Molecular Characterization of Parental Lines and Validation of Snp Markers for Disease Resistance in Common Bean

The implementation of molecular tools that help the early selection of genotypes carrying target alleles increases eciency and reduces the time and costs of breeding programs. The present study aimed the molecular characterization and validation of SNPs targeting disease resistance alleles for assisted selection. A total of 376 common bean lines with contrasting responses for anthracnose and angular leaf spot resistance were used, as well as 149 F 2 plants from the cross between BRS Cometa x SEL 1308 (carrying the Anthracnose resistance gene Co-4 2 ). Seven of the ten SNP markers evaluated showed potential for assisted breeding: snpPV0025 (Phg-2), snpPV0027 (Phg-5), snpPV0079 (Phg-5), snpPV0046 (Co-u), snpPV0068 (Co-4 2 ), snpPV0070 (Co-4 2 ) and snpP8282v3-817 (Co-4 2 ). Markers snpPV0070 and snpP8282v3-817 showed high eciency of selection (99.7 and 99.8%, respectively). These markers exhibit great potential to assist in the selection at different stages of the breeding program and may be readily incorporated into marker-assisted selection. 349, MAB 351, MAB 352, MAB 353, MAB 354 and MAB 484 lines, with high angular leaf spot resistance, contain the G10474 line, source of Phg-2, as one of their parents (Gil et al. 2019). The results of this study indicate that the Phg-2 allele is present in elite germplasms developed by Embrapa, the Federal University of Lavras, IAC, IAPAR, Agropecuária Terra Alta and CIAT, and have been detected in 72 lines/cultivars (Table S5). However, as pointed out by Gi et al. (2019), care should be taken when using the snpPV0025 (ALS_08_62193174) for MAS, since it has been used to tag and introgress the Phg-2 locus from Mesoamerican MAB sources into Andean breeding lines, and do not tag specic meso alleles. Nay et al. (2019a) shed greater light on this by identifying pathotype-specic haplotypes at the Phg-2 and offering new molecular markers to be tested and used in MAS. This paper is a cooperative contribution on validation of SNP markers linked to disease resistance genes for their effective use in the routine of Embrapa (Brazilian Agricultural Research Corporation) common bean breeding program. The main goal was to characterize and select elite parent lines according the presence of disease resistance alleles, as well as validate SNP markers for MAS of superior genotypes from populations derived from these parent lines. It reports an elegant example of cross validation and effective use of SNP markers for MAS in an applied breeding program of a very social important orphan crop (common bean or dry bean).


Introduction
Brazil is the world's largest producer and consumer of common beans (Phaseolus vulgaris), an important nutritional source and socioeconomically signi cant crop (Kotue et al. 2018; Embrapa Rice and Beans 2021). This legume is widely cultivated in the country, being produced in three growing seasons and grown by farmers with different technological pro les, at different scales and production systems (Brusamarello et al. 2017). In this scenario, numerous challenges limit production potential, one of the main causes being biotic stresses (Basavaraja et al. 2020).
The common bean is host to a wide range of diseases caused by different pathogens that affect, to a greater or lesser extent, all cultivars recommended by genetic improvement programs ( In Brazil, at least 15 epidemiologically important diseases can potentially cause severe damage (Wendland et al. 2018). Among the diseases that affect common bean shoots, anthracnose (Colletotrichum lindemuthianum) and angular leaf spot (Phaeoisariopsis griseola Sacc.) stand out due to the signi cant production losses in major producing regions worldwide (Singh and Schwartz 2010). These diseases are caused by pathogens that exhibit pathotype diversity and a monogenic and/or quantitative inheritance pattern of resistance (Padder et al. 2017;Nay et al. 2019b), which hinder the development of genetically resistant cultivars. Depending on the level of susceptibility of the cultivar, these diseases can cause losses of 80-100% (Singh and Schwartz, 2010).
The main objective of this study was to evaluate several SNP markers previously associated with alleles linked to anthracnose (Co-4 2 and Co-u) and angular leaf spot resistance (Phg-1, Phg-2 and Phg-5). These SNPs were evaluated in a diverse panel of common bean genotypes, including important sources of resistance and parent plants used in Embrapa's (Brazilian Agricultural Research Corporation) bean breeding program, in order to characterize elite parent lines for the presence of disease resistance alleles, as well as validate or indicate SNP markers to be incorporated into MAS.

Genetic material
We used 376 diverse common bean genotypes from several national and international breeding programs that show contrasting resistance responses to anthracnose and angular leaf spot, including cultivars, elite parent plants and sources of resistance (donor parent plants). Information on the institution of origin, gene pool, grain type and reaction to anthracnose and angular leaf spot are presented in Table S1. Phenotypic information regarding the reaction to the two diseases was obtained for 240 of the 376 lines in this study, of which 139 were phenotyped for anthracnose and 101 for angular leaf spot (Table S1).
Ten seeds from each line were deposited on sheets of germitest paper, moistened with autoclaved distilled water and then placed in a germinator (Mangelsdorf) at a constant temperature of 25°C and 27% humidity for seven days. Two leaf discs obtained from the primary leaves with a puncher were individually collected and transferred to 96 sterile deep well plates. The plates were stored in a freezer at -80°C for 24 hours and then submitted to lyophilization for six hours using a freeze dryer (Liotop® model L101). Finally, the plates were sent to Intertek Agritech (Sweden) for genotyping. This genotyping service provider performs KASP marker analysis (Rasheed et al. 2016).

Molecular analysis with SNP markers
Nine SNPs associated with disease resistance in common bean were selected from a portfolio of molecular markers made available by the "High Through-Put Genotyping (HTPG)" project (Bohar et al. 2020) [https://cegsb.icrisat. org/marker-panels-2/]. Three of these are associated with anthracnose (two with the Co-4 2 allele and one with the Co-u genes) (

Segregating population and contrasting lines
The TaqMan® SNP markers associated with anthracnose (Co-4 2 ) were validated using an F 2 population of 149 plants from the cross between BRS Cometa (female parent, susceptible to pathotype 73) and SEL 1308 (male parent, harboring the allele Co-4 2 , resistant to C. lindemuthianum pathotype 73) (Table S2), which was phenotyped for the reaction to C. lindemuthianum pathotype 73. The TaqMan® SNP marker associated with angular leaf spot (Phg-2) was validated in a set of 30 contrasting common bean genotypes for the angular leaf spot reaction (Table S3), evaluated under eld conditions in different tests performed in the Embrapa Rice and Beans breeding program.

Obtaining Colletotrichum lindemuthianum inoculum
The C. lindemuthianum isolate CL1869 (pathotype 73) was used to inoculate 149 F 2 plants from the cross between BRS Cometa x SEL 1308. F 2 seeds, as well as ten seeds from each parent plant and the susceptible control (Rosinha G2) were sown in polystyrene seedling trays. Plants were inoculated seven days after sowing, at stage V2 (fully expanded primary leaves) (Pastor-Corrales et al. 1992). The spore solution (1.2 × 10 6 spores mL-1 ) was applied to the abaxial and adaxial surfaces of the primary leaves with the aid of a hand sprayer (De Vilbiss, No. 15). After inoculation, the plants were incubated in a humidity chamber for 48 hours, at 20±2ºC, with relative humidity of around 95%, controlled by a misting system, and a 12-hour light/dark photoperiod. Next, misting was interrupted and the inoculated plants were kept in a controlled environment under the same temperature and photoperiod described above, where they remained until the disease symptoms were evaluated.
Symptoms were assessed seven days after inoculation, based on a grading scale proposed by Pastor-Corrales and Tu (1989), in which grade 1 represents no symptoms and 9 dead plants due to fungal disease. Plants with grades between 1 and 3 were considered resistant and the others susceptible (Table S2).

DNA extraction and determination of target alleles
Genomic DNA from 149 F 2 plants (BRS Cometa x SEL 1308) (Table S2) and from the set of 30 contrasting genotypes related to angular leaf spot reaction (

Genetic-statistical analysis
The phenotypic data and the genotypes of the anthracnose-associated markers in the F 2 generation (BRS Cometa × SEL 1308) were submitted to the chi-square test ( 2) to test the 3R-:1rr and 1RR:2Rr:1rr segregation hypotheses (R: resistant; rr: susceptible), respectively, adopting a 5% signi cance level. Linkage analysis between the marker locus and the Co-4 2 allele was performed using the OneMap package (Margarido et al. 2007) and the estimated recombination frequency converted into genetic distance (cM). All analyses were conducted using R software, version 4.1.0 (R Development Core Team 2021).
The selection e ciency (SE) for codominant markers was estimated according to the methodology described by Liu (1998), using the following estimator: where rf is the recombination frequency.

Molecular analysis with SNP markers
Eight of the nine SNPs that were selected out of the HTPG project data and genotyped at Intertek (88.8%) were polymorphic and were considered suitable for genotyping analysis. The snpPV0071 marker, associated with the Phg-2 locus (Nay et al. 2019a), was the only one that displayed a monomorphic pro le for the susceptible associated allele (G:G) and was discarded from the analysis. This marker is speci c to tag the resistance Phg-2 allele at G10474 line, not genotyped in this study. The snpPV0051 (Phg-1) marker is located in a region that contains repeats in the genome (identi ed via RepeatMasker). Of the seven markers that exhibit a techncally adequate genotyping pro le, snpPV0033 did not amplify the target allele in the sources carrying the Phg-2 allele.
The target allele was ampli ed by the snpPV0051 (A:G, where the resistance associated allele is the rst in bold) marker in only 103 (27.4%) of the 376 lines evaluated. This SNP is located in a repetitive region of the common bean genome, identi ed using the RepeatMasker tool (Table 3), which may explain why the fragment was not ampli ed well. Of the 103 genotyped lines, the "A" allele associated with Phg-1 was ampli ed in 11 lines (Table S4) (Table S4).     (Table S5) (Table S4) and the differential varieties of anthracnose pathotypes G2333, G2858 and PI 207262 (Co-u + Co-4 2 ) (Table S6). This demonstrates the need to obtain lines that simultaneously combine alleles that confer resistance to anthracnose and angular leaf spot, in addition to the other agronomic characteristics demanded by the market.

SNP markers associated with the Co-4 and Co-u loci
The Co-4 locus is located close to a telomeric region of the chromosome Pv-08, characterized by containing about 18 copies of the COK-4 gene and described as being associated with anthracnose resistance in the common bean (Oblessuc et al. 2015). In the present study, four SNP markers associated with the anthracnose-resistant locus were analyzed. In case of the markers snpPV0068 (G:C) and snpPV0070 (G:T), the "G" alleles (of both SNPs) were identi ed in the G-2333, SEL 1308, PI 207262, K-10, K-13, and CNFC 5547 lines (Table S6) (Table S6). Some of these lines, already characterized in terms of resistance/susceptibility, are part of the differential varieties of anthracnose pathotypes or originated in the anthracnose resistance breeding program (Table S6). The presence of "G" alleles in the two SNPs also coincided with the polymorphisms identi ed in the snpP8282v3-817 marker (GRAF1, A:G) (Cieslak et al. 2015) in detecting the Co-4 2 allele of the Co-4 locus in lines K-10, K-13 and CNFC 5547 (Table S6). However, there was no "A" allele ampli cation of the snpP8282v3-817 marker in the 22 black bean lines ampli ed by snpPV0070 (Table S6). This result indicates that snpPV0070 is not speci c for the Co-4 2 allele, but capable of detecting resistance at the Co-4 locus. An alternative to snpPV0070 are the markers snpPV0068 and snpP8282v3-817, both ampli ed in lines that are known to carry the Co-4 2 allele (Table S6) For snpPV0046 (A:G, Co-u locus), located in the exon of the Phvul.002G328300 gene, the "A" allele is associated with the Co-u resistant allele, which also confers resistance to anthracnose (Zuiderveen et al. 2016;Oblessuc et al. 2014;Geffroy et al. 2008). In this study, the "A" allele was identi ed in 51 of the 376 lines genetically characterized with snpPV0046, including the BAT 93 breeding line (Table S7), which contains the parental genotype PI 207262 (Geffroy et al. 2008) and is a source of the Co-u allele (Geffroy et al. 2008). In the present study, the "A" allele was ampli ed in PI 207262, suggesting that Co-u may come from this parent or from a mutation that occurred during the evolutionary process of BAT 93. Of the 51 lines that contain the "A" allele, 13 are from the black bean group, 4 from the Carioca group and the remainder belong to different commercial classes, such as brindle, jalo, white, calima (Table S7), which are detected in Andean and Mesoamerican gene pools. Of these, 12 are resistant cultivars that belong to differentiating varieties of the anthracnose pathotypes (Table S7). The Co-u allele is located in the Pv-02 linkage group, very close to I locus, conferring resistance to important common bean viruses, such as the bean common mosaic virus, potyviruses and comovirus (Meziadi et  In addition, the introgression of resistance alleles from different gene pools (Andean and Mesoamerican) in the same line through assisted selection by molecular markers is an important strategy for developing bean cultivars with broad and long-lasting resistance

Simple linear regression analysis of marker effects
Of the eight SNP markers analyzed, ve (snpPV0046, snpPV0068, snpPV0070, snpP8282v3-817 and snpPV0025) showed potential for indirect selection of common bean genotypes containing the Co-4 2 and Phg-2 alleles and were therefore submitted to simple linear regression analysis. Only for the marker snpPV0046 the regression model was signi cant (Table S8), but they do not explain much of the variability (low R-square of 3%). Although marker effects were not signi cant in the sample set of lines containing information regarding the reaction to anthracnose and angular leaf spot (snpPV0046 = 133; snpPV0068 = 134; snpPV0070 = 115; snpP8282v3-817 = 46 and snpPV0025 = 78), all lines carrying the target SNPs showed resistance, suggesting that these alleles can be monitored from these markers in the lines of interest. The absence of signi cance in the regression test is certainly due to the fact that many lines, even though resistant, did not exhibit the target alleles of this study, suggesting the presence of other anthracnose and angular leaf spotresistant alleles. When the regression analysis was applied in a subset of genotypes contrasting for the resistance/susceptibility to the anthracnosis and ALS diseases (Table S9), the Co-4 2 and Phg-2 marker effects were signi cant (Table 4) and the slope values were negative for all markers (Table 4), revealing an association between SNPs and resistance to anthracnose and ALS. The introgression of resistance alleles into elite bean germplasms assisted by molecular markers is a promising strategy in breeding programs, given that it reduces time and costs in the initial selection stages (Sakiyama et al. 2014). Fig. 1 illustrates the importance of resistance alleles Co-u ("A"), Co-4 2 ("G") and Phg-2 ("G") in reducing mean phenotypic values in the lines that contain them. The alleles in bold are associated with resistance; (1) Contrast considered in the regression analysis between marker locus alleles and the severity of anthracnose or angular leaf spot infection; (2) Angular coe cient of the linear regression equation. The negative sign on the slope indicates that the allele is associated with resistance.
Additionally, gene annotation revealed that snpPV0046 (Co-u), snpPV0068 (Co-4 2 ), snpPV0070 (Co-4 2 ) and snpP8282v3-817 (Co-4 2 ) are located in gene regions that encode defense proteins in plants ( Table 2). The snpPV0046 marker is found in the gene that encodes the Mitogen-Activated Protein Kinase (MAPK) protein, which interacts with salicylic acid, a plant hormone known to play a role in plantacquired resistance against pathogen infection (Jagodzik et al. 2018). The snpPV0068 marker is located in the MYB transcription factor (MYB Transcription Factor) coding region, playing an essential role in the control of cellular processes in response to biotic and abiotic stresses (Ambawat et al. 2013 (Table 5), segregating as expected for codominant markers. Linkage analysis revealed that the snpPV0070 and snpP8282v3-817 markers used in F 2 population genotyping (BRS Cometa x SEL 1308) are strongly linked to the Co-4 locus, with a recombination frequency of 0.026 (2.6 cM) and 0.019 (1.9 cM), respectively (Table 5). Markers snpPV0070 and snpP8282v3-817 showed selection e ciency (SE) of 99.7% and 99.8% respectively, indicating the high potential value of molecular markers in strong linkage disequilibrium in MAS programs. Total The alleles in bold are associated with resistance; rf Recombination fraction; SE Selection e ciency; 1 Distance in centiMorgans; P-value associated to the null hypothesis not rejected (1:2:1 for molecular markers data and 3:1 for phenotype data); snpPV0070: "G:G" dominant homozygous for the Co-4 2 allele, "G:T" heterozygous for the Co-4 2 allele and "T:T" recessive homozygous for the Co-4 2 allele; snpP8282v3-817: "A:A" dominant homozygous for the Co-4 2 allele, "A:G" heterozygous for the Co-4 2 allele and "G:G" recessive homozygous for the Co-4 2 allele.
The TaqMan® probe derived from snpPV0025 (G:T) associated with the Phg-2 allele was evaluated in a set of contrasting elite lines for resistance to angular leaf spot (Table S3). This target SNP was able to detect the Phg-2 locus, despite its ampli cation in varieties that are susceptible to angular leaf spot (Table S3). This is in accordance with previously reported that snpPV0025 would only effectively tag Phg-2 in Andean backgrounds. Additional SNP markers have been identi ed as associates with Phg-2 and should be tested (
The markers snpPV0025 and snpPV0070 associated with the Phg-2 and Co-4 loci, respectively, are indicated to monitor the presence of target alleles in crosses involving well-characterized parental lines, such as Mexico 54 (Phg-2) and SEL1308 (Co-4 2 ).
The genotyping systems based on hydrolysis probes developed in this study (TaqMan® SNP) for the snpPV0070, snpP8282v3-817 (Co-4 2 ) and snpPV0025 (Phg-2) markers enabled the speci c ampli cation of target alleles and are therefore suitable for use in MAS activities for the common bean.
The snpPV0070 and snpP8282v3-817 markers showed high selection e ciency (99.7 and 99.8%, respectively) for the allele that confers anthracnose resistance located in the Co-4 locus and may considerably improve e ciency in identifying superior genotypes in the common bean breeding program for resistance to anthracnose and angular leaf spot. This paper is a cooperative contribution on validation of SNP markers linked to disease resistance genes for their effective use in the routine of Embrapa (Brazilian Agricultural Research Corporation) common bean breeding program. The main goal was to characterize and select elite parent lines according the presence of disease resistance alleles, as well as validate SNP markers for MAS of superior genotypes from populations derived from these parent lines. It reports an elegant example of cross validation and effective use of SNP markers for MAS in an applied breeding program of a very social important orphan crop (common bean or dry bean).

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Relevant data are included in this paper and its associated Online Resources.
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