene of locally collected Musaceae family species; (Njalipoovan), Sample B3. Authors own work

Figure 23. BLAST search for matK gene of locally collected Musaceae family species; (Robusta), Sample C1. Authors own work

Figure 24. BLAST search for matK gene of locally collected Musaceae family species; (Robusta), Sample C2. Authors own work

Figure 25. BLAST search for matK gene of locally collected Musaceae family species; (Robusta), Sample C3. Authors own work

Figure 26. Multiple sequence alignment of partial rbcL gene of locally collected Musaceae species. Authors own work

Figure 27. Multiple sequence alignment of partial rbcL gene of locally collected Musaceae species. Authors own work

Figure 28. Partial sequence rbcL gene of locally collected Musaceae species by UPGMA method. Authors own work

Figure 29. Partial sequence rbcL gene of locally collected Musaceae species by Maximum likelihood method. Authors own work

Figure 30. Pairwise phylogenetic distance matrix based on partial rbcL gene of Musaceae species. Authors own work

Figure 31. Partial sequence of matK gene of Musaceae species by UPGMA method. Authors own work

Figure 32. Partial sequence of matK gene of Musaceae species by UPGMA method. Authors own work

Figure 33. BLAST search for matK gene of locally collected Musaceae species; sample A3, Njalipoovan. Authors own work

Table of tables

Table 1. Different vernacular names of Musaceae in India

List of abbreviations

illustration not visible in this excerpt

Studies on genetic relationships among locally cultivated Musaceae varieties in Kerala employing rbcL and matK gene using PCR technique and RFLP markers

Prem Jose Vazhacharickal1*, Sajeshkumar N.K1, Jiby John Mathew1 and Betty Sebastian1

* premjosev@gmail.com

Abstract

The classification of the Musaceae (banana) family species and their phylogenetic inter-relationships remain controversial, in part due to limited nucleotide information to complement the morphological and physiological characters. In this work the phylogenetic relationships within the Musaceae family were studied locally using 3 species. DNA sequences obtained from nine unlinked nuclear genes. Musa species grow in a wide range of environments and have varied human uses, ranging from the edible bananas and plantains of the tropics to cold-hardy fibre and ornamental plants. There are five taxonomic sections in the genus Musa, two of which contain edible bananas. For studying phylogeny of locally collected Musaceae (banana) family varieties using rbcL gene and matK gene. For this study DNA was extracted by using CTAB method. This extracted DNA was analysed by spectrophotometry method for checking purity of DNA. The samples were gel electrophoresed by 1% agarose gel electrophoresis at 80 volts. After electrophoresis the gel is examined in gel documentation system. The DNA band was observed under UV light looking florescent orange red colour. The extracted DNA was amplified by PCR method and PCR sample was applied for electrophoresis for checking DNA bands. After all this analysis PCR sample send for DNA sequencing for checking the nucleotide. Comparing the sequenced nucleotide for checking phylogeny of locally collected banana varieties. The locally collected gene sequence-based phylogeny presented here provides support for the early studies of speciation within the Musaceae. An understanding of the main phylogenetic relationships between banana species will help to fine-tune the taxonomy of Musaceae.

Keywords: Anti-oxidant; matK; PCR; RFLP; Musaceae.

1. Introduction

Musa is a member of the monocot order Zingiberales, a lineage of Commelinids that diverged from the lineage leading to rice (Poales) in the mid Cretaceous period over 100 million years ago. The banana plant is a large perennial herb with leaf sheaths that form trunk-like pseudostems. These are tree-like, herbaceous plants that live in warm countries. They can be grown in hot houses in colder areas, or outside during the summer months for the tropical beauty of their large, paddle-shaped leaves. The "stems" of these plants are made of overlapping leaf sheaths that die after flowering. The common Banana is Musa paradisiaca subspecies sapientum. It can grow to 15 or 20 feet tall and produce seedless, nourishing fruits. There are many different kinds; most having yellow skin, but some are red. Some of the better-known varieties are: Gros Michel, Red Jamaica, Apple, Orinoco and Champa (Lady-Finger Banana). The Plantain, M. paradisiaca, is a species of Musa that grows about 30 feet high and produces green or greenish-yellow seedless fruits that can be eaten after they have been cooked. It is grown in most tropical countries and is a part of the staple diet. It's probably a native of India (Chia, 1981).

Flower development is initiated from the true stem underground (corm) 9 - 12 months after planting. The inflorescence (flower stalk) grows through the center of the pseudo stem. Flowers develop in clusters and spiral around the main axis. In most cultivars, the female flowers are followed by a few "hands" of neuter flowers that have aborted ovaries and stamens. The neuter flowers are followed at the terminal ends by male flowers enclosed in bracts. The male flowers have functional stamens but aborted ovaries. Fruits mature in about 60 - 90 days after flowers first appear. Each bunch of fruits consists of variable numbers of "hands" along a central stem. Each "hand" consists of two transverse rows of fruits ("fingers"). The fruit quality is determined by size (finger length and thickness), evenness of ripening, freedom from blemishes and defects, and the arrangement of the clusters. Bananas contain about 74% water, 23% carbohydrate, 1% protein, and 0.5% fat (Chia, 1981). A 4-ounce banana without the peel is a good source of vitamin B6, potassium, and fibre.

Banana fruit may be eaten raw or as a cooked vegetable. The fruit can also be processed for a number of food products. Ripe fruits can be pulped for puree for use in a variety of products including ice cream, yogurt, cake, bread, nectar, and baby food. Ripe bananas can be dried and eaten, or sliced, canned with syrup, and used in bakery products, fruit salads, and toppings. Green (unripened) bananas can be sliced and fried as chips. Whole green fruits can also be dried and ground into flour. Vinegar and alcoholic beverages can be made from fermented ripe bananas. Other parts of the banana plant are consumed besides the fruit. The heart of the growing pseudo stem is eaten in India. In Southeast Asia, the male bud is eaten as a boiled vegetable. The banana leaves are not eaten but may be used for wrapping food in cooking. The banana foliage and pseudo stems are used as cattle feed during dry periods in some banana producing areas. Culled bananas are used to feed cattle and hogs. Bananas are a good energy source but need to be supplemented with protein. There are hundreds of varieties of banana found growing in different parts of the world. In Kerala, there are about 50 cultivars of Banana.

Palayam kodan – this is the most widely cultivated variety of banana in Kerala, very soft when fully ripe. It has a cooling effect on places of very hot and humid conditions. Annaan – there are different cultivars under this name, each type offering a distinct flavor. Morris or Robusta – the banana that retains its green color even after ripening, it comes from a dwarf variety of banana plants, cultivated throughout Kerala. Kappa vazha (red banana) – is characterized by its red skin. Fairly large sized fruit turns its color from deep brown to dark red as it ripens. The edible part is very soft. Many varieties of banana plants in Kerala have been imported from countries like Brazil, African countries, USA, Australia, etc. The Agricultural University of Kerala too has developed new cultivars of bananas combining traces of different variety.

The core stem of the plant, called vazhappindi is used to prepare a side dish called thoran, which is recommended for persons with diabetes. The preparation is very tasty too. Similarly vazha koombu (vazha chundu), the flower or cone of plantain plants is also used as a nicely tasting vegetable. The cone is collected after the flower ceases to produce new fruits.

Species important for domestication/cultivation:

Musa acuminata (wild and cultivated bananas)

Musa balbisiana (wild banana)

Musa paradisiaca (plantains)

1.1 Objectives

The objectives of this study was PCR based amplification, and sequencing of Musa species and determination of the phylogenetic relationships based on matK gene and rbcL gene. The development of DNA-based genetic markers has had a revolutionary impact on plant genetics. It is theoretically possible to observe and exploit genetic variation in the entire genome of organisms with DNA markers. RuBisco (Ribulose-1,5- bisphosphate carboxylase oxygenase), is an enzyme which encodes in chloroplast of plant cells that functions in the Calvin cycle to fix carbon dioxide (CO2) and help drive the synthesis of glucose. RuBisco is an important enzyme in photosynthesis because without it plants would not be able to form glucose and other necessary metabolites to sustain life. Research has shown that decreases in RuBisco can dramatically alter plant size and thus leading to smaller crop yields. The other gene used in this study is matK gene. The matK gene is also located on the cpDNA and encodes a maturase involved in splicing type II introns from RNA transcripts. The matK gene is encoded by the chloroplast trnK intron (Sugita et al. 1985). Since matK has a relatively fast mutation rate, it evolves faster than the rbcL gene therefore; matK analysis should be useful for studying the phylogeny of different genera. The matK gene, formerly known as orfK, is emerging as yet another gene with potential contributions to plant systematics and evolution and DNA markers in plant improvement (Kumar et al., 2009).

For this experiment primer pairs of rbcL and matK genes were selected for the partial amplification of these genes of the plants stated above and their sequences were analyzed for similarities and dissimilarities to check the phylogenetic relationship between them. The development of DNA-based genetic markers has had a revolutionary impact on plant genetics. It is theoretically possible to observe and exploit genetic variation in the entire genome of organisms with DNA markers.

According to modern evolutionary theory, all organisms on earth have descended from a common ancestor, which means that any set of species, extant or extinct, is related. This relationship is called a phylogeny, and is represented by phylogenetic trees, which graphically represent the evolutionary history related to the species of interest. Phylogenetics infers trees from observations about existing organisms using morphological, physiological, and molecular characteristics. The “tree of life” represents a phylogeny of all organisms, living and extinct. Other, more specialized species and molecular phylogenies are used to support comparative studies, test biogeographic hypotheses, evaluate mode and timing of speciation, infer amino acid sequence of extinct proteins, track the evolution of diseases, and even provide evidence in criminal cases (Baldauf, 2003).

1.2 Objectives of the study

The objectives include

1. To generate a base line information on the phylogenetic relatedness between the locally available Musa varieties viz. Robusta, Njalipoovan, Palayam kodan etc.
2. To understand the variations among the said varities of Musa and also with other selected members of the Musaceae family.

Gene specific primers used here are matK and rbcL gene. The matK gene was first identified by Sugita et al. (1985) from tobacco (Nicotiana tabacum) when they sequenced the trnK gene encoding the tRNA (Lys) of the chloroplast and rbcL .The rbcL gene is a valuable tool for assessing phylogenetic relationships. This gene is found in the chloroplasts of most photosynthetic organisms. These genes are analysed by PCR amplification and sequence analysis. This is based on the principle that denaturation at 94°C, annealing at 54°C and extension at 72°C .Temperature and time may depend on the primer used (Chen and Janes, 2002).

1.3 Taxonomical classification

Kingdom: Plantae-- planta, plantes, plants, vegetal Subkingdom: Tracheobionta -- vascular plants Division: Magnoliophyta -- angiosperms, flowering plants, phanerogames Class: Liliopsida -- monocotyledones Subclass: Zingiberidae Order: Zingiberales Family: Musaceae - banana Genus: Musa L Species: Musa acuminate/Musa colla/Musa paradiscica.

Bananas and plantains belong to the genus Musa, of the family Musaceae. The genus has five sections namely Eumusa (x=11), Rhodochlamys (x = 11), Australimusa (x- 10), Calimusa (x = 10) and incerte sedis (x= 7, 0). The vast majority of cultivated bananas and plantains belong to section “Euromusa” and have originated in tropical region of South East Asia from two wild species - Musa acuminate Colla and Musa balbisiana Colla. Depending upon their ploidy level they posses 22, 23 or 44 chromosomes (x = 11). The most widely cultivated clones of commerce are the triploids (2n = 3x = 33) which have more vigorous growth characteristics and higher yield than diploids (2n=2x=22). Tetraploid (2n = 4x = 44) clones are rather rare but diploids are often cultivated in tropical areas for local consumption and some are valued for their good flavoured fruit. The development of edible bananas initially resulted from the human selection of diploid M. acuminate varieties that were parthenocarpic. Later, selection was for female infertility which resulted in fruits with few or no seeds. Diploid (AA) cultivars gave rise through nuclear restitution during meiosis, to triploid (AAA) cultivars.

2. Review of literature

Musa, a plant genus of extraordinary significance to human societies, produces the fourth most important food in the world today (after rice, wheat, and maize), are bananas and plantains. Musa species grow in a wide range of environments and have varied human uses, ranging from the edible bananas and plantains of the tropics to cold-hardy fiber and ornamental plants. These large, perennial herbs, 2–9 m (6.6–30 ft) in height, evolved in Southeast Asia, New Guinea, and the Indian subcontinent, developing in modern times secondary loci of genetic diversity in Africa, Latin America, and the Pacific. Musa species attained a position of central importance within Pacific societies: the plant is a source of food, beverages, fermentable sugars, medicines, flavorings, cooked foods, silage, fragrance, rope, cordage, garlands, shelter, clothing, smoking material, and numerous ceremonial and religious uses (Bailey and Sood, 1987)

There are five taxonomic sections in the genus Musa, two of which contain edible bananas. It belong to the Family Musaceae (banana family) (www.traditionaltree.org August 2006 ver. 2.2). There are two types of molecular markers have been used for the study of phylogenetics of Musa spp.

Molecular phylogenetics applies a combination of molecular and statistical techniques to infer evolutionary relationships among organisms or genes. The similarity of biological functions and molecular mechanisms in living organisms strongly suggests that species descended from a common ancestor. Molecular phylogenetics uses the structure and function of molecules and how they change over time to infer these evolutionary relationships. This branch of study emerged in the early 20th century but didn’t begin in earnest until the 1960s, with the advent of protein sequencing, PCR, electrophoresis, and other molecular biology techniques. The primary objective of molecular phylogenetic studies is to recover the order of evolutionary events and represent them in evolutionary trees that graphically depict relationships among species or genes over time (Christelová et al., 2011)

2.1 Uses and importance

There are many healing and medicinal properties of bananas. The high content of iron in bananas increases the production of hemoglobin in the blood therefore they are very good for anemia. They regulate bowel movement whether it is constipation or diarrhoea. Eating a banana after every meal improved digestion significantly. Cold milk soothes the stomach lining and bananas with honey build up depleted blood sugar levels. Bananas are exceedingly good for students as the rich source of potassium can make a person very alert; the fruit is often called a brain tonic. Bananas work well as a snack for people who have high blood pressure as they are wholesome with low salt levels.

For those suffering from depression, bananas are good as they contain a protein called serotonin, which is also called the ‘happy hormone’ as it makes one feel happy and relaxed. Bananas can be eaten frequently to treat ulcers as they neutralize acidity in the stomach. This soft and smooth fruit cannot irritate the stomach walls. For pregnant women suffering from morning sickness, eating bananas in between meals helps immensely in settling the queasiness in the stomach. Eating bananas helps people give up smoking as this fruit is rich in vitamin C, A, B6 and B12. Vitamin B6 in bananas acts as an anti-inflammatory agent that helps ward off cardiovascular disease, type II diabetes, as well as obesity. B6 also contributes to the maintenance of the lymphoid glands that ensure the production of healthy white blood cells that protect the body from infection. Finally, the vitamin B6 in bananas plays a pivotal part in cell formation and proper nervous system function, making one banana a day a healthy and delicious choice.

Bananas contain potassium and magnesium as well, which help the body recover from nicotine withdrawal. Bananas have an antacid effect, so people who experience heartburn find relief on eating a banana. Potassium is a vital mineral which normalizes heartbeat while regulating the body’s water balance. As banana is a rich source of potassium, it re-balances disturbances of fluids in the body. For weight watchers, banana is an excellent snack in place of crisps and chocolates. Research found that food craving during high pressure work could be assuaged safely and in a healthy manner by eating a banana every 2-3 hourly as it is a high carbohydrate food that controls blood sugar levels. Topically, the peel of a banana with the yellow side on top can be taped to a wart. It will shrivel and fall off. The peel of a banana fruit can be rubbed on a mosquito bite with good effect, the stinging sensation stops and the swelling also reduces. A ripe banana mashed and applied on the face is great at moisturizing and nourishing tired and dry skin. The probability of developing kidney cancer in female subjects decreased by 50% when eating bananas four to six times a week (Slavin and Lloyd, 2012; Mudgil and Barak, 2013).

Along with other fruits and vegetables, consumption of bananas associated with a reduced risk of colorectal cancer and in women, breast and renal cell carcinoma Individuals with a latex allergy may experience a reaction to bananas. Bananas contain considerable amounts of vitamin B6, vitamin C, and potassium. The latter makes them of particular interest to athletes who use them to quickly replenish their electrolytes In India, juice is extracted from the corm and used as a home remedy for jaundice, sometimes with the addition of honey, and for kidney stones (Prasobh et al., 2010).

2.2 Banana stem and flower

Both are used as a vegetable and eaten along with rice. The banana stem is rich with fibre and this is very beneficial for those on a weight-loss program. It is also a rich source of potassium and vitamin B6 which helps in the production of insulin and hemoglobin. Eating banana stem once a week keeps high blood pressure in control. Banana stem also maintains fluid balance within the body. It is a diuretic and helps detoxify the body. The banana flower is rich in vitamins, flavonoids and proteins. The flower has been used in traditional medicine to treat bronchitis, constipation and ulcer problems. It eases menstrual cramps. The extracts of banana flower have anti oxidant properties that prevent free radicals and control cell and tissue damage.

2.3 Banana Leaves:

The leaf makes an excellent platter and food served on these leaves tastes delicious. The leaves are not eaten but while steaming food some of the polyphenols are imparted to the food.

2.4 Environmental status:

Banana is essentially a tropical plant requiring a warm humid climate, but it is adapted to a wide range of climatic conditions ranging from wet tropical to dry sub-tropical. It can grow successfully from sea level to an altitude of 1,500 metres. Temperature plays an important role as the rate of new leaf formation and fruit growth is largely governed by it. It grows well at a temperature range of 10 to 40°C. However, a mean temperature of 26.7°C and 100 mm of rain per month are considered satisfactory. Frequent rains and humid weather are preferable (Chia, 1981).

Low temperature, frost and wind storm are the limiting factors in successful banana cultivation. Low temperature below 10°C is harmful and repeated occurrence of cold waves during the winter seriously interferes with the normal growth of the plants. Storms and winds are also very hazardous and sometimes result in complete uprooting of or breakage of the pseudo stem. Hot winds during summer months shed and desiccate the leaves (Chandy, 1989).

2.5 Molecular markers

The development of DNA-based genetic markers has had a revolutionary impact on animal genetics. It is theoretically possible to observe and exploit genetic variation in the entire genome of organisms with DNA markers. Allozymes, mitochondrial DNA, RFLP, RAPD, AFLP, microsatellite, SNP, and EST markers are the popular genetic markers employed in genomic studies.

One of the main questions at the beginning of any genome study is what type of marker is most suitable for the given project and to the species of interest. There is no simple answer to this question, and much depends on the specific objectives of the study. Depending on the problem addressed, on the available resources, time and costing, some molecular markers can be more appropriate than others for studying a given problem. For example, DNA sequencing provides a resolution appropriate to phylogenetic and population-level studies but microsatellites are more appropriate to studies of parentage and breeding.

2.5.1 Chloroplast DNA analysis

In this study two genes, namely, rbcL and matK were used to elucidate the phylogenic relatedness of the selected species. The gene rbcL encodes Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known by the abbreviation RuBisCO, is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy rich molecules such as glucose . The rbcL gene, located on the chloroplast DNA (cpDNA) (Penjor et al., 2013). Compared to most genes encoded in the cpDNA, the rbcL gene has a relatively slow nucleotide substitution rate. In plants, algae, cyanobacteria, and phototrophic and chemoautotrophic proteobacteria, the enzyme usually consists of two types of protein subunit, called the large chain (L, about 55,000 Da) and the small chain (S, about 13,000 Da).

The large-chain gene is part of the chloroplast DNA molecule in plants. There are typically several related small-chain genes in the nucleus of plant cells, and the small chains are imported to the stromal compartment of chloroplasts from the cytosol by crossing the outer chloroplast membrane The enzymatically active substrate (ribulose1, 5-bisphosphate) binding sites are located in the large chains that form dimmers in which amino acids from each large chain contribute to the binding sites (Josh et al., 1999; Mohan et al., 1997). A total of eight large-chains (= 4 dimmers) and eight small chains assemble into a larger complex of about 540,000Da .In some proteobacteria and dinoflagellates, enzymes consisting of only large subunits have been found. Magnesium ions (Mg2+) are needed for enzymatic activity. Correct positioning of Mg2+ in the active site of the enzyme involves addition of an "activating" carbon dioxide molecule (CO2) to a lysine in the active site (forming a carbamate). Formation of the carbamate is favored by an alkaline pH. The pH and the concentration of magnesium ions in the fluid compartment in plants, the stroma of the chloroplast increases in the light. The rbcL gene is a valuable tool for assessing phylogenetic relationships. This gene is found in the chloroplasts of most photosynthetic organisms. It is an abundant protein in leaf tissue and very well may be the most abundant protein on earth. Thus this gene exists as a common factor between photosynthetic organisms and can be contrasted with the rbcL genes of other plants in order to determine genetic similarities and differences. It codes for the large subunit of the protein ribulose-1, 5-biphosphate carboxylase/oxygenase (Rubisco) (Geilly and Taberlet, 1994).

The most common gene used to provide sequence data for plant phylogenetic analysis is the plastid-encoded rbcL gene (Chase et al., 1993; Wojciechowski et al., 1993). This single copy gene is approximately 1,430 base pairs in length, is free from length mutations except at the far 3' end, and has a fairly conservative rate of evolution. The function of the rbcL gene is to code for the large subunit of ribulose 1, 5 phosphate carboxylase/oxygenase (Rubisco or RuBPCase). The sequence data of the rbcL gene are widely used in the reconstruction of phylogenies throughout the seed plants. However, it is apparent that the ability of rbcL to resolve phylogenetic relationships below the family level is often poor (Doebley et al., 1990). Thus, interest exists in finding other useful DNA regions that evolve faster than does rbcL to facilitate lower-level phylogenetic reconstruction.

The other gene used for this study is matK gene. The matK gene is also located on the cpDNA and encodes maturase enzyme involved in splicing type II introns from RNA transcripts. The matK gene is encoded by the chloroplast trnK intron. Since matK has a relatively fast mutation rate, it evolves faster than the rbcL gene .Therefore; matK analysis should be useful for studying the phylogeny of the genera included Aurantioideae. The matK gene, formerly known as orfK, is emerging as yet another gene with potential contributions to plant systematic and evolution (Johnson and Soltis, 1994, Johnson and Soltis, 1995; Steele and Vilgalys, 1994; Liang and Hilu, 1996). The gene, ˜ 1500 base pairs (bp), is located within the intron of the chloroplast gene trnK on the large single-copy section adjacent to the inverted repeat. There have been several studies using the matK gene sequence in phylogenetic reconstruction. These studies involved six families. In Saxifragaceae, matK was found to evolve approximately three-fold faster than rbcL (Johnson and Soltis, 1994; Johnson and Soltis, 1995; Soltis et al., 1996). The sequences of matK in Polemoniaceae varied at an overall rate twice that of rbcL sequences (Steele and Vilgalys, 1994; Johnson and Soltis, 1995). Substitutions at the third codon position predominated in rbcL sequences, while in matK substitutions were more evenly distributed across codon positions. Recently, the matK gene sequences have been also used in Orchidaceae tribe Vandeae (Jarrell and Clegg, 1995), Myrtaceae (Gadek et al.1996), Poaceae (Liang and Hilu 1996), Apiaceae (Plunkett et al. 1996), and flowering plants (Hilu and Liang, 1997). According to the detailed analysis of the matK sequence data available in Gene Bank and preliminary studies (Liang and Hilu, 1996; Hilu and Liang, 1997), matK has higher variation than any other chloroplast genes. Although the variation is slightly higher at the 5’ region than at the 3’ region, approximate even distribution was observed throughout the entire gene. In addition, the high proportion of tranversion of the matK gene might provide more phylogenetic information. These factors underscore the usefulness of the matK gene in systematic studies and suggest that comparative sequencing of matK may be appropriate for phylogenetic reconstruction at subfamily and family levels.

3. Hypothesis

The current research work is based on the following hypothesis

1) Morphological variations among Musaceae varieties in Kerala would be reflected in genetic diversity among them
2) Gentetic relationships among Musaceae varieties could be established using ‘matk gene’.
3) RFLP and other bioinformatics tools would provide a better knowledge and establish relationship among different morphologically identified Musaceae varieties.

[...]


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

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Pages
58
Year
2016
ISBN (eBook)
9783668482616
ISBN (Book)
9783668482623
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3.5 MB
Language
English
Catalog Number
v370768
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Mar Augusthinose College
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1,5
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studies musaceae kerala rflp

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Title: Studies on genetic relationships among locally cultivated Musaceae varieties in Kerala employing rbcL and matK gene using PCR technique and RFLP markers