Morphological, agronomical and molecular characterization in irradiated Cowpea (Vignaunguiculata (L.) Walp.) and detection by start codon target markers

ABSTRACT Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important foods and economic vegetable crops in the world. Plant breeders resorted to using mutagenesis, especially gamma radiation to improve the yield production and protein content. Two Egyptian cowpea varieties Dokii 331 and Kaha 1 were exposed to different doses of gamma rays (0, 50, 100, 150, 200, 250 and 300 Gy) to obtain mutations of economic value and to identify genetic variation by SCoT markers. The exposure of cowpea CVs. Dokii 331 and Kaha 1 to these gamma radiation doses had a positive effect on the early flowering, increase of the number of primary branches number of flowers per plant, pods per plant and the number of seeds, and consequently crop quantity. The dose of 150 Gy was effective in obtaining a dwarf mutation in cv. Kaha 1 where the length of the plant reached 33.4 cm. While in cv. Dokki 331, the dose of 250 Gy induced a change in size and pod color and increase number of pods per plant. While the effect of dose 300 Gy caused to change the color of flower from white to violet and plant length until it reached to 277.4 cm. However, the dose 300 Gy caused changing in the flower color and plant length reached 277.4 cm. Genetic variation in both irradiated cultivars was determined by SCoT markers analysis. The isolated DNAs from irradiated plants were amplified by fifteen SCoT primers. It was obtained 219 bands identified in both cultivars. Dakii 331 (105) Band was given by Polymorphic band number 46 (43.81%). Kaha 1 was 114 Band by Polymorphic band number 55(48.25%). Some SCoT primers were able to generate with a total of 26 specific markers. The cluster analysis based on Jacquard’s similarity coefficients and UPGMA algorithms were calculated.


Introduction
Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important vegetable legume crops in the world in general and particularly in Egypt. The world's cultivated area is about 16 million hectares, so the cultivated area in Egypt is about 2832.8 hectares with a productivity 36,772 hg/ha (www.faostat.fao.org/faostat). Millions of people depend on cowpea as an important food source because of its high nutritional value. The grains contain a high protein content of 20-30%. This protein is rich in amino acids lysine and tryptophan (Steele, 1976;Yasmin, Arulbalachandran, Soundarya, & Vanmathi, 2019).
The genetic improvement of crops is one of the main factors for solving food shortage problems worldwide (Ronald, 2011), to meet the needs of continuous population growth (FAO, 2009;Ray, Mueller, West, & Foley, 2013;Tester & Langridge, 2010). In the absence of sufficient natural variations, in this case, these variations can be induced through chemical or physical mutagens.
Mutation breeding is one of the oldest breeding programs. Due to the limitations of some biotechnologies such as hybridization, cross-breeding, and genetically modified plants, breeding mutations program has become more common and widespread among plant breeders Oladosu et al. (2016). Physical mutagens (X-rays, UV light, neutrons-alpha-beta particles, fast and thermal neutrons, gamma rays) are more widely used than chemical mutagens ethyl methanesulfonate (EMS) because physical mutagens are more precise, safer, and cheaper than chemical mutagens (Jain, 2010;Singh & Krishna, 2006). Plant breeders turned to the use of mutagenic agents either chemical or physical to cause an increase in the rate of genetic variation. Gamma rays are the most common and used radiation in the mutation breeding program Beyaz and Yildiz (2017). Numerous studies have been carried out on radiation and have an impact on genetic, morphological and biological changes and the consequent different applications in many fields including agriculture, pharmacy, and medicine. These variations help breeders to crop improvements and acquire new varieties, as it is difficult to rely on spontaneous mutations in improvements to slow their occurrence (Tester & Langridge, 2010;Olasupo, Ilori, Forster, & Bado, 2016, 2018. The induced mutations have played a large and effective role in the improvement of plants worldwide and their effect has been clear and noticeable on the increase in the productivity of some crops, although most of these mutations are recessive and their impact harmful. The effect of mutation methods has already been evaluated to crop improvement through several publications (Jain, 2010;Maluszynski, Ahloowalia, & Sigurbjornsson, 1995;Micke, Donini, & Maluszynski, 1990;Rutger, 1992).
The mutation induced in plants has used a lot of functional genomics in model organisms and crops. There is a new method of plant genetic marker developed where it depends on the short conserved region flanking the ATG start codon in plant genes. Start codon targeted (SCoT) markers polymorphism is a simple technique for generating gene-targeted markers in plants (Collard & Mackill, 2008;Xiong et al., 2011). The amplification profile of SCoT-PCR indicated that the SCoT marker is a dominant marker as RAPD and ISSR. The objective of this study is induced genetic variation in cowpea (Vigna unguiculata (L.) Walp.) by using gamma irradiation and the selection of some crop characteristics of economic value. As well as the identification and evaluation of these genetic changes using the SCoT genetic marker.

Seed material
Cowpea Seeds, Dokii 331 and Kaha 1 cultivars obtained from the Faculty of Agricultural, Horticulture Department, University of Minia.

Gamma radiation treatments
Irradiation was carried out with the 137 Cs source at the dose rate 1 Gy/2 min 14 sec, at National Center for Radiation Research and Technology, Cairo, Egypt.
Both cowpea cultivars Dokii 331 and Kaha 1 are exposed to different gamma radiation doses (50,100,150,200,250 and 300 Gy). The un-irradiated (control) and irradiated seeds in both cultivars were cultivated in a Randomized Complete Block Design (RCBD) at Experimental Farm of Horticulture Department, Faculty of Agriculture, Minia University, Minia, Egypt. The experiments were done in two seasons in April of 2017 and 2018 to get the first (M 1 ) and second (M 2 ) mutated generation. Morphological measurements for both mutated generations M 1 and M 2 were recorded and included % Emergence, number of branches, peduncle number per plant, plant heights (cm), fresh weight (g), pods number per plant, pod length (cm), seeds number per pod, hundred seed weight (g), and leaf length and width (cm).

Statistical analysis
To compare the varieties and their irradiated treatments, data were analyzed using ANOVA as described by Gomez and Gomez (1984) using MSTAT-C software version 4.

Genomic DNA extraction
Weighed about 1.5 g from irradiated plant leaves samples with different gamma radiation doses (0,50,100,150,200,250 or 300 Gy) and ground by liquid nitrogen to obtain a fine powder. Total genomic DNA was isolated from leaves of each the two cowpea varieties and their irradiated treatments according to the protocol described by Anderson, Ogihara, Sorrells, and Tanksley (1992) with a few modifications intended to improve the quality of DNA: two consecutive extractions with phenol: chloroform (1:1) were carried out by an additional wash of 97% (left at −20°C for one hour) an 70% pre-cooled ethanol, respectively. The yield and quality of DNA were assessed by spectrophotometer and gel electrophoreses.

SCoT -PCR amplification
Fifteen (SCoT) primers were selected according to Collard and Mackill (2009), Table 1. Amplification reactions were carried out in a total volume of 25 µl, which contained 250 μM of each primer, 0.2 mM of each deoxynucleotide, 1.5 mM MgCl 2 , 1 unit Taq polymerase, and 50-100 ng of template DNA. All reaction volumes were 25 µl overlaid with a drop of mineral oil. The thermocycling program used was: one cycle at 94°C for 3 min, 35 cycles at 94°C for 50 sec,1 min at 50°C, 2 min at 72°C, and the final extension step of 7 min at 72°C. Electrophoresis was done to visualize the PCR amplified product. It was carried out on 1.0% agarose gel and amplified fragments were visualized by staining with ethidium bromide.

Data analysis
Fragment sizes of both SCoT and ISSR were determined with PyElph 1.4 software Pavel and Vasile (2012) comparison with the marker. Amplified products were scored as present (1) or absent (0) to form a binary matrix.
In order to measure the informativeness of the markers to differentiate between genotypes, polymorphism information content (PIC) and marker index (MI) were calculated. PIC was calculated according to the formula of Anderson et al. (1992), as PIC = 1-AEp¡ 2 Table 1. SCoT primers code and nucleotide sequences.

Morphological characterization
Gamma radiation doses have caused numerous morphological changes in both cultivars as shown in Figure

Agronomical characterization
Radiation had a major role in producing agronomical changes that can be summarized in

SCoT analysis
All fifteen SCoT primers used in the analysis of the cowpea varieties and the six radiation treatments for each were able to form polymorphic fingerprint patterns Figure 2. Fifteen primers produced 105 DNA fragments with an average of 7 bands per primer in Dokki 331 cultivar, however in the Kaha 1 cultivar; the primers produced 114 DNA fragments with an average 7.6 per primer (Table 3). The polymorphism number in the Dokki 331 cultivar was 46 (43.81%) with an average of 3.06 polymorphic bands per primer. The highest polymorphic band observed was with the SCoT-22 primer (7), while the lowest polymorphic band was one observed with the SCoT-33 primer. In the Kaha 1 cultivar, out of the total 114 amplified fragments, 55 (48.25%) were polymorphic with an average of 3.6 polymorphic bands per primer. The SCoT-12 primer had the ability to produce higher polymeric bands (6)

Genetic diversity assessment by SCoT markers
Genetic diversity summarizes in Table 4

Treatments specific markers
Some of the fifteen SCoT primers did not obtain specific markers, two primers with the Dokki 331 cultivar and eight primers with the Kaha 1 cultivar. However, the other SCoT primers succeeded to generate a total of 26 specific markers which ranged from one to two specific markers. There were some similarities in specific markers in both cultivars with primers SCoT-1, SCoT-5, SCoT-13, SCoT-14 and SCoT-33 Table 5.

Genetic relationships
The genetic identity and genetic distance of 12 gamma radiation treatments in both cowpea cultivars Dokii331 and Kaha 1 are presented in Table 6. The Nei's genetic identity was the highest (0.9958) in treatments pairs 50 Gy in cv. Kaha 1. As well, the lowest genetic identity was (0.8263) in treatments pairs 100 Gy in the same cultivar. On the other hand, the highest Nei's genetic distance was (0.1555) between the un-irradiated cv. Dokii 331 and 100 Gy in cv. Kaha 1. The lowest Nei's genetic distance was (0.0043) within irradiated Dokii 331 with dose 200 Gy and irradiated Kaha 1 with dose 50 Gy. Principle component analysis was used to illustrate genetic relationships between two cowpea genotypes and their gamma radiation treatments as shown in Figure 3. Of the total polymorphism, the first two components accounted for only 69.599%, this proves that the SCoT markers used in this study have an appropriate dispersion of markers in the genome. The two cowpea cultivars Dokii 331 and Kaha 1 and their gamma radiation treatments were clustered into four groups. The first group includes Kaha 1 cultivar and its radiation treatment of 100, 250 and 300 Gy, as well as radiation treatments of the Dokii 331 cultivar 150, 250 and 300 Gy. The second group includes the irradiation treatment of 150 Gy for the Kaha 1 cultivar and 50 Gy for the Dokii 331 cultivar. The third group includes irradiation of 50 and 200 Gy of the Kaha 1 cultivar. The fourth group includes both Dokii 331 and its radiation treatments 100 and 200 Gy. The analysis of principle components thus is largely compatible Shannon's information index (I) 0.5585 ± 0.08 0.5586 ± 0.07 0.5586 ± 0.07 0.6306 ± 0.07 0.5310 ± 0.06 0.6235 ± 0.06 0.6372 ± 0.05 0.6035 ± 0.2 0.5440 ± 0.07 0.6251 ± 0.2 0.5879 ± 0.07 0.5641 ± 0.07 0.6365 ± 0.05 0.6264 ± 0.1 with those from cluster analysis obtained from UPGMA.

Discussion
Of the results obtained, it is clear that the gamma radiation treatments used in this experiment, 50, 100, 150, 200, 250 and 300 Gy have led to many morphological and agronomical changes. It was noted that of the doses of gamma radiation that made the most change, dose 150 Gy in the Kaha 1 cultivar was the most significant, as this dose led to changes in leaf size and shape, increase peduncles number and dwarf plants. The increase of peduncles due to the increase in the numbers of pods resulted in an increase in the yield. The irradiated plant with gamma-ray induced genetic variation and mutation leading to qualitative and quantitative alterations depend on the strength and duration of the gamma irradiation dose exposure, (Gnanamurthy, Mariyammal, Dhanavel, & Bharathi, 2012;Kim, Kim, Lee, Baek, & Kim, 1998;Wi et al., 2005). Gamma Radiation had a positive effect on the early flowering, increase the number of primary branches number of flowers per plant, pods per plant and the number of seeds, and consequently crop quantity, (Jan, Parween, Siddiqi, & Mahmooduzzafar, 2010;Khan et al., 2000) in legumes. However in French beans (Phaseolus vulgaris L.), gamma radiation had a negative impact on seed maturity, flowering, plant height, seed yield per plant, prolonged the growth period and retarded plant height (Svetleva & Petkova, 1992;Yousaf, Raziuddin, & Ahmad, 1991).  These results are in agreement with the results of (Ogidi, Omosun, Markson, & Kalu, 2010;Horn & Shimelis, 2013;Badr et al., 2014;Olasupo et al., 2016Olasupo et al., , 2018, where they found that doses more than 100 Gy lead to a reduction of cowpea leaf size and plant height. On the other hand, in cv. Dokii 331, the highest doses 200 Gy showed the morphological and agronomical alterations. It was observed that the number of branches increased with the dose of 200 Gy. The plant height, number of pods per plant, pods length, and the number of seeds per pod, as well as the fresh weight, were increased with the dose 300 Gy. These results are agreement with (Horn, Ghebrehiwot, & Shimelis, 2016;Singh & Krishna, 2006), this dose also caused changes in the flower color and the color of the pods. These results are consistent with (Horn et al., 2016;Olasupo et al., 2018). The Start codon-targeted markers (SCoT) technique has had a major role in the identification, characterization and genetic comparison between many plant varieties. These results were in accordance with Huang et al. (2014) for Hemarthria, Que et al. (2014) for sugarcane, Gajera, Bambharolia, Domadiya, Patel, and Golakiya (2014) for mango, Gao et al. (2014) for Lycoris, Fang-Yonga and Ji-Honga (2014) for Myrica rubra, Jiang et al. (2014) for orchard grass, Satya et al. (2015) for ramie (Boehmeria nivea L. Gaudich.), Zhang, Xie, Wang, and Zhao (2015) for Elymus sibiricus, Vivodík, Gálová, Balážová, and Petrovičová (2016) for maize.
Radiation is one of the best and most successful mutagens used in plant breeding programs. It has the ability to make large genetic changes in a safe and conclusive manner in a short time. Thus, the plant breeder has a large area of selection and improvement of plants. This has had a significant impact on the cowpea, where highvalue mutations such as the dwarf mutant, highly productive obtained in kaha 1 cultivar resulting from irradiation treatment 150 Gy. The use of SCoT marker has been of great importance in the characterization and identification of genetic variation between varieties and radiation treatments with high accuracy.