Genetic improvement of faba bean (Vicia faba L.) genotypes selected for resistance to chocolate spot disease Received for publication: April 14, 2022. Accepted for publication: August 4, 2022. Doi: 10.15446/agron.colomb.v40n2.102128 1 Food Legumes Research Section, Field Crops Research Institute, Agricultural Research Center, Giza (Egypt). 2 Genetics Department, Faculty of Agriculture, Zagazig University, Zagazig (Egypt). 3 National Gene Bank, Agricultural Research Center, Giza (Egypt). * Corresponding author: ehab.mahdy@arc.sci.eg Agronomía Colombiana 40(2), 186-197, 2022 ABSTRACT RESUMEN Inter-varietal hybridization is a powerful tool for genetic improvement and production of new genotypes for a trait of interest. Four parents of faba beans (Vicia faba L.) were hybridized using agromorphological and molecular charac- terization to obtain genotypes resistant to the chocolate spot disease. The study was done at the Nubaria Research Station, Giza, Egypt. Eight traits including resistance to chocolate spot, days to f lowering, plant height (cm), number of branches/ plant, number of pods/plant, number of seeds/plant, 100-seed weight (g), and seed yield/plant were estimated during the three growth seasons of 2016/2017, 2017/2018, and 2018/2019. Genetic parameters revealed by RAPD and ISSR markers assessed the genetic variation of genotypes with their generations. Crosses 1 (P1 “Nubaria-1” x P2 “Sakha-1”), 2 (P1 “Nubaria-1” x P3 “TW”), and 3 (P1 “Nubaria-1” x P4 “Camolina”) revealed high resistance to disease with high yield. Markers patterned specific loci of resistant parents at a length of 360, 470, 450, 660, and 140 bp in RAPD and 1100, 810, 650, 700, 480 bp in ISSR. Inter-varietal hybridization between the resistant and susceptible genotypes is considered one of the most promising methods to obtain germplasm with resistance and high yield. La hibridación intervarietal es una herramienta poderosa para el mejoramiento genético y la producción de nuevos genotipos prometedores para un rasgo de interés. Cuatro progenitores de haba (Vicia faba L.) fueron cruzados para obtener genotipos resistentes a la enfermedad de la mancha chocolate mediante caracterización agromorfológica y molecular. El estudio de campo se llevó a cabo en la Granja Experimental de la Estación de Investigación de Nubaria, Giza, Egipto. Se estimaron ocho características, incluidas el grado de resistencia a la mancha chocolate, los días a f loración, la altura de planta (cm), el nú- mero de ramas/planta, el número de vainas/planta, el número de semillas/planta, el peso de 100 semillas (g) y el rendimiento de semillas/planta, durante las tres temporadas de crecimiento de 2016/2017, 2017/2018 y 2018/2019. Los parámetros genéticos se estimaron mediante marcadores RAPD e ISSR para evaluar la variación genética de los genotipos con sus generaciones. Los cruces 1 (P1 “Nubaria-1” x P2 “Sakha-1”), 2 (P1 “Nubaria-1” x P3 “TW”) y 3 (P1 “Nubaria-1” x P4 “Camolina”) revelaron alta resistencia a la enfermedad de la mancha chocolate con alto rendimiento. Los marcadores modelaron loci específicos de padres resistentes a una longitud de 360, 470, 450, 660 y 140 pb en RAPD y 1100, 810, 650, 700, 480 pb en ISSR. La hibridación intervarietal entre los genotipos resistentes y susceptibles es considerada uno de los métodos más promisorios para obtener germoplasma con resistencia y alto rendimiento. Key words: hybridization, ISSR, genetic resistance, plant breeding, RAPD, yield components. Palabras clave: hibridación, ISSR, resistencia genética, fitomejoramiento, RAPD, componentes de rendimiento. Genetic improvement of faba bean (Vicia faba L.) genotypes selected for resistance to chocolate spot disease Mejoramiento genético de genotipos de haba (Vicia faba L.) seleccionados con resistencia a la enfermedad de la mancha chocolate Hany Elsayed Heiba1, Elsayed Mahgoub2, Ahmed Mahmoud2, Mostafa Ibrahim1, and Ehab Mawad Badr Mahdy3* Introduction Faba beans (Vicia faba L.; 2n=12) are one of the most com- mon field pulses in Egypt (Bakry et al., 2011; Mohamed et al., 2012) and one of the oldest crops cultivated worldwide (Link et al., 1995; Zong et al., 2009). They belong to the Fabaceae family, the subfamily of Papilionoideae, and the tribe of Viceae (Duc, 1997). Faba beans are consumed for their green pods and dried seeds (Duc et al., 2010). They are considered a main source of cheap protein and energy in Africa and some parts of Asia and Latin America, where many people cannot afford a meat source of protein (Duc, 1997; Alghamdi, 2009). https://doi.org/10.15446/agron.colomb.v40n2.102128 mailto:ehab.mahdy@arc.sci.eg 187Heiba, Mahgoub, Mahmoud, Ibrahim, and Badr Mahdy: Genetic improvement of faba bean (Vicia faba L.) genotypes selected for resistance to chocolate spot disease A fungal disease called chocolate spot, caused mainly by Botrytis fabae (Sardina) and by B. cinerea (Pers.), is one of the most damaging diseases to this plant (Harrison, 1988; El-Komy et al., 2015; Haile et al., 2016; Aguilar-Luna et al., 2021). Symptoms oscillate from some spots on leaves to complete covering of the plant. Under severe condi- tions, the disease spreads from affected leaves into stems, f lowers, and pods, causing damages (Bernier et al., 1993; Rahman et al., 2002; Villegas-Fernández et al., 2012; Haile et al., 2016). Various approaches are employed to control the disease, including genetic improvement (Wilson, 1937; Sahile et al., 2008; Abou-Zeid & Hassanein, 2000; Aguilar- Luna et al., 2021). Quantitative trait loci (QTL) utilizes our knowledge of the effect of genetic control tools for selection in crop breed- ing programs. Various approaches are used for selecting promising traits, especially those for adapting to the local environment (Mahdy & El-Sharabasy, 2021). Phenotypic- revealed markers assess genetic diversity and performance of germplasm versus the attribute (Mahdy, 2012). These markers are based on visual observations (El-Sharabasy et al., 2021; Mahdy & El-Sharabasy, 2021) and screen the quantitative traits to increase field crop production. PCR (polymerase chain reaction) - based markers are applied for genetic improvement and breeding, genetic diversity, and genetic relationships (Chen et al., 2008; Tomás et al., 2016). PCR is fast, reproducible, simple, and low-cost procedure. PCR-based markers are very practical in multi-disciplines, including genetic diversity and genetic improvement programs (González et al., 2005). Various PCR-based markers were applied on crops, i.e., Random Amplified Polymorphic DNA (RAPD) (Link et al., 1995), Inter-simple Sequence Repeats (ISSR) (Terzopoulos & Be- beli, 2008; Aguilera et al., 2011; Abdel-Razzak et al., 2012; Mahdy, 2012; Asfaw et al., 2018) on jew’s mallow, Restric- tion Fragment Length Polymorphism (RFLP) (Torres et al., 1993), Start Codon Target (SCoT) (Mahdy et al., 2021) on cowpea, and Conserved DNA-Derived Polymorphism (CDDP) (Ghazzawy et al., 2021) on date palm. We chose four faba bean parents for (1) improving genetic resistance against the chocolate spot, (2) determining the genetic variations, (3) evaluating the performance under the infection of chocolate spot, (4) measuring genetic distance, and (5) generating a molecular profile using agromorphological traits and RAPD markers. Materials and methods Faba bean materials, planting, and field experiment Four parents, as shown in Table 1, were sown on the experimental farm at the Nubaria Research Station, Agricultural Research Center (ARC), Egypt, during the seasons of 2016/2017, 2017/2018, 2018/2019, to evaluate their performance via the measurement of agromorpho- logical traits. There is a high incidence of chocolate spot disease in Nubaria; moreover the disease is spread widely in the northern region of the Nile Delta of Egypt, with low temperatures and high relative humidity (Khalil et al., 1993). Four parents were hybridized in 2016/2017 to secure F1 hybrid seeds in the 2016/2017 season. In the 2017/2018 season, parents were re-hybridized; their F1 hybrids were grown in a randomized complete block de- sign with three replicates under insect-free cages. In the 2018/2019 season, parents with F1 and F2 generations were artificially inoculated with Botrytis fabae fungus, under insect-free cages, then purified and identified according to Morgan (1971). Each plot comprised six rows 3 m long, with 0.60 m distance between rows, and 0.2 m between mounds with two seeds in each. Measurement of agromorphological traits Eight traits were measured: plant height (cm), number of branches/plant, days to f lowering, seed number per plant, pod number per plant, seed yield per plant (g), 100-seed weight (g), and reaction to chocolate spot disease. An assessment scale of response to chocolate spot disease was estimated using a quantitative scale of 0-5, where 0 (very highly resistance) indicates no visible chocolate spot, 1 (high resistance) indicates a few chocolate spots, 2 (resistance) indicates increased and scattered spots, 3 (moderately resistance) indicates larger spots , 4 (suscep- tible) indicates necrotic spots reaching half of the leaf, 5 (highly susceptible) indicates majority of necrotic spots and leaf abscission (ICARDA, 2005). Homogeneity of the TABLE 1. Pedigree and origin of four parents. ID Parents Origin Pedigree Botanical group Foliar disease reaction P1 Nubaria1 Egypt Single plant selection form Giza Blanka Large Resistant P2 Sakha1 Egypt 620/283/85x716/724/88 Medium Resistant P3 TW Sudan Sudan Medium Susceptible P4 Camolina Spain Imported from Spain Small Susceptible https://bnrc.springeropen.com/articles/10.1186/s42269-019-0145-3#Tab1 188 Agron. Colomb. 40(2) 2022 variance across environments was tested according to the Bartlett test (Steel & Torrie, 1980). Parents and their F1 and F2 hybrids were evaluated for the experiment traits and measurements without reciprocal. Molecular analysis DNA extraction DNA was extracted from twenty samples of the faba beans (4 parents + 6 hybrids F1+ 10 hybrids F2) using the DNeasy Plant Kit (Qiagen, Germany). Nanodrop was used to de- termine the DNA concentration and quality. PCR analysis Six RAPD primers and five ISSR primers were used to de- tect the polymorphism among the twenty samples, which were synthesized by Metabion Corp., Germany (Tab. 2). The amplification reaction was done in 25 μl reaction vol- ume containing 12.5 μl Master Mix (sigma), 2.5 μl primer (10 pcmol), 3 μl template DNA (10 ng), and 7 μl dH2O, ac- cording to Ibrahim et al. (2019). The PCR was processed with a Perkin-Elmer/GeneAmp® PCR System 9700 (PE Applied Biosystems, USA) adjusted to fulfill 40 cycles. The initial denaturation cycle was for 5 min/94ºC. Each was at 94ºC in 45 s for the denaturation step and 72ºC in 60 s for the elongation step. The annealing temperature was adjusted as in Table 2 for 50 s. The extension was adjusted at 72ºC in 7 min in the final cycle. The amplified prod- ucts were run in a 1.5% agarose gel containing ethidium bromide (0.5 µg ml-1) in 1X TBE buffer at 95 V. A 100 bp DNA ladder (Promega, USA) was standardized to deter- mine the PCR product sizes. Gel images were visualized using a UV transilluminator and photographed using a Gel Documentation System (BIO-RAD 2000, USA). A binary matrix as present (1) or absent (0) was scored for the PCR products. The final data sets included both polymorphic and monomorphic bands. Statistical analysis A randomized complete block design (RCBD) with three replicates was used according to Gómez and Gómez (1984) and analyzed using MSTATC computer software. Average values and analysis of variance were conducted for all stu- died traits of 20 faba bean genotypes (four parents: (1) P1, (2) P2, (3) P3, (4) P4, six F1 generation: (5) P1xP2, (6) P1xP3, (7) P1xP4, (8) P2xP3, (9) P2xP4, (10) P3xP4, and ten F2 gene- ration: (11) P1xP2, (12) P1xP3(R), (13) P1xP3(S) (14) P1xP4(R), (15) P1xP4(S) (16) P2xP3(R), (17) P2xP3(S), (18) P2xP4(R), (19) P2xP4(S) and (20) P3xP4 (S×S)) in each generation according to Steel et al. (1997). The least significance difference (LSD) test (P≤0.05) was calculated (Steel & Torrie, 1980). We calculated the number of total bands, unique bands, polymorphic bands, and the percent of polymorphism. We also estimated some genetic parameters. Shannon informa- tion index was calculated {I = -1 × (p × Ln (p) + q × Ln(q))} according to Shannon (1948). The observed number of alleles (Na), effective number of alleles {Ne = 1 / (p 2 + q2)}, Nei genetic diversity {h = 1 – Σ (p2 + q2)}, and Unbiased Diversity {uh = (N / (N - 1)) × h)}; where p = band frequency and q = 1 – p estimated according to Hartl and Clark (1997) and Liu and Muse (2005). Nei’s genetic identity and dis- tance used Nei (1972) and Nei (1978). Nei’s genetic identity was calculated with the following formula: NeiI = Jxy √JxJy , where Jxy = ∑i=1k Pi xPiy, Jx = ∑i=1k Pi x 2 , Jy = ∑i=1k Piy 2 . Nei genetic distance was estimated with the formula NeiD = −lnI, where I is the genetic identity. F-test was de- termined trait-marker associations. Power marker software V3.0 was fed. The association analysis selected markers with a high P-value (P>0.01). Results and discussion Phenotype-based traits The average values of the agromorphological traits were calculated (Tab. 3). The results showed broad significance, as evidenced by the characteristic ranges for chocolate spot disease (1.30 to 5.63, resistant to susceptible, respectively), days to f lowering (40.80 to 63.66), plant height (93.30 to 131.66 cm), number of branches per plant (3.10 to 10.80), number of pods per plant (25.46 to 111.36), number of seeds per plant (83.13 to 295.83), 100 seed weight (47.30 to 137.20 g), and seed yield per plant (56.37 to 199.96). TABLE 2. List of primers and their nucleotide sequence. No Name Sequence Temperature, oC 1 OPA-07 5’-GAAACGGGTG-3’ 36 2 OPA-10 5’-GTGATCGCAG-3’ 36 3 OPA-17 5’-GACCGCTTGT-3’ 36 4 OPB-05 5’-TGCGCCCTTC-3’ 36 5 OPG-19 5’-GTCAGGGCAA-3’ 36 6 OPG-20 5’-TCTCCCTCAG-3’ 45 7 ISSR-1 5’-AGAGAGAGAGAGAGAGTC-3’ 45 8 ISSR-2 5’-AGAGAGAGAGAGAGAGTG-3’ 45 9 ISSR-3 5’-ACACACACACACACACAT-3’ 45 10 ISSR-4 5’-ACACACACACACACACTG-3’ 45 11 ISSR-5 5’-GTGTGTGTGTGTGTGTAG-3’ 45 189Heiba, Mahgoub, Mahmoud, Ibrahim, and Badr Mahdy: Genetic improvement of faba bean (Vicia faba L.) genotypes selected for resistance to chocolate spot disease As shown in Table 3, Nubaria 1 recorded the highest mean values of 100-seed weight, seed yield/plant, and plant height (cm). Meanwhile, Camolina recorded a higher number of pods/plant, seed/plant, and branches/plant than the other parents. On the other hand, TW recorded the lowest mean values of the number of pods and seeds per plant. The two parents, Nubaria 1 and Sakha 1, gave the highest mean values of plant height. The parents, Nubaria 1 and Sakha 1, were considered highly resistant; their estimated mean values were (1.30 and 1.80) for chocolate spot infection. The parents TW and Camolina had the highest susceptible values (5.63 and 5.40) for the chocolate spot. F1 generation The mean values of the sex-tested hybrids were calculated (Tab. 3). Results indicated that the cross (P1xP2) had the highest mean value of 100-seed weight (137.20 g). The two crosses, P1xP3 and P1xP4, produced the last cross. For plant height, the crosses, P1xP3 and P2xP4, had the highest mean values (127.47 cm and 126.10 cm, respectively) of plant height and the lowest value (103.83 cm) obtained from the cross (P3xP4). The genotypes Camolina, Sakha1, and TW were the parents which f lowered quickest. Also, cross P1xP2 (41.90 d) was the cross which flowered quickest, followed by the cross P3xP4 with a mean value of 42.06 d. Concerning the number of branches/plant, the results showed that three crosses (P1xP4), (P2xP4), and (P1xP2) had the highest values. Concerning the number of pods per plant, the parental variety Camolina (P4) showed the highest mean value (46.56), whereas cross (P1xP2) gave the lowest number of pods/plant. For the number of seeds per plant, the cross P2xP4 gave the highest mean value (295.83), followed by the two crosses P3xP4 (265.96) and P1xP4 (250.86). The two crosses (P1xP2) with 137.20 g and (P1xP3) with 93.26 g had the highest mean of 100-seed weight and highest seed yield per plant. The highest seed yield per plant could be attributed to the high number of seeds and seed weight/plant. The two men- tioned crosses were the most promising for yielding ability and tended to combine high seed yield and its components. TABLE 3. Performance of growth and yield characteristics. Genotypes Chocolate spot Days to flowering Plant height (cm) No. of branches/plant No. of pods/plant No. of seeds/plant 100-seed weight (g) Seed yield/plant Parents P1 1.30 63.66 130.80 10.80 44.50 152.33 129.30 187.16 P2 1.87 42.93 112.20 4.50 42.33 131.73 100.36 132.70 P3 5.63 42.56 102.63 3.10 38.73 104.03 55.20 56.73 P4 5.40 40.80 102.17 5.10 46.56 137.80 47.30 64.93 F1 generation P1×P2 1.86 41.90 125.80 6.53 40.73 119.00 137.20 155.66 P1×P3 2.73 51.83 127.47 5.63 44.33 132.13 93.26 129.00 P1×P4 2.20 44.96 126.10 7.56 82.46 250.86 78.10 199.96 P2×P3 2.30 46.63 119.13 5.90 67.46 205.20 71.83 147.96 P2×P4 2.86 44.63 111.90 6.96 98.50 295.83 62.53 181.20 P3×P4 3.86 42.06 103.83 5.53 111.36 265.96 54.53 134.06 LSD 0.05 1.18 3.33 5.35 1.03 9.27 35.34 7.61 30.35 F2 generation P1×P2 (R×R) 1.73 50.20 131.66 6.90 47.40 147.30 132.13 172.36 P1×P3 (R) 2.44 49.63 125.38 5.42 44.33 132.13 93.26 129.00 P1×P3 (S) 3.53 47.00 122.20 4.20 25.46 83.13 76.40 62.60 P1×P4 (R) 2.22 49.43 136.18 7.56 73.58 242.08 78.16 169.88 P1×P4 (S) 3.53 48.63 132.43 6.56 53.86 175.73 76.40 123.06 P2×P3 (R) 2.34 45.83 117.73 5.92 65.77 203.82 71.93 149.76 P2×P3 (S) 3.40 42.93 116.63 4.46 55.86 173.13 70.46 119.80 P2×P4 (R) 2.48 45.13 113.82 5.87 91.73 276.91 61.84 171.68 P2×P4 (S) 4.50 42.86 111.06 4.60 73.06 158.90 60.03 95.76 P3×P4 (S×S) 5.20 43.33 93.30 5.36 61.40 181.70 51.90 93.50 LSD0.05 1.01 4.10 6.13 1.44 8.07 32.92 6.52 29.82 190 Agron. Colomb. 40(2) 2022 F2 generation The cross (P1xP2) gave the highest mean values of the num- ber of seeds per plant, seed yield per plant, and 100-seed weight and gave the lowest infection value for chocolate spot (1.73) (Tab. 3). The cross (P1xP3) had the lowest mean values for number of pods per plant and number of seeds per plant and the highest infection value of the cross P1xP2 (3.53). The cross (P3xP4) had a low mean value for 100-seed weight, and the reaction for chocolate spot gave the highest susceptible value (5.20). Three crosses, P2xP3, P2xP4, and P3xP4, were the earliest crosses for the f lowering date. The cross (P2xP4) gave the highest number of pods per plant, number of seeds per plant, and the highest susceptible value for reaction choco- late spot (4.50). The lowest values were scored by the two crosses, P1xP4 and P2xP3, which also gave the lowest value for chocolate spot infection (3.53 and 3.40, respectively). Those genotypes considered promising for chocolate spot resistance and the best performing F1 crosses had mean values slightly better than those of F2 crosses in most of the studied traits. Significant differences among faba bean genotypes in all studied traits were considerable evidence for the existence of a suitable amount of genetic diversity valid for further as- sessments (Ahmed et al., 2016; Abdalla et al., 2017; Hamza & Khalifa, 2017; Abou-Zaid & El-Gendy, 2019; El-Abssi et al., 2019). The results suggest that these genotypes carry genes for resistance to chocolate spot disease, coming from their parents according to their pedigree (Tab. 1). Similar results have been reported for faba bean yield traits and compo- nents, as well as for disease resistance traits (Abid et al., 2015; Zakaria et al., 2015; Beyene et al., 2016; Eldemery et al., 2016; Belal et al., 2018; El-Rodeny et al., 2020). Polymorphism revealed via RAPD markers Performance among twenty faba bean genotypes was analy- zed by PCR-based markers, RAPD and ISSR (Figs. 1-2). Table 4 summarizes the data resulting from all analyzed loci that resulted from 148 amplified fragments ranging from 60 for ISSR and 88 for RAPD. The RAPD markers ranged from 130-2000 bp while the ISSR markers oscillated from 160-1500 bp. The polymorphism averaged a score of 74%, oscillating from 57% for ISSR to 74% for RAPD. RAPD markers revealed a high percentage of polymorphism com- pared with ISSR. The RAPD markers scored four positive unique bands by the primer OPA-07. Unique bands were at 1350 and 140 bp for P1xP2 (F1), 920 and 130 bp for P1xP4 S and P1xP3 S (F2), respectively. On the contrary, the ISSR revealed negative unique bands across the F2 generation. The primer ISSR-1 scored a negative unique band at a dis- tance of 160 bp (P1xP4 R); ISSR-3 scored at 650 bp (P2xP4 R), 360 (P1xP3 S), and 250 bp (P2xP3 R); and ISSR-5 scored 529 bp (P2xP4 R). TABLE 4. Polymorphism revealed by ISSR and RAPD markers. Primers MW (bp) Total (AF) Mb Ub+ Ub- Pb P (%) RAPD OPA-07 130 -1350 19 5 4 0 14 74 OPA-10 160 - 1500 14 3 0 0 11 79 OPA-17 140 - 860 14 5 0 0 9 64 OPB-05 140 - 820 10 3 0 0 7 7 OPG-19 160 - 2000 15 2 0 0 13 87 OPG-20 180-1800 16 5 0 0 11 69 Total RAPD 130-2000 88 23 4 0 65 74 ISSR ISSR-1 1530 -1100 14 4 0 1 10 71 ISSR-2 170 - 800 12 6 0 0 6 5 ISSR-3 200 - 1500 14 4 0 3 10 71 ISSR-4 160 - 530 11 8 0 0 3 27 ISSR-5 239 - 950 9 4 0 1 5 56 Total ISSR 160 - 1500 60 26 0 5 34 57 Total 148 49 4 5 99 67 MW=molecular weight, AF=amplified fragment, Mb=monomorphic bands, Ub+=positive unique bands, Ub-=negative unique bands, Pb=polymorphic bands, P=polymorphism percentage. 191Heiba, Mahgoub, Mahmoud, Ibrahim, and Badr Mahdy: Genetic improvement of faba bean (Vicia faba L.) genotypes selected for resistance to chocolate spot disease Table 5 summarizes specific loci in parents segregated dur- ing subsequent generations. These loci can be used directly for the breeding program for resistance to chocolate spot disease. RAPD markers patterned specific loci of resistant parents at a length of 360 bp for OPA-07, 470 bp (P2), and 450 bp (P1 and P2) for OPA-17, 660 and 140 bp (P1 and P2) for OPB-05. Primers, OPA-10, OPG-19, and OPG-20, patterned specific loci of susceptible P4 except the primer OPA-17 owing to a specific locus for susceptible parent (P3) at a distance of 400 bp. ISSR patterned resistant specific loci at a length of 1100 & 810 bp by ISSR-1, 650 bp by ISSR- 2, and 700 & 480 bp by ISSR-3 for parents 1 and 2. The primers ISSR-2 and ISSR-5 patterned susceptible specific loci at 800 & 170 bp for P3 and P4 and 390, 281 and 239 bp, respectively. The primer ISSR-1 characterized P3 only at a length of 200 bp. We found the RAPD markers showed higher polymor- phism than the ISSR, in contrast to other results detected in wheat (Nagaoka & Ogihara, 1997) and vigna beans (Ajibade et al., 2000). Terzopoulos and Bebeli (2008) found 68% OPA 17 OPB-05 OPG-20 OPG-19 OPA-07 OPA-10 M 151413121110987654321 16 20191817 ISSR-1 ISSR-2 ISSR-4 ISSR-3 ISSR-5 M 151413121110987654321 16 20191817 FIGURE 1. Profiles of twenty faba bean genotypes revealed via RAPD. FIGURE 2. Profiles of twenty faba bean genotypes revealed via ISSR. polymorphism between the Greek faba bean populations using eleven ISSR primers. Khalaf et al. (2015) found 73% polymorphism between seven faba bean genotypes using fifteen ISSR primers. Genetic parameters, identity, and distance Genetic parameters are vital for the efficiency of a mar- ker technique used in discriminatory objects based on polymorphism. The number of genetic parameters was estimated to evaluate the informative and discriminatory power during subsequent faba beans generation, as shown in Table 6. Heterozygosity (h) averaged 0.18, oscillating from 0.16 to 0.20 for RAPD, from 0.15 to 0.23 for ISSR, and from 0.16 to 0.21 for both markers. h reveals impor- tant polymorphism information content (PIC), with high informative marker value. The Shannon index (I) revealed an average of 0.271 for both markers used. The RAPD markers ranged from 0.229 for Parents to 0.304 for F1, with an average of 0.275. The ISSR markers oscillated from 0.23 (F1) to 0.33 (F2), with an aver- age of 0.266. The effective number of alleles (Ne) of RAPD markers ranged from 1.275 (Parents) to 1.311 (F1), while ISSR revealed the range from 1.26 (F1) to 1.1 (F2), with a grand average of 1.306. 192 Agron. Colomb. 40(2) 2022 TABLE 5. Specific loci related to performance of resistance and susceptible genotypes. Primer MW PR PS F1 F2 OPA-07 360 P1, P2 P1xP3 P1xP2, P2xP3(R), P2xP3 S, P3xP4 OPA-10 1200 --- P4 P3xP4 P1xP3S 810 --- P4 P1xP4 & P3xP4 P1xP3S & P1xP4S & P2xP4R & P3xP4 OPA-17 470 P2 --- ----- P2xP4S & P3xP4 450 P1 & P2 --- P1xP4 & P2xP3 & P3xP4 P1xP2 & P1xP3R & P1xP3S & P1xP4R & P2xP4R 400 P3 P1xP2 P1xP3S & P1xP4R & P1xP4S OPB-05 660 P1 & P2 --- P1xP2 & P2xP3 P2xP4S & P3xP4 140 P1 & P2 --- P1xP2 & P2xP3 P1xP2 & P1xP3R & P2xP4S & P3xP4 OPG-19 2000 --- P4 ----- P2xP4R 840 --- P4 P2xP4 & P3xP4 P1xP3R & P1xP3S & P2xP4R & P2xP4S 600 --- P4 P2xP4 P1xP3S OPG-20 1500 --- P4 P3xP4 P1xP2 & P2xP4R & P2xP4S 1050 --- P4 P3xP4 P2xP4R & P2xP4S & P3xP4 210 --- P4 P1xP4 & P3xP4 ----- ISSR-1 1100 P1 & P2 --- P1xP2 P1xP4R 810 P1 & P2 --- P1xP2 & P2xP4 P1xP2 & P2xP4R & P2xP4S 200 --- P3 P1xP3 & P1xP4 & P2xP3 & P2xP4 & P3xP4 P1xP3R & P1xP4S & P2xP3R & P2xP4R & P3xP4 ISSR-2 800 --- P3 & P4 P3xP4 P1xP3R & P1xP3S & P1xP4R & P1xP4S & P2xP3R & P2xP3S & P2xP4R & P2xP4S 650 P1 & P2 --- P1xP2 & P2xP4 P1xP2 & P2xP4R & P2xP4S 170 --- P3 & P4 P3xP4 P3xP4 ISSR-3 700 P1 & P2 --- P1xP3 & P1xP4 & P2xP3 & P2xP4 & P3xP4 P1xP2 & P1xP3R & P1xP3S & P1xP4R & P1xP4S & P2xP3R & P2xP3S & P2xP4R & P2xP4S 480 P2 --- P2xP3 & P2xP4 P1xP2 & P1xP3R & P1xP4R & P2xP3R & P2xP3S & P2xP4S ISSR-5 390 --- P3 & P4 P2xP3 & P3xP4 P1xP3R & P1xP3S & P1xP4R & P1xP4S & P2xP3R & P2xP3S & P2xP4R & P2xP4S & P3xP4 281 --- P3 & P4 P2xP4 & P3xP4 P1xP3S & P1xP4S & P2xP3S & P2xP4R & P2xP4S & P3xP4 239 --- P3 & P4 P3xP4 P1xP3S & P1xP4S & P2xP3S & P2xP4S & P3xP4 TABLE 6. Genetic parameters calculated among parents and their generations. Generation Na Ne I He uHe %P RAPD P 1.148 1.275 0.229 0.16 0.178 38 F1 1.568 1.311 0.304 0.20 0.213 64 F2 1.568 1.297 0.292 0.19 0.197 64 Total 1.428 1.294 0.275 0.18 0.196 56 ISSR P 1.33 1.30 0.24 0.17 0.19 0.38 F1 1.38 1.26 0.23 0.15 0.17 0.42 F2 1.57 1.41 0.33 0.23 0.24 0.57 Total 1.428 1.324 0.266 0.18 0.199 45.56 Grand markers P 1.223 1.286 0.232 0.16 0.183 39.19 F1 1.493 1.291 0.272 0.18 0.194 54.73 F2 1.568 1.342 0.309 0.21 0.216 60.81 Total 1.428 1.306 0.271 0.18 0.197 51.58 Na=number of alleles, Ne=effective number of alleles, I=Shannon index, He=expected heterozygosity, uHe=unbiased expected heterozygosity, and %P=percentage of polymorphism. 193Heiba, Mahgoub, Mahmoud, Ibrahim, and Badr Mahdy: Genetic improvement of faba bean (Vicia faba L.) genotypes selected for resistance to chocolate spot disease Duc et al. (2010) reported an enormous genetic variability for faba beans, useful for breeding purposes. The results of h agree with those of Suresh et al. (2013), who recorded PIC values of 0.45 in faba beans genotypes revealed by developing 55 novel polymorphic cDNA–SSR markers. Also, Oliveira et al. (2016) recorded PIC values from 0.07 to 0.66, with an average of 0.33. Hemeida (2008) used PIC to evaluate the primer efficiency of ISSR and established the relationships, and successfully discriminated among the genotypes tested. The difference between the mean diversity (h) of both markers and between markers was undoubtedly due to mir- ror inbreeding or selection method against heterozygotes. The nature of used markers might be due to the level of observed heterozygosity resulting in the non-detection of homozygotes from heterozygotes because of the presence of null alleles. The heterozygosity represents the direct count of heterozygosity in the population and is estimated based on the allele frequency of individuals in that population according to the Hardy-Weinberg equilibrium. The PIC evaluates the informative potential of markers in different germplasm (Grativol et al., 2011; Mahdy, 2018). The h value of a marker with many amplicons desirable for variation splits into three main classes based on Botstein et al. (1980). The PIC values are more than 0.5 for highly informative markers, between 0.25-0.5 for reasonably informative markers, and less than 0.25 for slightly informative mark- ers. The Shannon information index (I) is one of the most important genetic diversity measurements (Sherwin et al., 2006). The effective number of alleles (Ne) is a reciprocal of gene homozygosity (Hartl & Clark, 1997). The Ne is used as a corollary to h; when h is high, Ne will be the high. Table 7 summarizes the estimates of genetic distance and genetic identity between generations and their parents according to the Nei coefficient (Nei, 1972; Nei, 1978). It ref lects the genetic relationships and the direction of the genetic improvement process. Results show that the RAPD markers revealed high genetic identity and distance values. The highest genetic distance value scored by RAPD was 0.089 on parents versus F1, and the lowest value scored 0.028 on F1 versus F2. Genetic identity oscillated from 0.914 (par- ent versus F1) to 0.972 (F1 versus F2), as revealed by RAPD. Nei genetic identity ranges from 0 to 1. Consequently, Nei genetic distance ranges from 0 to infinity (Nei 1972; Nei, 1978). ISSRs are more efficient markers for polymorphism and potent for intra- and inter-genomic diversity than other arbitrary markers like RAPDs (Zietkiewicz et al., 1994). Both markers target different portions of the genome. Genetic variations in genotypes of interest may be more directly due to polymorphism detected by technique rather than which techniques are employed. Cluster analysis and similarity between twenty genotypes Cluster analysis derived from both markers based on UPG- MA in accordance with Jaccard (1908; Fig. 3). The twenty genotypes divided into two main groups at a distance of 0.65. Cluster I (right cluster) divided into two subgroups at a distance of 0.668. The first subgroup (left subgroup) consists of P1 and P2 in the sub-sub group and P1xP2 (F1) only in a sub-sub group. The second subgroup (right sub- group) split further into sub-sub groups, which include the genotypes of P1xP2(F2) and P2xP4(F1) grouped in a cluster, P1xP3(F1) only in a group, P2xP3(F1) only in a group, and P2xP3 S(F2) and P2xP3R(F2) together in a group. Cluster II (left cluster) separated into two subgroups at a distance of 0.672. Each split further into sub-sub groups. The first sub-sub group (right one) includes P1xP2R(F2) and P2xP4 R(F2) together, P4 and PxP4(F1) in a cluster, and P1xP4 R(F2) and P1xP4 S(F2) in a group. The second one consists of P2xP4 S(F2), P1xP3 S(F2), and P3xP4(F2) in a separated cluster for each and P3 and P3xP4(F1) in a cluster. El-Ghadban et al. (2017) and Mahdy et al. (2021) reported similar results. The differences in the clustering pattern of genotypes may be due to marker sampling error, the level of polymorphism, or the number of loci and their coverage across the genome (Loarce et al., 1996). TABLE 7. Genetic and identity distance among generations and parents. Parameter P versus F1 P versus F2 F1 versus F2 RAPD Nei genetic identity 0.924 0.914 0.972 Nei unbiased genetic identity 0.947 0.932 0.989 Nei genetic distance 0.079 0.089 0.028 Nei unbiased genetic distance 0.055 0.070 0.011 ISSR Nei genetic identity 0.951 0.942 0.954 Nei unbiased genetic identity 0.973 0.964 0.969 Nei genetic distance 0.050 0.059 0.047 Nei unbiased genetic distance 0.028 0.037 0.031 All Nei genetic identity 0.935 0.925 0.964 Nei unbiased genetic identity 0.957 0.945 0.981 Nei genetic distance 0.067 0.077 0.036 Nei unbiased genetic distance 0.044 0.057 0.020 194 Agron. Colomb. 40(2) 2022 Association analysis Trait-marker association was analyzed using a single locus F-test module in Power maker software (Fig. 4). The results show the property of one trait for one locus characteri- zed. The characterized locus generated by OPG-20 was associated uniquely with seed yield per plant. Moreover, several traits were associated with one or more than one locus. Amplified loci of OPA-07 were associated with the different characterized chocolate spots, days to f lowering, the number of branches per plant, and seed yield per plant. Previous results have suggested the high potential use of molecular markers in search for genes affecting crop pro- ductivity and resistance to chocolate spots. Also, defense mechanisms against biotic and abiotic stress factors iden- tified statistical associations between the genetic markers and the traits of interest. Marker-trait associations were generally diverse in the amount of genetic variation valid for assessment. The results exhibited that a greater number of primers was pos- sibly involved in controlling traits at resistance to chocolate spot disease. This variation described by identified associa- tions for each trait may be attributed to the role of many minor genes controlling the trait, performance, and reac- tion of faba bean genotypes to chocolate spot disease, mark- ers exhibiting minor quantitative effect, rare alleles, and complex allelic interactions (Yang et al., 2010; Debibakas et al., 2014). These results correspond with the findings of Lou et al. (2015) and Sun et al. (2015). Association data can be used for faba beans breeding, especially in terms of its resistance to biotic and abiotic stresses. Conclusion The parents Nubaria1, Sakha1, TW, and Camolina could be considered good for crossing for resistance to foliar chocolate spot disease. Crosses 1, 2, and 3 (P1xP2 Nubaria-1 x Sakha-1, P1xP3 Nubaria-1 x T.W, P1xP4 Nubaria-1 x Camo- lina, respectively) showed resistance to disease during both generations, F1 and F2 with high values for yield and its components, especially date of f lowering and 100-seed weight per plant. Both markers exhibited interest specific loci relating to performance of chocolate spot that are 360 bp (OPA-07), 450 and 470 bp (OPA-17), 140 and 660 bp (OPB-05), 810 and 1100 bp (ISSR-1), 650 bp (ISSR-2), and 480 and 700 bp (ISSR-3). The genetic distance valued by RAPD was 0.089 on parents versus F1, and the lowest value scored 0.028 on F1 versus F2. Genetic identity oscillated from 0.914 (parent versus F1) to 0.972 (F1 versus F2), as revealed by RAPD. Estimating genetic relationships and differences generated by both markers could be moderately clarified by the product number of PCR, the number of bands, and coverage along the genome with the association of agromorphological traits. The current research could provide information on the morphological and molecular characteristics of faba beans chocolate spot disease. Inter- varietal hybridization (resistant and susceptible) is still considered one of the most promising methods for obtain- ing germplasm with resistance and high yield. 19 13 20 3 10 12 18 4 7 14 15 5 1 2 9 11 6 8 16 17 0.650.700.750.800.850.900.951 2.50 2.00 1.50 1.00 0.50 0 O PA 7- 64 0 O PA 7- 35 0 O PA 7- 86 0 O PA 7- 30 0 O PA 17 -3 90 O PA 17 -9 20 O PA 17 -4 90 O PA 10 -3 60 O PA 10 -3 10 O PA 7- 83 0 O PG 20 -3 20 O PG 19 -6 50 O PG 19 -7 30 O PG 19 -2 60 IS SR 3- 35 0 IS SR 3- 29 0 IS SR 5- 75 0 IS SR 3- 76 0 IS SR 3- 73 0 IS SR 5- 53 0 Log 10 No. of pods per plants No. of seeds per plants 100-seed weight Chocolate spot Days to flowering Plant height FIGURE 3. Dendrogram tree constructed using UPGMA according to Jaccard similarity. Cluster analysis based on RAPD and ISSR markers; (1)-P1,(2)-P2, (3)-P3, (4)-P4, “six F1” (5)-P1xP2, (6)-P1xP3, (7)-P1xP4, (8)-P2xP3, (9)-P2xP4, (10)-P3xP4, “ten F2” (11)-P1xP2, (12)-P1xP3 (R), (13)-P1xP3 (S) (14)-P1xP4 (R), (15)-P1xP4 (S) (16)-P2xP3 (R), (17)-P2xP3 (S), (18)-P2xP4 (R), (19)-P2xP4 (S) and (20)-P3xP4 (S×S)”. FIGURE 4. Association analysis. Trait-marker association was achieved through F-test analysis. Power marker software V3.0 was used and markers with high P-value (P>0.01) were selected in the association analysis. X and Y axes refer to -Log P values and the name of primers associated with certain traits. 195Heiba, Mahgoub, Mahmoud, Ibrahim, and Badr Mahdy: Genetic improvement of faba bean (Vicia faba L.) genotypes selected for resistance to chocolate spot disease Acknowledgments We deeply acknowledge Dr. Mohamed Hamdy Dawood, professor of genetics, Faculty of Agriculture, Al-Azhar University, for help in writing and completing this study. 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