Molecular evaluation of pumpkin and zucchini genotypes using molecular markers associated with diseasesABSTRACT
This study evaluated disease resistance in Cucurbita spp. using specific molecular markers. The authors evaluated 47 accessions of C. moschata and C. pepo in order to identify regions which confer resistance to Zucchini Yellow Mosaic Virus (ZYMV), Papaya Ringspot Virus Watermelon (PRSV-W), and Powdery Mildew (Erysiphe spp.). Molecular markers were amplified and analyzed according to their references. The presence of the resistance allele to ZYMV was observed in 82.6% of the C. pepo genotypes and 95.8% of the C. moschata genotypes, using SCAR and CAPS markers. In relation to resistance to PRSV-W, 62.5% of the C. pepo genotypes showed the corresponding allele. Regarding powdery mildew, 47.8% of the C. pepo genotypes were classified as resistant, whereas 62.5% of the C. moschata genotypes presented the resistance allele, based on In-Del and CAPS markers. The variations in resistance rates observed between the markers suggested a polygenic genetic control for these diseases. The results found in this study highlighted the importance of an integrative approach to marker selection for a comprehensive analysis of genetic resistance, which is essential for developing more resistant and efficient cultivars and hybrids.
Keywords:
Cucurbita spp.; molecular selection; resistance; polymorphism
RESUMO
Este estudo investigou a resistência a doenças em Cucurbita spp. por meio de marcadores moleculares específicos. Foram analisados 47 genótipos, reunindo as espécies Cucurbita moschata e C. pepo, para identificar regiões de resistência ao Zucchini Yellow Mosaic Virus (ZYMV), ao Papaya Ringspot Virus Watermelon (PRSV-W) e ao oídio (Erysiphe spp.). Os marcadores moleculares foram amplificados e analisados de acordo com suas referências. A presença do alelo de resistência ao ZYMV foi detectada em 82,6% dos genótipos de C. pepo e em 95,8% dos genótipos de C. moschata, utilizando marcadores SCAR e CAPS. Em relação à resistência ao PRSV-W, 62,5% dos genótipos de C. pepo apresentaram o alelo correspondente. Para o oídio, 47,8% dos genótipos de C. pepo foram classificados como resistentes, enquanto 62,5% de C. moschata mostraram a presença do alelo de resistência, com base em marcadores In-del e CAPS. As variações nas taxas de resistência observadas entre os marcadores sugerem um controle genético poligênico para essas doenças. Esses resultados enfatizam a importância de uma abordagem integrativa na seleção de marcadores para uma análise abrangente da resistência genética, o que é essencial para o desenvolvimento de cultivares e híbridos mais resistentes e eficientes dessas espécies.
Palavras-chave:
Cucurbita spp.; seleção molecular; resistência; polimorfismo.
Cucurbitaceae family consists of numerous grown species of great economic importance, mainly due to its fruits, like pumpkin and zucchini (Cucurbita spp.) (Hernandez et al., 2023). Nowadays, the genus Cucurbita has global economic relevance, China is the largest worldwide producer, reaching average annual production of 30 million tons (Kesh & Yadav, 2022). Considering the genus, three global economic important species are widely cultivated, such as C. maxima, C. moschata, and C. pepo (Chomicki et al., 2020; Kaźmińska et al., 2017; Ferriol & Picó, 2008).
Pumpkin breeding program is essential for modern agriculture, aiming to increase the productivity, quality and resistance of the crops to biotic and abiotic stresses. Species like C. pepo, C. maxima and C. moschata are widely grown and show great importance both for human and animal feeding, as it also has a significant impact on the global agriculture economy (Lebeda et al., 2024). However, pathogens like Zucchini Yellow Mosaic Virus (ZYMV), Papaya Ringspot Virus Watermelon (PRSV-W) and powdery mildew (Erysiphe spp.) threaten pumpkin and zucchini crops in most Brazilian regions (Brandão Filho et al., 2018). Genus Cucurbita presents about 27 species, considering that C. moschata and C. pepo stand out in Brazil (Amaro et al., 2021). These are diploid species, showing 20 pairs of chromosomes (2n=2x=40), the genus, as a whole, is of tetraploid origins, though (Amaro et al., 2021; Lira-Saade, 1995). The genetic diversity of these species is remarkable, reflecting variations concerning adaptation to different environments, phenological cycles, growth habits, morphological traits, nutritional properties and levels of disease resistance (Zhang et al., 2021). The use of genome markers is one strategy for searching genetic resistance (Lebeda et al., 2024; Capuozzo et al., 2017).
The use of molecular markers, such as SCAR, CAPS, SNP and In-del, has been revolutionizing Cucurbita spp. Breeding program (Sabharwal et al., 2024; Ma et al., 2022). These markers can accurately identify the genes associated with resistance to diseases, nutritional quality and other agronomic traits. The use of these markers accelerates the development of cultivars better adapted to varied environmental conditions and more resistant to diseases like ZYMV, PRSV-W and powdery mildew. In addition to improving the efficiency of genetic breeding, these advances contribute to the sustainability of agricultural production and global food security (Yoo et al., 2023).
Some studies have reported the use of marker-assisted selection of genotypes resistant to ZYMV (Kim et al., 2016; Capuozzo et al., 2017; Sawazaki et al., 2018; Shrestha et al., 2021; Amoroso et al., 2022), PRSV-W (Sawazaki et al., 2018; Martin-Hernandez & Picó, 2021; Shrestha et al., 2022) and powdery mildew (Holdsworth et al., 2016; Park et al., 2020; Wang et al., 2021b; Alavilli et al., 2022). The Cucurbita spp. breeding program is able to establish strategies to select and use several sets of molecular markers aimed at selecting resistant genotypes for more than one disease. Thus, the aim of this study was to evaluate the molecular polymorphism in tropical C. pepo and C. moschata genotypes, which means, adapted to the Brazilian weather conditions, by the use of molecular markers associated with resistance to ZYMV, PRSV-W and powdery mildew.
Genotypes of pumpkin and zucchini tested for molecular markers associated with diseases. Araras-SP, UFSCar, 2024.
MATERIAL AND METHODS
Plant material and DNA extraction
In this study, the authors used 47 Cucurbita spp. genotypes, being 24 C. moschata and 23 C. pepo (Table 1). All the genotypes are from a germplasm bank (Banco de Germoplasma de Hortaliças da Universidade Federal de São Carlos (UFSCar)), located at Centro de Ciências Agrárias (CCA UFSCar) in Araras-SP (22º18'S, 47º23'W, 687 m altitude). The seedlings were produced in 128-cell trays filled with Carolina Soil substrate for 15 days. Then, they were transplanted into 0.9-L pots and kept in a greenhouse for about 30 days. Approximately, 200 mg of frozen leaf tissue (-80°C) were macerated in a mortar, and the total DNA was extracted according to Al-Janabi & Martinez (1997). The samples were quantified using a spectrophotometer NanoDrop®One (Thermo Scientific), and, afterwards, the samples were stored at -20°C.
Amplification of disease-associated markers
To evaluate genomic region associated with ZYMV resistance in C. pepo, we used the SCAR markers proposed by Capuozzo et al. (2017) and Sawazaki et al. (2018) and, to evaluate C. moschata genotypes, we used CAPS marker proposed by Kim et al. (2016). To evaluate the resistance to PRSV-W, only in the accessions of C. pepo, the authors used the SCAR marker proposed by Sawazaki et al. (2018). The presence of the region associated with the resistance to powdery mildew was evaluated using In-del and CAPS markers proposed by Wang et al. (2021a) in C. pepo, and also a CAPS marker for C. pepo and C. moschata, proposed by Holdsworth et al. (2016).
The amplification conditions of VirSq-F19 and SCAR SC522945 markers, associated with ZYMV, followed the methodology described by Sawazaki et al. (2018). The reaction consisted of 4 μL of genomic DNA (50 ng/μL), 1.5 μL of 10X buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 1.5 μL of MgCl2 (25 mM), 1.2 μL of dNTPs (2.5 mM), Taq DNA polymerase (5u/μL) and 1.5 μL of each primer, to a final volume of 15 μL. Amplifications were carried out sing a Thermo Fisher Scientific SimpliAmp™ thermal cycler. The amplification schedule was 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 54°C for 1 min, 72°C for 1 min and a final extension cycle of 72°C for 5 min. For 408PRSV/552PRSV marker, related to PRSV-W, the amplification condition was identical to VirSq-F19 and SCAR SC522945 markers, except for the annealing temperature, which was 44°C. The amplified products were analyzed in a 2% agarose gel using a 100bp and 1kb DNA ladder (Invitrogen). The gel was stained with 1% ethidium bromide, and the amplified fragments were visualized using ultraviolet light.
For SNP1/CAPS marker associated with ZYMV developed by Capuozzo et al. (2017), the amplification condition was also carried out as proposed by Sawazaki et al. (2018). The amplification products were submitted to digestion with the endonuclease BglII as follows: 1.5 mL of the amplified product, 2 mL of Cutsmart buffer and 0.5 mL of BglIII enzyme in a final volume of 20 uL and incubated at 37°C for 2 hours. The digestion product was analyzed in a 1.5% agarose gel, using a 100bp DNA ladder (Invitrogen), stained with 1% ethidium bromide and visualized using ultraviolet light.
The amplification of M2467704 marker, associated with powdery mildew, was carried out according to the proposed by Wang et al. (2021b): 4 μL of genomic DNA (50 ng/μL), 1.5 μL of 10X buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 1.5 μL of MgCl2 (25 mM), 1.2 μL of dNTPs (2.5 mM), Taq DNA polymerase (5u/μL) and 1.5 μL of each primer, totaling a final volume of 15 μL. The Thermo Fisher Scientific SimpliAmp™ thermal cycler was used with the following program: 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and a final extension cycle at 72°C for 7 min. The amplified products were submitted to digestion with the restriction enzyme HindIII at 37°C for 2 h. Afterwards, digestion products were revealed using 2% agarose gel, stained with 1% ethidium bromide, and visualized using ultraviolet light. The 1kb and 100 bp ladders were used as molecular weight markers.
For In-del M2454200 and M2475244 markers, also associated with powdery mildew and proposed by Wang et al. (2021b), the amplification reaction was carried out to a final volume of 15 uL, composed of 3 uL of genomic DNA (50 ng/uL), 1X buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 2 mM MgCl2, 0.2 mM dNTPs, Taq DNA polymerase (5u/μL) and 0.26 uM of each primer. The thermocycler program was 95°C for 5 min, followed by 30 cycles of 95°C for 40 s, 60°C for 30 s and 72°C for 45 s, with a final extension at 72°C for 6 min. The amplified products were analyzed in a 6% silver nitrate-stained polyacrylamide gel, according to Creste et al. (2001). The authors used 50 bp and 10 bp DNA ladders (Invitrogen).
CAPS NBS_S9_1495924 marker associated with powdery mildew and developed by Holdsworth et al. (2016) was amplified as the following: 4 μL of genomic DNA (50 ng/μL), 1.5 μL of 10X buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 1.5 μL of MgCl2 (25 mM), 1.2 μL of dNTPs (2.5 mM), Taq DNA polymerase (5u/μL) and 1.5 μL of each primer, totaling a final volume of 15 μL. The Thermo Fisher Scientific SimpliAmp™ thermal cycler was used according to the following program: 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, with a final extension cycle at 72°C for 7 min. Digestion of the amplified products was carried out with the restriction enzyme HaeIII at 37°C for 2 hours. The digested fragments were subjected to 2% agarose gel electrophoresis, followed by staining with 1% ethidium bromide and visualization using ultraviolet light. A 1Kb ladder (Invitrogen) was used as a reference for fragment size.
RESULTS AND DISCUSSION
The introgression of resistance against pathogens in Cucurbita spp. plants is a process that requires knowledge of genetic inheritance and complexity, often a time-consuming and costly process. Therefore, the most efficient approach is one that combines phenotypic selection and marker-assisted selection (Capuozzo et al., 2017). The use of molecular marker technology for association with resistance genes has been more suitable, fast and economically sustainable (Shrestha et al., 2021; Wang et al., 2021b; Verma et al., 2023). However, it depends on different levels of expression related to the allelic constitution (Ott et al., 2015).
In this study, molecular analyses of genomic regions associated with resistance to ZYMV in C. pepo were performed using SCAR SC522945 (Sawazaki et al., 2018) and SNP1/CAPS markers (Capuozzo et al., 2017). The evaluation of polymorphism using SCAR SC522945 marker revealed that of the 23 C. pepo genotypes, 82.6% presented the marker fragment. The results obtained using the SNP1/CAPS marker showed that 100% of C. pepo genotypes presented the allele of genetic resistance to the disease (Table 1). SCAR SC522945 marker is of the SCAR type, therefore with dominant inheritance, which was obtained from the study that performed a crossing between Px7051 (resistant) and La Belle (susceptible). Thus, SNP1/CAPS marker was developed through the conversion of a SNP (SNP1), identified from the genotyping of a progeny from the crossing of access 381 (resistant) and True French (susceptible). So, the marker SNP1/CAPS allowed the distinction, in the population sample, of homozygous resistant, homozygous susceptible and heterozygous genotypes, due to co-dominant inheritance. In the genomic region containing SNP1, CNL proteins (coiledcoil, leucine-richrepeat nucleotide binding sites) and ATP-dependent RNA helicase were identified. Co-segregation with Zym-1 gene, which is essential for resistance to ZYMV, was also observed in this region (Capuozzo et al., 2017). The results obtained in this study corroborate the results found by Amoroso et al. (2022), who used the same marker to evaluate the resistance to ZYMV in C. pepo population, showing correlation between phenotypic and molecular results of about 82%.
The high frequency of resistance alleles in C. pepo can be explained by the classical approach adopted by breeding programs for the development of new zucchini cultivars, considering that most commercial cultivars are hybrids, obtained from crossing between pure lines. The selection of parents for producing zucchini hybrids can be determined by several factors, including resistance and productivity. Therefore, these alleles are more likely to occur at higher frequencies among the offspring of these specific parents (Andolfo et al., 2021). Nevertheless, the possibility of them being present, although less frequently, in other genotypes cannot be ruled out (Amoroso et al., 2022).
Furthermore, the use of these markers, SCAR SC522945 and SNP1/CAPS, to detect polymorphic regions related to resistance through a simple PCR, stands out, providing a new approach in the management of varietal resistance, aiming at a more sustainable production aligned with the consumer preferences and for the benefit of the environment. Given the presence of these markers in most C. pepo genotypes and the consistency with the information described in the literature on the genetic purity of this species in its crossings, these markers can be used as valuable tools in selecting resistant genotypes (Kesh & Yadav, 2022).
Linkage disequilibrium between the genetic marker alleles and the respective resistance genes identified in the studies carried out by Sawazaki et al. (2018) and Capuozzo et al. (2017) showed a consistency in phenotype prediction, considering 82% of the marker data had corresponding results.
Although the SCAR SC522945 and SNP1/CAPS markers are associated with ZYMV resistance genes, the authors highlight that the genetic correlation between them is not necessarily a causal relationship. Further studies are necessary to validate and better understand the functional relationship between these markers and resistance to ZYMV. The markers, SCAR SC522945 and SNP1/CAPS, are located in different regions of the genome, however the similarity in the identification of resistant genotypes suggests that genotypes identified as resistant by one marker tend to be consistently identified as resistant by another marker, which strengthens confidence in the conclusions about genetic resistance in the C. pepo population (Kaur et al., 2022).
In this study, genetic resistance to ZYMV was also evaluated in C. moschata genotypes using VirSq-F19 marker. The results showed that 95.8% of C. moschata individuals showed the allele associated with the resistance to the disease. VirSq-F19 marker was developed using a segregating Korean population (SP213 x SP203) by the conversion of RAPD marker to SCAR, showing dominant inheritance as described by Kim et al. (2016).
Resistant genes found in C. moschata are extremely important for interspecific crossing between C. maxima and C. moschata, aiming at the production of Tetsukabuto hybrid. The source of disease resistance in these hybrids comes from C. moschata (Kaur et al., 2022). The resistant genotypes identified by the dominant marker of Kim et al. (2016) are promising candidates for this crossing.
In this study, molecular evaluation of resistance to PRSV-W was performed using primers 408 PRSV/552 PRSV. The results showed that 62.5% of C. pepo individuals presented a resistant allele (Table 1). The 408 PRSV/552 PRSV marker developed by Sawazaki et al. (2018) was evaluated in Px7051 and La Belle genotypes, and also in 110 samples of zucchini provided by HortecSeeds. These authors also proposed one molecular marker associated with resistance to ZYMV (SCAR SC522945). When evaluating, at the same time, PRSV-W and ZYMV (SCAR SC522945) markers, we verified that, of the genotypes resistant to ZYMV (SCAR SC522945), 68.4% were also resistant to PRSV-W.
The relationship between molecular resistance to ZYMV and PRSV-W showed that the molecular resistance to a disease is not automatically related to resistance to another disease. This indicates a moderate correlation between resistance to the two diseases in this species (Sawazaki et al., 2018). These results highlighted the complexity involved in resistance to different diseases and the need for specific strategies for management and genetic improvement in relation to each pathogen. Furthermore, resistance to the potyviruses ZYMV and PRSV has been developed from the C. moschata 'Nigerian Local' and 'Menina' lines, and the exploration of new genetic backgrounds, such as in C. pepo, can expand strategies searching for new alleles for resistance to these viruses (Grumet et al., 2021).
Ultimately, the convergence of these markers suggests an integrated and comprehensive approach to genetic analysis in pumpkins, considering that different sets of markers can be used in combination to provide more complete information on genetic diversity, cultivar purity, and the potential for developing new resistant and productive lines (Martín-Hernandez & Picó, 2021).
Resistance to powdery mildew was evaluated using specific molecular markers. The NBS_S9_1495924 marker proposed by Holdsworth et al. (2016) was used to evaluate the genetic resistance to powdery mildew. For C. moschata, approximately 62.5% of the population was identified as genetically resistant to powdery mildew, whereas in C. pepo resistance was observed in approximately 47.8% of individuals. The region of chromosome 9 where marker NBS_S9_1495924 is located revealed that both C. moschata and C. pepo share similar alleles that confer resistance to powdery mildew (Holdsworth et al., 2016). This pattern can be explained by the horizontal nature of powdery mildew resistance, which is influenced by multiple genes and genetic interactions that result in varying susceptibility or resistance phenotypes. The balanced distribution between resistant and susceptible species is crucial for genetic variability and continued adaptation to environmental changes and selective pressures (Kaur et al., 2023).
These data are in accordance with the results showed by Alavilli et al. (2022), who developed a marker which co-segregates with the populations and is located close to the region of NBS_S9_1495924 marker. This marker focuses on the CmoAP2/ERF gene. Similar results obtained by different molecular markers developed for the same disease and, which are physically located nearby these regions contribute to a more complete and personalized approach in the development of powdery mildew-resistant C. moschata and C. pepo lines.
The identification of individuals that do not have the markers associated with resistance genes, or that present some type of genetic alteration, is also important for the development of breeding strategies for genetic improvement, selecting individuals that are resistant or seeking alternative sources of resistance (Beraldo-Hoischen et al., 2021; Zhang et al., 2021).
Other markers tested for powdery mildew were M2454200, M2475244 and M2467704, developed by Wang et al. (2021b), to verify the presence of the resistance allele in C. pepo. These molecular markers are CAPS and In-del type and flank the CpPM10.1 gene area on chromosome 9.
Data of markers close to the CpPM10.1 gene were analyzed based on the method developed by Wang et al. (2021b), with little alteration. Wang et al. (2021b) verified that the presence of just one of the three markers was sufficient to classify a genotype as resistant to powdery mildew, as this method correctly identified resistance in 98% of the genotypes tested in the field. However, as in this study field data were not collected, the authors chosed a more strict criterion: a genotype would be classified as resistant only if it presented all three markers analyzed, and as susceptible if it did not amplify any of the three markers. Based on these criteria, the results indicated that 43.4% of the C. pepo population was resistant to powdery mildew.
Although the markers proposed by Holdsworth et al. (2016) and Wang et al. (2021b) are associated with powdery mildew resistance in C. pepo, a slight variation is observed in the presence rates of resistance alleles, which are 47.8% and 43.4%, respectively. The small percentage difference suggests that the markers are identifying alleles in genetically related regions. Furthermore, the distance between markers may allow genetic recombination within the mapped region. This dynamic may be associated with polygenic inheritance. Thus, a marker can be anchored in only one of the genes with an additive effect, whereas other genes can also contribute to the effective resistance in plants. This genetic complexity highlights the importance of a comprehensive approach to understanding powdery mildew resistance in C. pepo (Holdsworth et al., 2016; Zhang et al., 2021; Wang et al., 2021b).
The evaluated genotypes have potential to be used in C. pepo and C. moschata breeding programs.
ACKNOWLEDGMENTS
GDO received a master's scholarship from CAPES (Coordination for the Improvement of Higher Education Personnel - Finance Code 001).
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The data will be made available upon request to the corresponding author.
