Transgenic Elite Lines of Carioca Seeded Common Bean (Phaseolus Vulgaris L.) With Multiple Resistance to Viruses Reduce Cowpea Mild Mottle Virus Transmission

The most important viruses infecting common bean (Phaseolus vulgaris L.) in Brazil are BCMV, BGMV and CPMMV, the last two transmitted by the whitey Bemisia tabaci, occurring simultaneously and causing severe yield losses. Genetically modied progenies of common bean, from carioca market class and multiple virus resistance (BCMV, BGMV and CPMMV), have been developed using conventional breeding and molecular tools. Agronomic performance and virus disease severity (VS) evaluated in two eld trials, selected 39 elite progenies out of 477. Molecular analyses identied the presence of BCMV and BGMV resistance alleles in plants. CPMMV resistance was measured on mechanically inoculated plants using a VS scoring scale. Among the lowest VS average scores, ve progenies showed resistance to BCMV, BGMV and CPMMV, and upright plant architecture, resistance to plant lodging and carioca market class grains, presenting potential to be developed into a new transgenic cultivar, with multiple virus resistance. Additionally, the resistant progenies may also contribute to reduce virus spread in the eld, as they were a less ecient inoculum source of CPMMV in insect transmission assays. of the event Embrapa 5.1 TaqMan hydrolysis probes (ThermoFisher). probes designed Primer Express TM software (ThermoFisher). as previously described, to be used as CPMMV inoculum sources. Twenty days after inoculation, systemic infection was conrmed by RT-PCR. Total RNA was extracted using the Promega SV Total RNA isolation system, and cDNA was synthesized using the Promega Go-script reverse transcription system, with oligo-dT primers. PCR reactions were performed using CPMMV specic primers CPMMV-cpF (5’- GCATGTGGCTGAGGTTCT -3’) and CPMMV-cpR (5’- AGGCGGCAATCACTGAATCA -3’). Then, virus-free adult whiteies were transferred to the CPMMV-infected plants for virus acquisition for 1 hour. After that, insects were carefully transferred to three groups of test plants, one from each bean line (CNFCT 19119 or CNFCT 19120, BRS Sublime and BRS FC401 RMD) in individual cages (5 insects/plant and 40 replicates per treatment), for a 2h inoculation period. After 20 days, virus disease severity was evaluated using a 1-4 scoring scale for the level of symptoms (1=none, 2=light, 3=moderate and 4= strong). The homocedasticity Levene test and residual normality Kolmogorov-Smirnov test were performed for data analysis. The inoculum sources were compared using the Pearson’s Chi-square test for the proportion of plants showing each symptom score (category). resistant lines 19120), the proportions of plants within each grade of the scoring scale were compared using the Equality of Proportions Hypothesis test (p<0.05).


Introduction
Common bean (Phaseolus vulgaris L.) is the most directly consumed legume worldwide. This crop has a social and economic impact, its grain being an excellent source of proteins, minerals, bers and vitamins. In addition, it has a wide geographic distribution, being cultivated mainly in countries in Latin America, Africa and Asia (Brigide et al., 2014;Martins et al., 2016;Yi et al., 2016). In Brazil, the common bean crop is grown in most states, by small, medium and large farmers, which use different technological levels (L. C. Faria et al., 2013;Martins et al., 2016; L. C. Melo et al., 2017). The country is the world's largest producer and consumer of common bean (FAOSTAT, 2020). In the 2018/19 harvest, Brazil produced approximately 1.88 million tons of common bean in 1.31 million hectares, with an average yield of 1.44 kg ha −1 (Embrapa Arroz e Feijão, 2019). The average per capita consumption in Brazil is approximately 17.0 kg/person/year (Embrapa Arroz e Feijão, 2019), with national preference for grains from the carioca market class (around 70% of consumption), followed by black beans (20%), and other commercial classes (10%) (Anderson et al., 2016;Barili et al., 2016;Barros & Prudencio, 2016; L. C. Faria et al., 2013. Despite the genetic progress obtained in the crop yield in recent years, around 17.4 kg ha −1 year increase for carioca beans (L. C. Faria et al., 2013), common bean still presents grain yields below their productive potential (L. C. Faria et al., 2013. One of the factors that compromises the quality and, above all, the grain yield, is the large number of diseases that affect the crop (Assefa et al., 2019;Meziadi et al., 2016;Miklas et al., 2006), with emphasis on virus diseases (Alves-Freitas et al., 2019;Meziadi et al., 2016;Souza et al., 2018). Among the most important virus diseases of common beans in Brazil, there is the bean common mosaic virus (BCMV; family Potyviridae; genus Potyvirus) (Melotto et al., 1996;Meziadi et al., 2016), the bean golden mosaic virus (BGMV; family Germiniviridae; genus Begomovirus) (Bon m et al., 2007;Souza et al., 2018) and the cowpea mild mottle virus (CPMMV; family Beta exiviridae; genus Carlavirus). These last two viruses are transmitted by the white y (Bemisia tabaci; Hemiptera: Aleyrodidae) (Costa et al., 1983;Marubayashi et al., 2010) and can occur simultaneously in mixed infections (Alves-Freitas et al., 2019;. A low rate of seed transmission of different RMD, in 2.0 m long rows, spaced by 0.45 m, with 10 seeds per meter and a control row interspersed for every 10 progenies, without replications, totaling 525 plots, in which 48 were control lines. For the evaluation of the virus disease severity (VS), a scoring scale from 1 to 9 was used (L. Melo, 2009), where 1 is the score attributed to resistant plots and 9 to plots with susceptible plants with virus disease symptoms that affect seed yield. The elite progenies with the best performance were selected for further evaluations of agronomic performance in eld and greenhouse experiments.

Agronomic Performance Evaluation
Two experiments were carried out, in the rainy season/2016 (sowing in December) and in the dry season/2017 (sowing in March), for the evaluation of agronomic performance. A total of 39 elite progenies selected in the previous stage and three controls (BRS Estilo, BRS FC402 and BRS FC401 RMD) were evaluated. Among these 39 progenies, 10 were derived from the crossing BRS Estilo × CNFCT 16206, generations BC 4 F 4:6 and BC 3 F 5:7 (Figure 1), and 29 from the crossing BRS Sublime × BC3F1 (BRS Estilo × CNFCT 16206 RMD), generations F 4:6 and F 3:6 ( Figure 2). The experiments were carried out using a randomized block design with three replicates, each plot with 6 m 2 and density of 10-12 plants/m. The plots were conducted using the recommended management for the cultivation of beans (Arf et al., 2015), however, without the control of diseases and insect pests. The following variables were evaluated: seed yield in kg ha -1 , corrected at 13% humidity; 100-seed mass, in g, and seed appearance (scores 1 to 5): 1, commercial standard for carioca market class (light beige seeds with light brown stripes, opaque and not shiny); 5, lowest commercial quality or other market class. (Barili et al., 2016;Ramalho et al., 2012). Plant architecture (PA), Tolerance to Lodging (TL) and Virus disease Severity (VS) were evaluated at the end of the crop season. For PA and TL a scoring scale from 1 to 9 was used, where 1 refers to lines suitable for mechanized harvesting (short guides, high pods, closer branches, and without bedding) and 9, lines that are unable to be mechanically harvested (long guides, low pods, open branches, and lodged plants) (L. Melo, 2009). To evaluate VS, the natural occurrence of the virus diseases was assessed based on a scoring scale of disease severity, where 1 = no symptoms and 9 = plot with 80-100% of plant with disease symptoms, or 60-100% of infected tissue (L. Melo, 2009). BRS Estilo and BRS FC402 were used as the controls for BGMV infection; BRS FC401 RMD was used as the control for CPMMV infection.

Genetic and Statistical Analysis for Agronomic Traits
Analysis of individual variances (ANOVA) were estimated for all traits, in each environment, using the F-test. The joint analysis was also performed for all traits. Progeny and environment effects were considered xed. The averages were compared using the Scott-Knott test, at 5% probability (Scott & Knott, 1974). The experimental precision was also estimated through the selection accuracy (SA), as described (Resende & Duarte, 2007). Statistical analyses were performed using the GENES statistical software (Cruz, 2013).

Response to CPMMV infection at greenhouse
Superior progenies, within the 39 progenies selected in the eld experiments, were harvested individually and, thus, 20 plants per family, were grown in a greenhouse. Individual leaf tissue samples were collected 20 days after planting for DNA extraction and genotyping for the presence of resistance alleles (gene I for BCMV and gene ahas for the Embrapa 5.1 transgene event).
The individual plants were mechanically inoculated at eight days after sowing with a crude extract prepared by grinding leaves from CPMMV infected plants (isolate CPMMV: BR: GO: 14 -GenBank MK202583) in 0.1 M phosphate buffer containing sodium sul te, added of a pinch of carborundum (Alves-Freitas et al., 2019;Cheruku et al., 2017). The cultivars BRS Estilo and BRS Sublime ( Figures  1 and 2), BRS FC401 RMD and the line CNFCT 16207 were used as controls. At 35 days after inoculation, all plants were individually assessed for the severity of CPMMV symptoms, using a scoring scale from 1 to 9, with 1 = no symptoms and 9 = plants with severe mosaic and/or crinkling leaf symptoms. Plants with no symptoms or showing a light mosaic or even a mosaic with light leaf deformations were considered resistant (grades 1, 2 and 3, respectively). Plants with severe mosaic, with or without leaf deformations, severe leaf deformations or with prominent crinkling in most leaves, were considered susceptible (4 or higher).

CPMMV transmission in resistant common bean lines
Greenhouse and eld observations indicate that CPMMV can still replicate in the resistant progenies. Therefore, transmission assays were carried out to test how the resistant lines (CNFCT 19119 and CNFCT 19120, corresponding to the 422-39.1 and 554-15.1 progenies, respectively, Table 4), behave as inoculum source of CPMMV. In each experiment, eight-day old plants from each line (CNFCT 19119 or CNFCT 19120), BRS Sublime (resistant control) and BRS FC401 RMD (susceptible control) were mechanically inoculated with CPMMV, as previously described, to be used as CPMMV inoculum sources. Twenty days after inoculation, systemic infection was con rmed by RT-PCR. Total RNA was extracted using the Promega SV Total RNA isolation system, and cDNA was synthesized using the Promega Go-script reverse transcription system, with oligo-dT primers. PCR reactions were performed using CPMMV speci c primers CPMMV-cpF (5'-GCATGTGGCTGAGGTTCT -3') and CPMMV-cpR (5'-AGGCGGCAATCACTGAATCA -3'). Then, virus-free adult white ies were transferred to the CPMMV-infected plants for virus acquisition for 1 hour. After that, insects were carefully transferred to three groups of test plants, one from each bean line (CNFCT 19119 or CNFCT 19120, BRS Sublime and BRS FC401 RMD) in individual cages (5 insects/plant and 40 replicates per treatment), for a 2h inoculation period. After 20 days, virus disease severity was evaluated using a 1-4 scoring scale for the level of symptoms (1=none, 2=light, 3=moderate and 4= strong). The homocedasticity Levene test and residual normality Kolmogorov-Smirnov test were performed for data analysis. The inoculum sources were compared using the Pearson's Chi-square test for the proportion of plants showing each symptom score (category). Fixing the inoculum source as one the resistant lines (CNFCT 19119 or CNFCT 19120), the proportions of plants within each grade of the scoring scale were compared using the Equality of Proportions Hypothesis test (p<0.05). Table 4 Summary of the combined analysis of variance for the agronomic traits seed yield, 100-seed mass (100M), plant architecture (PA), tolerance to lodging (TL), seed appearance (SaP) and virus disease severity (VS), evaluated in 39 common bean elite progenies and three controls in Santo Antônio de Goiás, Goiás, Brazil, during the rainy growing season/2016 and dry growing season/2017. df, degrees of freedom; MS: mean square; Yield, seed yield in kg ha −1 , corrected to 13% moisture content;100M, 100-seed mass in g.; Plant architecture (PA) and tolerance to lodging (TL) (scores 1 to 9): 1, lines suitable for mechanized harvesting (short guides, high pods, closer branches, and without bedding); 9, lines that are unable to be mechanically harvested (long guides, low pods, open branches, and lodged plants); SaP, seed appearance (scores 1 to 5): 1, commercial standard for carioca market class; 5, lowest commercial quality or other market class; VS, Virus disease severity (scores 1 to 9): 1 = no symptoms and 9 = plot with 80-100% of plant diseases, or 60-100% of infected tissues (Melo, 2009); BGMV -bean golden mosaic virus, evaluated in the conventional controls (BRS Estilo and BRS FC402) and CPMMV -cowpea mild motle virus, evaluated in the elite progenies and in the transgenic control (BRS FC401 RMD).

Results And Discussion
Thirty nine out of 477 elite progenies have been selected in the eld experiments based on their agronomic performance, considering the best scores for virus disease severity (VS), seed yield, plant architecture (PA), tolerance to lodging (TL), 100-seed mass (100M) and seed appearance (SaP) (Table 1). Overall, the VS average among the selected progenies ranged from 2.3 to 5.8, con rming there is high genetic variability for the resistance to viruses under eld conditions (Table 1). As expected, conventional controls (BRS Estilo and BRS FC402) were susceptible to BGMV, with a virus disease severity (VS) average of 5.8 (Table 1). Because CPMMV symptoms can be totally suppressed by the symptoms of BGMV (Alves-Freitas et al., 2019;Souza et al., 2018), it was not possible to distinguish the severity of CPMMV symptoms from the BGMV symptoms in the conventional controls, which are not resistant to BGMV. Thus, VS average in the elite progenies, regarding CPMMV, were compared only to that of the transgenic control (BRS FC 401 RMD), being signi cantly lower (Table 1). Twelve out of the 39 elite progenies (31%) had average scores for VS ≤ 3.0 (Table 1), indicating that they were highly resistant/tolerant to the virus diseases, including CPMMV. Sixteen out of 39 elite progenies (41%) showed average seed yield statistically equal to the transgenic control, and among those, there were 8 of the 12 elite progenies selected as the most tolerant to CPMMV (VS ≤ 3.0) ( Table 1). The superiority of the elite progenies, along with the transgenic control, in comparison with the conventional controls, corroborates the results reported by Souza et al. (2018), indicating that the presence of the Embrapa 5.1 transgene increased seed yield stability in elite GM common bean lines. Yield, seed yield in kg ha-1, corrected to 13% moisture content.
Plant architecture (PA) and tolerance to lodging (TL) (scores 1 to 9): 1, lines suitable for mechanized harvesting (short guides, high pods, closer branches, and without bedding); 9, lines that are unable to be mechanically harvested (long guides, low pods, open branches, and lodged plants).
SaP, seed appearance (scores 1 to 5): 1, commercial standard for carioca market class; 5, lowest commercial quality or other market class.
BGMV -bean golden mosaic virus, evaluated in the conventional controls (BRS Estilo and BRS FC402) and CPMMV -cowpea mild motle virus, evaluated in the elite progenies and in the transgenic control (BRS FC401 RMD).
Means followed by the same letter in the Yield, seed yield in kg ha-1, corrected to 13% moisture content.
Plant architecture (PA) and tolerance to lodging (TL) (scores 1 to 9): 1, lines suitable for mechanized harvesting (short guides, high pods, closer branches, and without bedding); 9, lines that are unable to be mechanically harvested (long guides, low pods, open branches, and lodged plants).
SaP, seed appearance (scores 1 to 5): 1, commercial standard for carioca market class; 5, lowest commercial quality or other market class.
BGMV -bean golden mosaic virus, evaluated in the conventional controls (BRS Estilo and BRS FC402) and CPMMV -cowpea mild motle virus, evaluated in the elite progenies and in the transgenic control (BRS FC401 RMD).
Means followed by the same letter in the table columns are not signi cantly different according to the Scott-Knott method at 5% probability.
Phenotypic and molecular analyses were applied to discriminate for the presence of resistance alleles in plants from the 12 selected progenies, after mechanical inoculation with CPMMV in a controlled environment. The dominant allele of the gene I, which confers resistance to BCMV, was detected in the 12 selected virus resistant progenies and in the controls (Table 2), using the SCAR SW13 marker. The line CNFCT 16206, used as the donor parent, presents a hypersensitivity reaction to the bean common mosaic necrosis virus (BCMNV), indicating the presence of gene I for resistance to BCMV . The transgene from the Embrapa 5.1 event, which confers resistance to the BGMV, was detected in 11 out of the 12 selected progenies. The successful transfer of the transgene to these 11 elite progenies, all resistant to BGMV, corroborates the previous agronomic and molecular characterization of the transgene (Aragão et al., 2013; J. C. . The transgene is inherited in a 3:1 Mendelian segregation (J. C.  with an intra-allelic relationship of complete dominance for the transgene and for the reaction to BGMV. A similar segregation pattern has been con rmed regarding the resistance to CPMMV in the cultivar BRS Sublime (J. C. Faria, unpublished data). Both recurrent parents, the cultivars BRS Estilo and Sublime are valuable sources of resistance and/or tolerance to CPMMV (J. C. Faria, unpublished data). In the present study, ve out of 12 progenies (41%) were considered resistant and/or tolerant to CPMMV, all ve of which were also resistant to BGMV and BCMV (  1 and 2). Of the ve progenies resistant to CPMMV, only the progeny 184-12.1 was derived from the cross between the cultivar BRS Estilo and the line CNFCT 16206. The others are from the cross (BRS Sublime × BRS Estilo GM) (Figures 1 and 2; Table 2). At this breeding stage, the progenies with multiple resistance to viruses were then considered as elite lines.  (Bon m et al., 2007;Dinon et al., 2012). d Bean common mosaic virus; Marker assisted selection using marker SCAR SW13 linked to gene I (Melotto et al., 1996). e CNFCT 16207, common bean genetically modi ed line, resistant to the golden mosaic (Event Embrapa 5.1; RMD technology) .
f BRS FC401 RMD, common bean genetically modi ed commercial cultivar, resistant to the golden mosaic (RMD) g BRS Sublime and BRS Estilo, common bean conventional cultivars developed by Embrapa.
Although CPMMV infection in the resistant lines primarily results in light symptoms, not affecting seed yield or other relevant agronomic traits, CPMMV replication is not suppressed in these common bean lines ( Figure S1). We conducted white y CPMMV transmission assays using two of the resistant lines (CNFCT 19119 and CNFCT 19120), the tolerant cultivar BRS Sublime and the susceptible cultivar BRS FC401 RMD, as both inoculum sources and test plants. Figures 3A and 3B show the proportion of test plants in each grade of the scoring scale, by inoculum source. The results show that when the inoculum source of CPMMV was the resistant line CNFCT 19119 ( Figure 3A), the majority of the infected plants were symptomless or presented only light symptoms, a signi cantly higher proportion than that observed for the other inoculum sources (BRS Sublime and BRS FC401 RMD) (Chi-square, p< 0.05). Additionally, no plants with strong symptoms of the disease were observed, when the inoculum source was line CNFCT 19119.
Remarkably, when the other resistant line, CNFCT 19120, was the inoculum source, over 80% of infected plants presented light virus disease symptoms ( Figure 3B). Fixing the inoculum source as one of the resistant lines, the proportion of test plants without symptoms or presenting light symptoms was signi cantly higher when the test plants were also from one of the resistant lines ( Figure 4). Likewise, a higher proportion of plants with moderate or strong symptoms was observed when the test plants were from a susceptible cultivar (BRS FC401 RMD). The reduced transmission of CPMMV, evidenced by the high proportion of plants presenting none or light symptoms in the transmission assays, indicates that these resistant lines represent a promising tool for reducing virus spread in the eld, as well as virus disease symptoms. Similar results have been reported for the selection of cassava breeding lines with effective resistance to cassava brown streak viruses (CBSVs) and cassava mosaic begomoviruses (CMBs) (Mukiibi et al., 2019), which are also transmitted by B. tabaci, showing signi cantly lower severity, compared to the controls.
Besides the trait virus disease severity, elite progenies were selected in eld experiments following the quality control usually applied for the selection of all other important agronomic traits in the Embrapa common bean breeding program. In the eld experiments, the coe cients of variation (CV%) ranged from 3.6 for 100-seed mass (100M) to 28.1 for seed yield (Table 3), similar to another study selecting common bean lines for pathogen resistance (Pereira et al. 2019). Estimates of selection accuracy (SA) of progenies were high (0.70 ≤ SA 0.90) for some traits (seed yield, 100M, SaP and VS), and moderate (0.50 ≤ SA 0.70) for others (PA and TL) ( Table 3). This indicates good experimental informativeness, similar to the SA values reported for VS of BGMV and CPMMV when the elite GM common bean lines were evaluated under eld conditions . SA represents the best parameter to assess experimental precision when evaluating plant lines (Resende & Duarte 2007), and also re ects environmental and genetic variations (Ramalho et al., 2012). The adequate experimental precision made it possible to discriminate the 39 elite progenies for all the agronomic performance traits, especially VS, in eld conditions, which allowed to select the best lines with the highest virus resistance. Table 3 Summary of the analysis of individual variance for the agronomic traits evaluated in 39 common bean elite progenies and three control cultivars, in Santo Antônio de Goiás, Goiás, Brazil, in the rainy growing season/2016 and dry growing season/2017. df, degrees of freedom; MS: mean square; Yield, seed yield in kg ha −1 , corrected to 13% moisture content;100M, 100-seed mass in g.; Plant architecture (PA) and tolerance to lodging (TL) (scores 1 to 9): 1, lines suitable for mechanized harvesting (short guides, high pods, closer branches, and without bedding); 9, lines that are unable to be mechanically harvested (long guides, low pods, open branches, and lodged plants); SaP, seed appearance (scores 1 to 5): 1, commercial standard for carioca market class; 5, lowest commercial quality or other market class; VS, Virus disease severity (scores 1 to 9): 1 = no symptoms and 9 = plot with 80-100% of plant diseases, or 60-100% of infected tissues (Melo, 2009); BGMV -bean golden mosaic virus, evaluated in the conventional controls (BRS Estilo and BRS FC402) and CPMMV -cowpea mild motle virus, evaluated in the elite progenies and in the transgenic control (BRS FC401 RMD); SA, selection accuracy. df, degrees of freedom; MS: mean square; Yield, seed yield in kg ha −1 , corrected to 13% moisture content;100M, 100-seed mass in g.; Plant architecture (PA) and tolerance to lodging (TL) (scores 1 to 9): 1, lines suitable for mechanized harvesting (short guides, high pods, closer branches, and without bedding); 9, lines that are unable to be mechanically harvested (long guides, low pods, open branches, and lodged plants); SaP, seed appearance (scores 1 to 5): 1, commercial standard for carioca market class; 5, lowest commercial quality or other market class; VS, Virus disease severity (scores 1 to 9): 1 = no symptoms and 9 = plot with 80-100% of plant diseases, or 60-100% of infected tissues (Melo, 2009); BGMV -bean golden mosaic virus, evaluated in the conventional controls (BRS Estilo and BRS FC402) and CPMMV -cowpea mild motle virus, evaluated in the elite progenies and in the transgenic control (BRS FC401 RMD); SA, selection accuracy.

Source of variation
On the other hand, there were signi cant differences for most traits within treatments and within progenies, indicating that, in general, there is genetic variability between treatments. There was no variability for VS within controls (Table 4), meaning that the controls did not differ for their response to the virus diseases. The interaction between treatments and environment (T x E) was signi cant (P ≤ 0.01) for most traits ( Table 4). The interaction between progenies and environment (P x E) was signi cant for VS, which can be explained by the weather differences between the growing seasons (Pereira et al., 2019) probably leading to differential virus pressure (J. Faria et al., 2016;Souza et al., 2018). The variation shown for seed yield by the signi cant interaction between progeny (P) vs environments (E) ( Common bean plants with upright architecture are preferred by farmers, as it allows mechanized harvesting in the eld, reducing grain losses and improving grain quality Barili et al., 2016). As a contribution to that demand, the development of modern cultivars by the Embrapa common bean breeding program has resulted in signi cant genetic progress for these two traits (PA and TL) over the years . In the present study, the majority of the elite progenies were statistically superior than the best controls (BRS Estilo and BRS FC402) for the traits PA and TL (95% and 92%, respectively) ( Table 1). The desirable patterns of PA and TL from both recurrent parents (Melo et al., 2010;Wendland et al., 2018) were successfully transferred to the 12 superior elite progenies selected for virus resistance. All the 12 selected progenies presented better PA scores than the best control (BRS Estilo, PA=5.3), and 10 out of 12 presented better TL scores (Table 1).
Last, but no least, the elite progenies have also been evaluated for grain quality traits. The trait seed appearance (SaP) refers to the commercial value of the carioca market class standard for grains, which are the most consumed in Brazil, representing about 70% of the national market (Barili et al., 2016;Souza et al., 2018). In the present study, seven out of the 12 selected progenies were among the best scores for this trait, and did not differ from the control BRS Estilo (Table 1). The other ve progenies did not differ from the average of the transgenic control, which also belongs to the carioca market class seeds (Table 1). In general, 56% of the elite progenies had SaP means statistically equal to the conventional control BRS Estilo (Table 1), indicating a good recovery of the desirable SaP scores. In the BC 4 F 4:7 and BC 3 F 5:8 ( Figure 1) and in the F 3:7 and F 4:7 generations (Figure 2), the analysis of genetic similarity using molecular markers showed that the relative genetic similarities between recurrent parents and their progenies were > 97.8% (data not shown). Additionally, larger grains, evaluated by the trait 100-seed mass (100M), are preferred among consumers Melo et al., 2017). The mean values of 100M ranged from 21.2g to 24g among the elite progenies (Table 1), being within the expected range for eld evaluations without disease or insect management, as reported for other cultivars developed by Embrapa (L. C. Melo et al., 2017;Souza et al., 2016;Wendland et al., 2018). Twenty six percent of the elite progenies had statistically higher 100M averages than the best control (RMD) ( Table 1) and among them, three out of the 12 selected virus resistant progenies.
The elite progenies selected for multiple virus resistance in the present study have potential for selection in future agronomic performance eld evaluations, aiming to develop a common bean cultivar combining high yield to desirable agronomic and grain quality traits, such as the standard carioca market class seeds and suitability to mechanized harvesting. The RMD technology has proved its value as a technology transfer tool in the common bean breeding program, by applying biotechnology and traditional selection methods (Assefa et al., 2019;Meziadi et al., 2016Meziadi et al., , 2017Patroti et al., 2019;Souza et al., 2018), which opens up new opportunities for strengthening a more sustainable bean production system in Brazil.

Figure 1
Diagram representing the breeding strategy used to develop genetically modi ed progenies of common bean resistant to viruses, by crossing the cultivar BRS Estilo and the transgenic line CNFCT 16206, carrier of the event Embrapa 5.1.

Figure 2
Representative diagram of the breeding strategy used in the development of genetically modi ed common bean progenies for resistance to viruses by crossing the BRS Sublime cultivar with BC 3 F 1 plants from the BRS Estilo x CNFCT 16206 crossing (Embrapa 5.1).