Screening of Rice Landraces of Coastal Areas for Salt Tolerance at Seedling Stage Using Molecular Markers

¹Shamsunnahar Mukta, *²Sumon M. Hossain, ²Dr. Khondoker M. Nasiruddin, ³Dr. Mirza Mofazzal Islam

¹Department of Plant and Environmental Biotechnology, Sylhet Agricultural University (SAU), Sylhet-3100, Bangladesh

²Department of Biotechnology, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

³Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh-2202, Bangladesh

Abstract

Background and Objective: The main objective of this study is to develop salt-tolerant rice varieties by identifying suitable parents and genetic diversity analysis. Salinity is becoming a serious problem in the world and a widespread soil problem in rice growing countries. The saline area is three times larger than land used for agriculture. The conventional methods of plant selection for salt tolerance are difficult because of the large effects of the environment. But DNA markers seem to be the best option for efficient evaluation and selection of plant material. SSRs markers have been proved to be ideal for making genetic maps, assisting selection and studying genetic diversity. Materials and Methods: The study was conducted under Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, Bangladesh. Initially 80 germplasms were used to evaluate the salinity tolerance at seedling stage at glass-house following IRRI standard protocol. Among them, 12 were found as salt tolerant, 13 were found as moderately tolerant, 29 were highly susceptible and 26 were susceptible by phenotypic analysis. Among them, 25 germplasms were used for molecular study, which carry all tolerant variety found in phenotypic study (Hogla, Jamai Naru, Dakhsail, Patnai, Kute Patnai, Holde Gotal, Bazra Muri, Ghunshi, Tal Mugur, Nona Bokhra, Kashrail and FL378), 7 were moderately tolerant, 5 were highly susceptible and 1 was susceptible. These germplasms were characterized by 3 SSR markers which are RM510, RM585 and RM336. Data was analyzed by POPGENE (version 1.31), Power Marker (Version 3.25) and NTSYS-PC (Version 2.2). Results: The number of alleles per locus ranged from 10 to 12, with an average number of alleles of 11 per locus and PIC values ranged from a low of 0.8533 (RM336) to a high of 0.8940 (RM585). The average gene diversity of overall SSR loci for the 25 genotypes was 0.8885, ranged from 0.9024 to 0.8672. Unweighted Pair Group Method of Arithmetic Means (UPGMA) dendrogram constructed from Nei’s (1972) genetic distance produced five distinct clusters of 25 rice genotypes. FL378 of IRRI was used as check variety. It can be concluded that Holde Gotal, Bazra Muri and Hamai were salt tolerant compared to FL 378. Conclusions: This scientific information could be used for solution of suitable parents, development of salt tolerant rice varieties, gene identification for salt tolerance and genetic diversity analysis.

http://scialert.net/qredirect.php?doi=ajbkr.2017.71.79&linkid=pdf

Molecular assessment of genetic diversity and relationship in selected mungbean germplasms


Saiful Islam Bhuyan, Md. Sanower Hossain, Mirza Mofazzal Islam, Shamsun Nahar Begum, Zannat Urbi & Md. Sumon Hossain

ABSTRACT

Mungbean is an important crop considering the nutritional supplementary of low fat, high fiber and protein but its production is very low compared to the daily requirements. Hence, the assessment of genetic diversity and relationships in the existing germplasms is a major concern for the development of high yielding variety of mungbean. In this study, Random Amplified Polymorphic DNA (RAPD) marker was used to analyse 7 exotic and 3 advance germplasms using 3 primers (OPA01, OPB06 and OPB07). On an average 6 amplified products/primer were formed with overall polymorphisms of 78.33%. The similarity coefficient was highest (0.93) between AVRDC-3 and AVRDC-4, indicating less divergence and was least (0.39) between AVRDC-5 and AVRDC-6, indicating more divergence. On the basis of UPGMA dandogram, genotypes AVRDC-5, AVRDC-6 and AVRDC-7 were found to be quite distinct and the simple matching coefficient varied from 0.1824 to 0.8109. These findings will be of significance in the development of intraspecies crossing variety of mungbean crop.

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DNA Fingerprinting and Genetic Diversity Analysis of Chilli Germplasm Using Microsatellite Markers

Sumon M. Hossain, U. Habiba, Saiful I. Bhuyan, M.S. Haque, S.N. Begum and Delwar M. Hossain

ABSTRACT

Microsatellite markers are useful tools for evaluating genetic diversity and DNA fingerprinting. The purpose of this study was to evaluate the genetic diversity within 22 chilli germplasm by using four microsatellite markers. All the microsatellite loci amplified by polymerase chain reaction (PCR) were found polymorphic in all studied germplasm. A total of 27 alleles were detected and the number of alleles per marker ranged from 4-13. Based on Nei’s genetic distance, the Unweighted Pair Group Method of Arithmetic Means (UPGMA) dendrogram, grouped 22 chilli germplasm into 3 clusters: fifteen varieties  Bogra morich, BD-2043, Balijuri morich, BD-2082, Kamranga morich, Tinn Tahori morich, Kalo morich, Angoor morich, Shada morich, Balujurii morich, BD-2011, BD-2035, BD-2005, Pepsicum morich, Dhani morich were grouped in cluster-1; Bindi morich, Altaf morich, Boro morich, BD-2025 were formed cluster-2; and Comilla morich, Sada gol morich and Ruma morich formed cluster-3. The values of pair-wise comparisons of Nei’s (1972) genetic distance (GD) between varieties were computed from combined data for the 4 primers and ranged from 0.704 to 0.926. The higher genetic distance indicated that these varieties were derived from different origin and could be utilized in breeding programme for traits of interest. From the difference between the highest and the lowest GD value, it was revealed that there were wide variabilities among 22 chilli varieties and genotypes. Higher genetic variability within varieties and significant difference between varieties indicate rich genetic material of a species.  The average gene flow value across all the loci (0.00) indicates that there was no genetic divergence among the germplasm. Thus microsatellite markers offer a potential, simple, rapid and reliable method to evaluate genetic variation and DNA fingerprinting among the chilli germplasm. The findings of the present study have the potential applications in future breeding programme for the genetic improvement of chilli.

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Biofortification of rice by targeted genetic engineering.

Globally, malnutrition, including both overt nutrient deficiencies as well as diet-related chronic diseases (e.g., heart disease, cancer stroke and diabetes) is responsible for more deaths than any other cause accounting for over 20 million mortalities annually(Kennedy et al., 2003; World Health Organization and Food and Agriculture Organization, 2003). Malnutrition also contributes to increased morbidity, disability, stunted mental and physical growth and reduced national socio-economic development (World Health Organization and Food and Agriculture Organization, 2003).  Micronutrient malnutrition alone afflicts over two billion people mostly among resource-poor families in developing countries with iron, iodine, Folic acid, zinc and vitamin A deficiencies most prevalent (Kennedy et al., 2003). 

Biofortification of Rice is a method of breeding rice variety to increase their nutritional value. This can be done either through conventional selective breeding, or through genetic engineering.

Conventional breeding has some problem as such either the desire gene should be or not introduce into the targeted rice plant. So , Genetic engineering is the way to confirm that the transgenes would be inserted into the targeted rice plant, because of the use of the marker gene with the transgenes.

Biofortification of Rice differs from ordinary fortification because it focuses on making rice foods more nutritious as the rice plants are growing, rather than having nutrients added to the rice foods when they are being processed. This is an improvement on ordinary fortification when it comes to providing nutrients for the rural poor, who rarely have access to commercially fortified foods.  As such, biofortification of rice is seen as an upcoming strategy for dealing with deficiencies of micronutrients in the developing world.

How can economically, nutritional rice plant food can be produced through genetic engineering?

The aim of the study is to explore the valuable nutrients into the rice plants through genetic engineering.

It has chosen the quantitative research method and prospective type of descriptive survey to carry out the answer of the research question. This study had been aimed to explore the state of biofortification of rice throw genetic engineering. 

Oryza sativa ssp. japonica is being transformed with the plasmid vector containing four transgenes. The transformation vector is constructed by cloning into pCAMBIA 1300 (Cambia, Canberra, Australia) the AtNAS, Pvferritin, fphytase and pmi genes encoding Arabidopsis thaliana NAS (At5g04950), Phaseolus vulgaris- ferritin (X58274), Aspergillus fumigates strain Af293 phytase (AFUA_4G08630) and Escherichia coli phosphomannose-isomerase, pmi- gene, respectively. The AtNAS and pmi genes are placed under the control of 35S promoters; the Pvferritin and Afphytase genes are placed under the transcriptional control of endosperm-specific globulin promoters. The pmi gene is used as a selectable marker. The transformation, selection and plant regeneration are conducted according to Lucca et al. (2001b) following a mannose selection regime. In brief, immature embryos are used as starting material and gene transfer is mediated by Agrobacterium tumefaciens strain LBA4404 (Hoekema et al., 1984). A total of 150 putative primary transformants is obtained after transformation and selection on mannose, of which 20 plants are regenerated and analysed by Southern blot as described by Poletti (2006). In order to exclude the possibility that null segregants dilute the results, all plants are checked by PCR for the presence of the transgenes prior to hydroponic culture using the following primers: Afphy-f, 5-AGCTGTCCGTGTCGAGTAAG-3; Afphy-r, 5-TGGAGACTAGAGTCGAGTTAG-3;Actin-f,5-TATGGTTGGGATGGGACA-3,Actin-r,5-AGCACGGCTTGAATAGCG-3.

In addition, RNA is extracted from the leaves of transgenic (NFP) and WT plants during the vegetative growth phase according to Poletti (2006). The transcription of the transgenes in the NFP line was checked by real time PCR.

Folic acid biosynthesis gene transfer into rice plant

Bangladesh is an agriculture based developing country. It has huge population, among them most of living under poverty and are suffer from hungry for food. Rice is our stale food. Every year the govt. export rice from the foreign country by the exchange of money to meet the hungry. Bangladeshi climate is suitable for the production of rice but the natural disaster is great problem for the production of more rice. Such disaster are: Flood, Storm and Saline water etc. Every year 25% of crop are being destroyed due to disaster. But it can be easily solve by using the modern technique of Biotechnology. Gene transformation is one of them. Many scientist have already produced this type of abiotic tolerant of rice plant. Beside this, diseases is also cause serious problem to produce much rice. This also overcome using gene transformation technique to produce biotic resistant variety of rice by the recombinant DNA technology.

So, I think it should be the best way to develop the rice variety to produce more and more rice in a small property of land. After all our population is increasing day by day but the land is still fixed.

Several methods are being used for gene transformation, such as:
 Agrobacterium mediated gene transfer
 Chemically mediated gene transfer
 Electroporation
 Microinjection
 Microprojectile bombardment method

Among those, Agrobacterium mediated gene transfer is more suitable than any other method. Because Agrobacterium tumefaciens is soil born natural genetic engineer. The transformation of dicotyledons by Agrobacterium is well established. But in the case of monocots it is not the general process. In the past monocots, particularly graminacious crop plants including important cereals like rice and wheat were considered to be recalcitrant to this technology and they were outside the Agrobacterium host range. However, transformation methods based on the use of Agrobacterium are still preferred in many instances because of the following properties:
 Easy to handle
 Higher efficiency
 More predictable
 pattern of foreign DNA integration, and
 Lowcopy number of integration.
This vector was the discovery that the T-DNA and the Vir region could be separated onto two different plasmids.

Using this technique I would like to produce GM rice by introducing the folic acid biosynthesis gene into the rice plant. Folate is a B-group vitamin, also called folacin, critical for normal cellular function, insufficient intake causes megaloblastic anemia and there are strong linkages to cardiovascular diseases, various cancers and cognitive decline. It is also vital raw materials for red blood cell production in the blood and also have brain function.
Folic acid can be found in the leafy green vegetable, yeast, mushrooms, papaya etc.
After expressing this gene into the rice genome then, when the people eat the GM rice they will meet the sufficient folic acid that is crucial for the preventing of above diseases.

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