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Cowpea as a Genetic model Organism. Models in Experimental Genetics

Hausarbeit 2020 10 Seiten

Biologie - Genetik / Gentechnologie

Leseprobe

1.0 Introduction

Cowpea (Vigna unguiculata) is a tropical grain legume widely distributed in sub-Saharan Africa, Asia, Central and South America as well as parts of southern Europe and the United States (Singh et al., 2002). Domestication of cowpea is presumed to have occurred in Africa given the exclusive presence of wild cowpea (Steele, 1976) although knowledge about the general region or regions of origin and number of domestication events within Africa is fragmented (Ng and Padulosi, 1988; Coulibaly et al., 2002). It is very important, widely adapted, and versatile grain legume of high nutritional value. Cowpea is mainly produced and consumed in Africa, where it provides a major low-cost dietary protein for millions of smallholder farmers and consumers, who cannot afford high protein foods, such as fish and meat. The seed protein content is reported to range from 23-32% of seed weight and therefore is often referred to as a “poor man’s meat” (Diouf and Hilu, 2005). In many parts of West Africa, cowpea hay is also critical as livestock feed, especially during the dry season (Wests and Francis, 1982). Being a legume, cowpea is nitrogen-fixing (Sanginga, 2003) and fits perfectly in the traditional intercropping systems that are common in Africa, especially given its ability to tolerate shade. The total area under cowpea cultivation is more than 12.5 million hectares worldwide, with an annual production of around 4.5 million metric tons (Singh et al., 2002).

Cowpea remains one of the most important summer adapted food grain legumes grown under rained conditions. Traditional selection methods in cowpea depended mainly on the observed morphological variations even though morphological characteristics are easily influenced by the environment (Meglic and Staub, 1996). The genetic diversity information is extremely important, accurate assessment of genetic variability is important for the preservation and utilization of germplasm resources (Huaqiang et al., 2012).There is an urgent need to undertake more detailed genetic characterization of cowpea germplasm in order to optimally exploit the resources for improved cowpea production in Sudan. Such analysis would also reveal the true origin of Sudanese cowpea germplasm and establish the extent at which the global cowpea collection at IITA would benefit the cowpea breeding programs in Sudan.

Cowpea is one of the most important food legumes in sub-Saharan Africa and this region accounts for over 80% of the 12.5 million ha of cowpea cultivated land worldwide (FAOSTAT, 2006). More than 80% of cowpea production in Africa comes from the drier savannas and the Sahelian region of West and Central Africa (WCA), which accounts for about 70% of cowpea’s worldwide production. Cowpea is a rich source of proteins; the grain contains about 25% protein, making it a cheap source of protein in the daily diet of rural and urban populations as well as it provides cash income for the farmers. The seeds also contain several minerals, vitamins and dietary fibre (Ng and Padulosi, 1988, Sanginga, 2003). Its haulms are important as nutritious fodder for livestock in the dry savannas (Singh et al., 2002, Tarawali et al. 1997). Despite the importance of the crop, the productivity of cowpea in sub-Saharan African farmers’ field is reduced considerable by biotic and abiotic stresses. Cowpea has been the subject of genetic research since the beginning of the 1900s (IITA, 2010).

2.0 The Genotoxic Effect of Lead and Zinc on Cowpea

In this study , the treatment effect of lead and zinc on the chromosomes of cowpea was investigated.

2.1 MATERIALS AND METHODS

Dr y seeds of Cowpea (Vigna unguiculata) accessions: TVU 3788 we r e collected from the International Institute of Tropical Agriculture (I.I.T .A.), Ibadan. The metal salts used were lead nitrate and zinc nitrate. The metal salts were purchased from Labio Scientific, Mushin, Lagos. Seeds were spread uniformly in Petri dishes lined with filter paper. The Petri dishes were divided into three replicates and the seeds were divided into two sets of metal treatment (Pb and Zn). Equal volumes of the different concentrations of lead and zinc nitrate solutions (25, 50 and 100 mg/L) respectively were administered while the control group had distilled water. The seeds were allowed to germinate within the Petri dishes and were treated with the different concentrations of each of the metals and distilled water respectively at a temperature of 25 C for 5 days. Growing root tips which were brittle, translucent and gently tapering were selected from the three plants grown in the effluents of different concentration and from the control.

About 2-3 mm terminal root tips were cut off using a sharp blade and then placed on a clean glass slide and macerated with the aid of two dissecting needles and the remaining portion discarded. A drop of 1N Hydrochloric acid (HCL) was added to the root tip and left for 5 minutes; this softens the root tissue breaking up the middle lamellae. The excess acid was sucked up with a filter paper and the softened tissue was further macerated with dissecting needles so that the cells easily absorb the stain and spread adequately for microscopic observation. Then, a drop of lactic acetic orcein stain (2%) was placed on the macerated root tip and allowed to stand for 20 minutes for clearer viewing of the mitotic stages using the microscope (Michelle et al., 2006). Each slide was covered with a cover slip and pressed down to allow the tissue spread out and also to allow the excess stain seep out at the edges of the cover slip . This was removed by placing the slide between the folds of the filter paper and maceration was done with the base of the dissecting set. All slides were examined under the light microscope with high power magnification ( X 40 objective); then the good slides were preserved by sealing the edges of the cover slip with nail varnish to prevent the stain from evaporating .

The photomicrographs of good slides were then taken under the oil immersion lens (X100 objective) using a WILD M20 microscope with MPS 55 photoautomat attachment. The mitotic index was calculated according to Balog (1982) using the formula:

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The results of the mitotic index were statistically evaluated by the Analysis of Variance at 5% significant level using Microcal Origin 5.0 software.

3.0 Assessing the genetic diversity of cowpea [Vigna unguiculata (L.) Walp.] accessions from Sudan using simple sequence repeat (SSR) markers.

Simple sequence repeat (SSR) markers are one of the most frequently used markers in the genetic diversity analysis of cowpea (Li et al., 2001; Ogunkanmi et al., 2008; Lee et al., 2009; Asare et al., 2010; Badiane et al., 2012). The earliest cowpea SSR research is conducted by Li et al. (2001) and 27 SSR primers have been developed. Comparative studies in plants have shown that SSR markers, which are single locus markers with multiple alleles, provide an effective means for discriminating between genotypes (Powell et al., 1996; Li et a l., 2001). This study assessed the genetic diversity of 245 Sudanese cowpea accessions alongside 22 global accessions obtained from IITA, Nigeria using SSR markers. The main objectives were to understand the extent of genetic variation and likely origin of Sudanese germplasm as well as create a mini-core collection based on.

3.1 MATERIALS AND METHODS

3.1.1 Plant materials

Seeds of 231 Cowpea (V. unguiculata L.) accessions obtained from Plant Genetic Resources Unit of the Agricultural Research Corporation of Sudan representing six different agro-ecological zones of Sudan that is, Northern, River Nile, South Kordofan, North Kordofan, Blue Nile and Bahr Eljabel State in addition to 36 global cowpea accessions obtained from International Institute of Tropical Agriculture (IITA)-Ibadan-Nigeria were used in the present study. These materials (267 accessions) were planted in the greenhouse of Bioscience Eastern Central Africa BecA-ILRI Hub-Kenya for seedling establishment. 15 accessions failed to germinate in the green house; a rest of 252 accessions was successfully grown and used.

3.1.2 DNA extraction

Young leaves sampling were taken eight days after sowing in 1.5 mL Eppendorf tube, frozen immediately in liquid nitrogen and stored in -80°C, then leaves samples were manually grinded using micropestle. Genomic DNA isolated from young seedlings leaves following ZR plant/seed DNA protocol. DNA quality and quantity check done using Nano-drop spectrophotometer and 1% Agarose gel electrophoresis stained with Gel red was used to run the gel. The DNA was normalized by adjusting its concentration to 25 ng μL-1 in an optical 96-well Reaction plates using sterile de-ionized water.

3.1.3 Microsatlite amplification

A total of 18 polymorphic SSR markers were used to screen 252 cowpea DNA samples. The forward and reverse primers for each of the 18 SSR markers were labeled at the 5´ end of the oligonucleotide using fluorescent dyes to enable detection. PCR reaction were performed in 10 µL final volume in a mixture containing (Tag DNA polymerase1U, dNTPs 1mM and Reaction buffer 1x) in bulk Polymerase chain reaction (PCR) premix, 5 mM reverse and forward primers, 2 µL DNA, 0.2 µL of 25 mM MgCl2 and 7.2 µL of double distilled water. The optimal annealing temperature varied according to the Tm of the primer pairs and was determined using gradient PCR. For each amplification process, an initial denaturation of DNA at 95°C for 3min was followed by 30 cycles consisting of 30 sec at 94°C, 30 s at 50 to 60°C for annealing temperature 2 min at 72°C extensions a final extension of 15 min at 72°C was performed and the amplification products analyzed on 2% agarose gels in Tris Borate buffer stained with Gel red for visualization to establish polymorphism.

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Details

Seiten
10
Jahr
2020
ISBN (eBook)
9783346228468
Sprache
Englisch
Katalognummer
v914607
Institution / Hochschule
University of Lagos
Note
4.81
Schlagworte
cowpea genetic organism models experimental genetics

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Titel: Cowpea as a Genetic model Organism. Models in Experimental Genetics