Revista Mexicana Ciencias Agrícolas   volume 10   number 7   September 28 - November 11, 2019

DOI: https://doi.org/10.29312/remexca.v10i7.1956

Article

Detection of genetic markers associated with resistance to pathogens
in ayocote beans from Puebla, Mexico

Régulo Ruíz-Salazar1, 2

María Luisa Patricia Vargas-Vázquez3

Sanjuana Hernández-Delgado1

José Socorro Muruaga-Martínez3

Netzahualcóyotl Mayek-Pérez1, 4§

1Center for Genomic Biotechnology-IPN. Boulevard del Maestro Esq. Elias Piña s/n, Col. Narciso Mendoza, Reynosa, Tamaulipas, Mexico. CP. 88710. (sanjuana70@hotmail.com). 2Reynosa-Aztlán-UAT Multidisciplinary Academic Unit. Calle 16 and Lago de Chapala s/n, Reynosa, Tamaulipas. CP. 88740. (regulo.ruiz@uat.edu.mx). 3Experimental Field Valley of Mexico-INIFAP. Los Reyes-Texcoco road km 13.5. Coatlinchán, Texcoco, Mexico. AP. 307. CP. 56250. (patricia-vargas-mx@yahoo.com; ymuruaga@hotmail.com). 4Mexico University North American AC. First s/n, Col. El Círculo, Reynosa, Tamaulipas, Mexico. CP. 88640.

§Corresponding author: nmayeklp@yahoo.com.mx.

Abstract

Ayocote beans (Phaseolus coccineus L.); 2n= 2x= 22) is a legume native to the highlands of Mexico, it is important in human nutrition and detection of improvement characteristics, it shows resistance to pathogens and certain abiotic factors that cause losses in the bean crop. With the premise of identifying Phaseolus germplasm with multiple resistance to biotic and abiotic adverse factors, this work was carried out with the objective of this work was to determine the resistance or susceptibility to two important pathogens: common blight [Xanthomonas axonopodis pv. phaseoli (Smith)] and anthracnose [Colletotrichum lindemutianum Sacc. & Magn (anamorph Glomerella lindemuthiana Shear)] based on the identification of sequences characterized by amplified regions (SCARs). The study included 117 accessions collected at different sites in Puebla, Mexico (region called Carso Huasteco), which were analyzed with 10 SCAR markers (six markers for common blight: SAP6, BAC6, SU91, LG5, R7313, R4865, four for anthracnose: SAS13, SBB14, SAB3, SH18) at the Center for Genomic Biotechnology in Reynosa, Tamaulipas. The SAS13 and SBB14 anthracnose resistance markers are present more frequently (89 and 74%), followed by the common blight resistance markers BAC6 and SU91 (74 and 42%). The germplasm of Zacapoaxtla and Tlatlauquitepec had a higher frequency of SCARs (Zacapoaxtla: 90% and 100% for SAS13 and SBB14; Tlatlauquitepec: 94% and 56%). Accessions with five SCARs could be used as a source of resistance to diseases in Phaseolus and early and late genotypes to flowering and physiological maturity, as well as variables in testa color, pod size and seed, but no association was detected between the presence of SCARs with the morphology of ayocote beans.

Keywords: Colletotrichum lindemuthianum, Phaseolus pathogens and Xanthomonas axonopodis pv. Phaseoli, Carso Huasteco.

Reception date: August 2019

Acceptance date: October 2019

Introduction

Ayocote beans (Phaseolus coccineus L.) in Mexico are mainly used as food and even in regions of Spain, the Netherlands and the United Kingdom, their consumption has replaced beans (P. vulgaris L.) (Rodiño et al., 2006). Of the domesticated species of the genus Phaseolus spp., the ayocote bean is the second species in economic importance (Al Hassan et al., 2016). Traditional ayocote bean cultivation occurs on a small scale (Schwember et al., 2017). In Central America, this bean is grown almost exclusively in association with corn (Zea mays L.) (Vargas-Vázquez et al., 2007).

Since beans are an important element in the self-supply of rural populations, the variation in forms of cultivation, use and forms of preparation for consumption is wide. Common blight is a disease with wide distribution in Mexico due to monoculture and the continuous exchange and reuse of grain as a seed, factors that increase the accumulation of the primary inoculum and the dispersion of the causative bacteria (Acosta-Gallegos et al., 2013). For its part, anthracnose also shows wide distribution in our country and can be a devastating disease if susceptible varieties are planted and, in addition, favorable climatic conditions occur for their development (Rodríguez-Guerra et al., 2006).

The ayocote bean has outstanding characteristics that can be exploited in programs of genetic improvement of the common bean such as: vigorous radical system, numerous floral knots, long peduncles, also stands out for its resistance to viral diseases such as the common mosaic virus and the virus of the golden mosaic; bacterial diseases such as halo blight (Pseudomonas syringae Van Mall) and common blight (Xanthomonas axonopodis pv phaseoli Smith.) (Duncan et al., 2006; Ruiz-Salazar et al., 2016), as well as fungal diseases such as anthracnose [Colletotrichum lindemuthianum (Sacc. & Magn) (teleomorph Glomerella lindemuthiana Shear)] (Ruiz-Salazar et al., 2016), presents resistance to abiotic factors such as low temperature and salinity (Rodiño et al., 2006; Al Hassan et al. 2016).

The use of simple and/or multiple genetic resistance to diseases of economic importance in beans is supported by the constant search for new sources of resistance, their incorporation into cross-breeding, evaluation and selection programs of segregating germplasm in field conditions and controlled; and obtaining germplasm with resistance to these adverse factors, which also shows high grain yields and with stability in the yield and quality of the superior grain (Acosta-Gallegos et al., 2007; Anaya-López et al., 2015a).

The genetic improvement identifies and selects genotypes based on phenotypes and the subsequent introgression of the desired characters for the development of superior germplasm. Thus, virtually all important agronomic features have been improved: grain yield and quality; phenology; resistance to diseases, pests, drought or salinity. The process can last for several years depending on the genetics of the trait and the response to the selection. Molecular marker assisted selection (SAMM) could reduce process time and costs (Bernardo, 2008; Collard and Mackill, 2008; Xu and Crouch, 2008).

SAMM quickly identifies germplasm that may contribute to the improvement of cultivated species of Phaseolus spp. (Svetleva et al., 2003). Unlike countries such as the United States of America, where SAMM supports and accelerates the incorporation of genes in the germplasm of interest (Miklas et al., 2006), in Mexico, only one variety released under this scheme is reported (‘Dalia’) (Acosta-Gallegos et al., 2014), with resistance to BCMV and BMCNV (common bean and black root mosaic virus or necrosis). Duncan et al. (2012) indicated; however, using a single SCAR is not enough to provide sufficient resistance to a pathogen, suggesting two or more SCARs under a scheme of resistance gene pyramidation (Anaya-López et al., 2015a, b).

The objective of this investigation was to determine the resistance or susceptibility to two important Phaseolus pathogens: common blight [Xanthomonas axonopodis pv. phaseoli (Smith)] and anthracnose [Colletotrichum lindemutianum Sacc. & Magn (anamorph Glomerella lindemuthiana Shear)] based on the identification of sequences characterized by amplified regions (SCARs).

Materials and methods

Extraction of DNA

The present work was carried out in the Plant Biotechnology Laboratory of the Center for Genomic Biotechnology of the National Polytechnic Institute (CBG-IPN) in Reynosa, Tamaulipas where 117 accessions of P. coccineus L. collected in the Carso Huasteco of Puebla subprovince were analyzed, more five accessions used as control group (Table 1), DNA extraction was carried out according to the protocol Doyle and Doyle (1987).

Table 1. Origin and number of samples of the populations of Phaseolus spp. studied.

SCAR marker amplification

From the sequences reported by the bean improvement program (BIC, 2010), ten SCAR markers were selected, of which six detect gene sequences that confer resistance to common blight (SAP6, BAC6, SU91, LG5, R7313 and R4865), and four to anthracnose (SAS13, SBB14, SAB3 and SH18) in beans (Table 2). The amplifications were carried out using a GeneAmp PCR System 9700 thermocycler (Applied Biosystems®) according to the indications cited for each of the SCAR markers (BIC, 2010).

Table 2. Information on SCAR markers evaluated in ayocote beans.

Δpb= base pairs of the expected product; GL= linkage group.

Visualization and statistical analysis

The products of each amplification were stained with 1 µL of SYBR Gold® and separated on 1% agarose gel, at 80 V and 50 mA. Each gel was then visualized with UV light and photo-documented (KODAK Digital Science 1D; Rochester, NY, USA). The revision of gels allowed to construct a binary matrix where 1= presence and 0= absence of band (amplicon) and that included all the amplified SCARs in each genotype. The data analysis was carried out with the Statistica program (StatSoft ™) version 7 (StatSoft Inc. 2004) and basically consisted of the calculation of frequencies, percentages and averages of occurrence of SCAR markers detected by location and type of marker generated.

Results and discussion

In this study, the SCARs with the highest amplification frequency were SAS13 and SBB14 with 89 and 74%, respectively, in the total accessions evaluated. The germplasm of Zacapoaxtla and Tlatlauquitepec, presented the detection frequencies of SCARs of resistance to anthracnose (34 of 38 accessions in Zacapoaxtla and 17 of 18 in Tlatlauquitepec for SAS13; 30 of 38 and 10 of 18 for SBB14), which suggests that these Accessions are candidates for use as parents in the genetic improvement of Phaseolus.

To date there were no reports of the identification of anthracnose resistant germplasm in P. coccineus L. by detecting SCARs such as SAS13 and SBB14, since these have only been used in P. vulgaris, but if SCARs of common blight resistance such as SAP6, BAC6 or SU91 (Ruiz-Salazar et al. 2016). Of the ten SCAR genomic sequences tested, only seven amplified PCR products in the ayocote bean germplasm evaluated.

The SCARs SAB3, SH18 and R7313 did not amplify in any accession. The SAS13 and SBB14 sequences were followed, in terms of higher amplification frequency by BAC6 (74%) and SU91 (42%), SCARs related to common blight resistance (Table 3). The locations with the highest number of accessions with SCARs for common blight correspond to Zacapoaxtla and Tlatlauquitepec (25 of 38 accessions and 11 of 18 accessions for BAC6; for SU91 17 of 38 accessions and 6 of 18 accessions, respectively) (Table 3). The highest proportion of SCAR markers in Zacapoaxtla and Tlatlauquitepec germplasm coincides with the case of common blight, coupled with the identification of SCARs associated with resistance to virosis (common mosaic virus and golden mosaic virus) and angular spot (Phaeoisariopsis griseola) (Ruiz-Salazar et al. 2016).

Table 3. SCAR amplification frequencies in ayocote beans from 16 locations in Puebla, Mexico.

n= number of accessions by location; = average SCAR amplified by location.

Several accessions were detected that have at least five resistance SCARs, which highlights the importance of these accessions for genetic improvement in common beans. This germplasm is more likely to be used as a source of multiple resistance to diseases in Phaseolus (Rodríguez-Guerra et al., 2006).

In spite of the high detection frequencies of SCARs in ayocotes of specific locations of the Carso Huasteco de Puebla, it should be considered that in addition to the ratification of the effective resistance in field conditions, said resistance may sometimes behave unstable, due to the high specificity of the interaction between the genotypes of the pathogen and the host (Rodríguez-Miranda and Rosas-Sotomayor, 2010).

However, it is notable to identify germplasm that has a high frequency of SCARs associated with resistance to two important diseases in central Mexico, which suggests that high selection pressures occur due to favorable environmental conditions for the higher incidence of diseases and inoculum. primary and greater pathogenicity (Francisco-Francisco et al., 2013; Rodríguez-Guerra et al., 2006) have forced farmers in the region to empirically select for disease resistance and, consequently, have achieved an increase in the frequency of resistance genes in them.

Mexico is considered the center of origin and diversity of beans and also the pathogens that cause its diseases exhibit wide pathogenic diversity, such as anthracnose (Rodríguez-Guerra et al., 2006) or common blight (Prudencio-Sains et al., 2008), unfortunately, in the case of common blight only the existence of such variability is assumed, but there is no updated information on its quantity and distribution in the beans regions of Mexico (Navarrete and Acosta, 2000; Prudencio-Sains et al., 2008).

In the case of anthracnose there is a more complete perspective, since more than 100 patotypes are recorded in the world, with at least 54 presents in Mexico (Rodríguez-Guerra et al., 2006). The wide genetic variability of the causative agents of both diseases hinders the development of resistant varieties. Also, it is common to observe that varieties with resistance in one region are susceptible in another (Acosta-Gallegos et al., 2013).

Sources of genetic resistance to common blight have been identified in P. vulgaris, P. acutifolius and P. coccineus. However, most resistance genes are inherited as quantitative character loci (QTL) and therefore exhibit variation in their genetic effects because they are influenced by the environment (Kelly et al., 2003; Miklas et al., 2006; Duncan et al., 2007). For anthracnose, a group of 21 genotypes with diverse characteristics were identified in terms of their genetic base, grain color, growth habit and response to different anthracnose patotypes in Mexico (González-Chavira et al., 2004).

Among them, Pinto Villa and Bayo Mecentral 90 stand out, commercially released with resistance to anthracnose and which have been cultivated in different regions and conditions in Mexico (Rodríguez-Guerra et al., 2006). Most studies have focused on the identification of molecular markers linked to genes with major (monogenic) effects in anthracnose (Miklas, 2002). For example, SCAR SAS 13 for anthracnose is linked to the Co-42 resistance gene that is located in linkage group 8, while SU91 is a QTL genomic region associated with common blight resistance and is also located in the group of linkage 8.

Then both markers could be more effective in improving by being more likely to co-segregate together in segregating bean populations. Emmalea and Kelly (2004) detected resistance to 33 of 34 C. lindemuthianum patotypes from nine countries in the Americas with the use of SCAR SAS13, because Co-42 was first detected in the differential variety Tu and also, in G-2333 The latter, also called 'Colorado de Teopisca' is originally from Chiapas, Mexico, was resistant to 380 anthracnose isolates from eleven Latin American countries because it has the Co-42, Co-5 and Co-7 genes (Pastor-Corrales et al., 1994; Vallejo and Kelly, 2009).

The SAP6, SU91 and LG5 SCARs, despite being located in different linkage groups, can be applied in the search for resistance to common blight. Ibarra-Perez and Kelly (2005) associated SU91 with resistance in the field, demonstrating the advantages that SAMM could provide to detect resistance to diseases in Phaseolus. SAMM, in the development of improved germplasm with disease resistance, has been successful because it allows indirect selection of desirable characteristics compared to conventional techniques.

Park and Yu (2004) identified SCAR SAS13 (linked to the Co-42, 0.39 cM gene) in the H4514 Navy, H4628 Dark Red Kidney, H4642 Navy and H4836 Dark Red Kidney bean lines, obtaining an increase in anthracnose resistance 43% (H4642 Navy), compared to H4628 (29%), H4514 (7%) and H4836 (7%), which were susceptible. Mukeshimana and Kelly (2003) studied Rwanda common bean germplasm and only detected SCAR SH18 on the RWV167 line.

Meanwhile, Miklas (2002) recorded low levels of resistance to common blight in ayocote beans, but high levels of resistance in tepary beans (P. acutifolius A. Gray) with SCAR R7313, which did not amplify in the germplasm of this job. According to Márquez et al. (2007), said SCAR has only been detected in the XAN159 line and in Tepary beans. Given the economic importance of common blight, lines that have broad spectra of resistance to said pathogen should be developed in the short term.

Accessions with up to five or six SCAR sequences include early and late accessions to flowering and maturity, as well as various testa colors, pod and seed size, so that no relationship was found between increased presence of SCARs with any particular morphological characteristic of the ayocote bean germplasm of Carso Huasteco (Prudencio-Sains et al., 2008; Ruiz-Salazar et al., 2016). Bitocchi et al. (2017) suggest that domestication acts on increasing functional diversity in particular loci, which probably control traits related to expansion and adaptation to new agroecological conditions (Table 4).

Table 4. Characteristics of ayocote bean accessions with five or six genomic sequences that confer resistance to anthracnose and common blight.

In the medium and long term, there are several options for the genetic improvement of beans in Mexico, assisted by Biotechnology. In principle, the strategy of handling two or more SCARs must be maintained at the same time or, better yet, emphasize resistance gene pyramid strategies (Anaya-López et al., 2015a, b). Also, it should not be overlooked that the efficiency of SAMM can be affected by the degree of dominance of the marker, the type of linkage between molecular markers and traits of interest, the need to develop progeny tests to corroborate resistance and changes in specificity of markers (commercial types of grain and/or genetic collections, for example) (Bello et al., 2014).

Finally, biotechnology progresses day by day, new molecular marker strategies are developed, which in the case of Mexico must be evaluated and validated. It is work with new generation sequencing strategies that take advantage of different types of polymorphisms, the combination of segregating analysis and their transcriptional profiles, the analysis of ‘in silico’ segregators and the structuring of high performance platforms for integral genomic analysis (BeanCAP , Langebio) (Bello et al., 2014; Bolger et al., 2014; Mukeshimana et al., 2014; Viteri et al., 2014; Saburido-Álvarez and Herrera-Estrella, 2015).

Conclusions

The ayocote bean germplasm of Carso Huasteco de Puebla analyzed with SCAR type markers presented regions associated with genes of resistance to common blight (X. axonopodis pv. phaseoli) and anthracnose (C. lindemuthianum), where the accessions of Zacapoaxtla and Tlatlauquitepec are those with the highest frequency of these markers.

Acknowledgments

R. Ruiz-Salazar (CVU: 229696) thanks the National Council of Science and Technology, the Government of Tamaulipas, the Institutional Program for Training of Researchers of the IPN and the ECOES-Santander Program for financing their doctoral studies at the CBG-IPN. Similarly, he thanks Dr. Guillermo Castañón-Najera of the Autonomous Juárez University of Tabasco for the support provided. This work was funded by the FOMIX-Government of the state of Veracruz (project 94070), CONACYT-Basic Science (181756) and IPN (project 1636).

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