EPAGRI hybrid apple cultivars resistant to Glomerella leaf spot have the R_gls resistance gene

Os cultivares híbridos de macieira da EPAGRI resistentes à mancha foliar de Glomerella possuem o gene de resistência R_gls

Abstract

Glomerella Leaf Spot (GLS) is the main disease of the ‘Gala’ apple cultivar in Brazil. Recent studies have suggested that the resistance of apple trees to GLS is controlled by a recessive gene called R_gls. This study investigated whether EPAGRI new apple cultivars have this resistance gene. The cultivars evaluated were ‘Fuji Suprema’ and ‘Gala Real’, which are standards of resistance and susceptibility to GLS, respectively, and the new apple cultivars selected by the phenotypic resistance to GLS: ‘Monalisa’, ‘Luiza’, ‘Venice’, ‘Isadora’ (hybrids) and ‘Gala Gui’ (spontaneous 'Gala' mutant). The DNA of cultivars was extracted and amplified using the SSR S0506206 molecular marker. RT-PCR was performed, and its products were evaluated by electrophoresis in 2% agarose gel. The new hybrid apple cultivars exhibited amplified DNA fragments similar to those of ‘Fuji Suprema’, indicating the presence of the R_gls gene. However, ‘Gala Gui’ cultivar showed amplified DNA fragments similarly to the ‘Gala Real’ susceptible cultivar. The results suggest that the genetic resistance of apple trees to GLS may be regulated by genes other than R_gls, particularly for spontaneous 'Gala' mutants that are phenotypically resistant to GLS.

Index terms
Colletotrichum spp.; genetic resistanc; molecular markers

Resumo

A Mancha Foliar de Glomerella (MFG) é a principal doença dos cultivares Gala no Brasil. Estudos recentes sugerem que a resistência das macieiras à MFG é controlada por um gene recessivo chamado R_gls. Este estudo investigou se os novos cultivares de macieira da EPAGRI possuem este gene de resistência. Os cultivares avaliados foram: Fuji Suprema e Gala Real, que são padrões de resistência e suscetibilidade à MFG, respectivamente, e os novos cultivares de maçã selecionadas pela resistência fenotípica à MFG: Monalisa, Luiza, Venice, Isadora (híbridos) e Gala Gui(mutante espontâneo de Gala). O DNA dos cultivares foi extraído e amplificado, usando o marcador molecular SSR S0506206. A RT-PCR foi realizada, e os produtos, avaliados poreletroforese em gel de agarose a 2%. Os novos cultivares híbridos de maçã exibiram fragmentos de DNA amplificados, semelhantes aos da 'Fuji Suprema', indicando a presença dogene R_gls.No entanto, a 'Gala Gui' mostrou fragmentos de DNA amplificados de forma semelhante ao cultivar suscetível Gala Real. Os resultados sugerem que a resistência genética das macieiras à MFG pode ser regulada por genes diferentes do R_gls, especialmente para mutantes espontâneos de 'Gala' que são fenotipicamente resistentes à MFG.

Termos para indexação
Colletotrichum spp.; resistência genética; marcadores moleculares

Introduction

The apple industry plays an important economic and social role in southern Brazil. It employs low-education workers, and apples are mostly produced on small family farms in regions with low human development index, particularly in the state of Santa Catarina, which accounts for more than half of Brazilian apple production. However, in the last decades, apple diseases such as Glomerella leaf spot (GLS), European pomaceous canker, fruit rot, and apple scab have increasingly impacted the profitability of the apple industry in this region.

GLS is the main summer disease of apple trees in Brazil (VALDEBENITO-SANHUEZA et al., 2002). It was first identified in 1983 at a warmer growing region in the state of Paraná (LEITE et al., 1988). Since then, it was disseminated throughout Santa Catarina, even in colder growing regions. The disease can cause early plant defoliation by up to 75% (KATSURAYAMA; BONETI, 2009), which affects the development of buds and flowers in the next harvest, resulting in low productivity and fruit quality (ANZANELLO et al., 2012).

‘Gala’ apple cultivar is the most produced in Brazil, accounting for about 60% of total production (ANUÁRIO..., 2019), despite being highly susceptible to GLS. The ‘Fuji’ apple cultivar, the second most produced in Brazil, has remained resistant to GLS since the beginning of its cultivation in the 1970s, indicating the possibility of being a long-lasting resistance.

The difficulty in preventing GLS is partly due to some of its epidemiological particularities (VALDEBENITO-SANHUEZA et al., 2002). Chemical treatments have not been effective enough in managing GLS, increasing production costs and the risk of environmental contamination. Therefore, cultivating apple cultivars resistant to GLS and other diseases is more economical and environmentally sustainable.

The EPAGRI (Agricultural Research and Rural Extension Company of Santa Catarina) apple breeding program has successfully developed new apple cultivars resistant to GLS (DENARDI et al., 2019) in the last decades.

Most of them are hybrids, while some are selections of spontaneous mutation (e.g.‘Gala Gui’ cultivar). These GLS-resistant apple cultivars were selected using the classic method of targeted inoculation with the Colletotrichum spp. complex under controlled conditions in apple seedlings. This technique was followed by a long-term assessment of GLS symptom incidence and severity in apple trees during scale-up trials and in commercial orchards. Each year, these cultivars are inoculated with a mixture of Colletotrichum spp. isolates collected from leaves showing GLS symptoms in various apple growing regions in Brazil to confirm their resistance.

Recent studies have indicated that the resistance of apple trees to GLS is governed by a single recessive gene named R_gls (LIU et al., 2016; LIU et al., 2017), and molecular markers have been developed to assist in the selection of resistant genotypes. For instance, the S0506206 SSR marker has genetic distance of 9.8 cM to the R_gls gene and is used for this purpose (LIU et al., 2016; LIU et al., 2017).

The present study assessed whether EPAGRI new apple cultivars with phenotypic resistance to GLS have the R_gls resistance gene and the efficiency of the molecular marker of this gene to assist in the selection of genotypes resistant to this disease in apple breeding programs.

Genetic analysis was performed in the tissue of young leaves harvested in the spring from mature apple trees of the following cultivars: ‘SCS417 Monalisa’, ‘SCS425 Luiza’, ‘SCS426 Venice’, ‘SCS441 Gala Gui’, ‘SCS443 Isadora’, and ‘Epagri 405-Fuji Suprema’, which are phenotypically resistant to GLS and ‘Gala Real’, which is susceptible to GLS.

Immediately after harvesting, leaf samples were crushed in liquid nitrogen and converted to a fine powder, which was the source for DNA extraction. The PureLink® Genomic DNA mini Kit (Invitrogen, USA) was used for DNA extraction and purification following the protocol provided by the manufacturer.

The amplification of the SSR molecular marker associated with the R_gls gene, known as S0506206, was performed using forward GCTGAGATTTCCCCCATT and reverse GCTGCGGACACTGCTTAG primers (LIU et al., 2017).

RT-PCR was performed with the following reaction mixture (components and reagents): 2.5 μL of buffer (200 mM Tris-HCl, pH 8.4 - 500 mM KCl, 1x concentrate), 0.5 μL of dNTPs (2.5 mM), 2.5 μL of each primer, 0.5 μL of MgCl2 (50 mM), 0.35 μL of Taq DNA polymerase (5 U μL^-1), 3.5 μL of DNA (10 ng μL^-1) and 12.65 μL of autoclaved sterilized ultrapure water. RT-PCR thermal cycles (35) comprised initial denaturation for 2 minutes at 95ºC followed by denaturation for 50 seconds at 95ºC, annealing for 50 seconds at 55ºC, extension for 50 seconds at 72ºC and final extension for 5 minutes at 72ºC. Agarose gel electrophoresis (2%) was used for the separation of amplicon (PCR products) and the stained DNA bands were visualized on an ultraviolet transilluminator and recorded with a digital image documenter.

A single DNA fragment from tissue samples of ‘Monalisa’, ‘Luiza’, ‘Venice’, and ‘Isadora’ new apple cultivars was amplified by RT-PCR, which are phenotypically resistant to GLS (DENARDI et al., 2019), as well as from tissue samples of ‘Fuji Suprema’ cultivar, which is the standard of resistance to GLS (DENARDI et al., 2019) (Figure 1). On the other hand, the ‘SCS441 Gala Gui’ new apple cultivar, which is phenotypically resistant to GLS, and the ‘Gala Real’ cultivar, which is the standard of susceptibility to GLS, exhibited an additional amplified DNA fragment in comparison to the other cultivars with size of 243 bp (Figure 1).

Figure 1
2% agarose gel with the S0506206 molecular marker linked to the R_gls resistance gene. M: 50-bp Marker. 1: ‘Gala Real’ (Susceptible), 2: ‘Epagri 405-Fuji Suprema’ (Resistant), 3: ‘SCS441 Gala Gui’ (Resistant), 4: ‘SCS426 Venice’ (Resistant), 5: ‘SCS425 Luiza’ (Resistant), 6: ‘SCS443 Isadora’ (Resistant), and 7: ‘SCS417 Monalisa’ (Resistant).

This difference in patterns of DNA fragment amplification between resistant and susceptible cultivars is the same as those described by Liu et al. (2016). These authors demonstrated that apple cultivars susceptible to GLS have RR and Rr alleles, while resistant cultivars contain rr alleles. Thus, in the current study, the codominant SSR S0506206 molecular marker efficiently distinguished resistant (rr) and susceptible (Rr) hybrid apple cultivars to GLS, characterized by the amplification of a single DNA band and two DNA bands by RT-PCR, respectively.

However, the ‘Gala Gui’ cultivar, selected as a spontaneous mutation for phenotypic resistance to GLS (FAORO, 2022), exhibited pattern of DNA fragment amplification equal to ‘Gala Real’ cultivar, which is the standard of susceptibility to GLS (DENARDI et al., 2019). This result suggests that the genetic resistance of apple trees to GLS may be regulated by genes other than Rgls, particularly for spontaneous 'Gala' mutants resistant to GLS.

In summary, the present study showed that the new Brazilian apple cultivars resistant to GLS have the Rgls gene and that resistance to GLS may not be regulated solely by this gene.

Acknowledgements

The authors thank Santa Catarina State Research Support Foundation (FAPESC/ 2021TR001408).

• The authors thank Santa Catarina State Research Support Foundation (FAPESC/ 2021TR001408).

• ANUÁRIO brasileiro da maçã 2019. Santa Cruz do Sul: Gazeta Santa Cruz, 2019. 54p.
• ANZANELLO, R.; SANTOS, H.P.; FIALHO, F.B.; MARODIN, G.A.B.; BERGAMASCHI, H. Evolução da endodormência em gemas de macieira em função da presença ou ausência de folhas no outono. In: CONGRESSO BRASILEIRO DE FRUTICULTURA, 22., 2012. Bento Gonçalves. Anais [...]
• DENARDI, F.; KVITSCHAL, M.V.; HAWERROTH, M.C. A brief history of the forty-five years of the Epagri apple-breeding program in Brazil. Crop Breeding and Applied Biotechnology, Viçosa, MG, v.19, n.3, p.347-55, 2019. https://doi.org/10.1590/1984-70332019v19n3p47
  » https://doi.org/10.1590/1984-70332019v19n3p47
• FAORO, I.D. (org.) Maçãs do grupo 'Gala' no Brasil Florianópolis: Epagri, 2022. 304p.
• KATSURAYAMA, Y.; BONETI, J. I. S. Mancha da gala. In: ENCONTRO NACIONAL SOBRE FRUTICULTURA DE CLIMA TEMPERADO, 11., 2009, Fraiburgo. Anais […] Caçador: Epagri, 2009. v.1, 226 p.
• LEITE, R. P.; TSUNETA, A. M.; KISHINO, A. Y. Ocorrência de mancha foliar de Glomerella em macieira no estado do Paraná Londrina: IAPAR, 1998. (Informe da Pesquisa, 81)
• LIU, Y.; LAN, J.; WANG, C.; LI, B,; ZHU, J.; LIU, C.; DAI, H. Investigation and genetic mapping of a Glomerella leaf spot resistance locus in apple. Plant Breeding, Oxford, v.136, n.1, p.119-25, 2017. https://doi.org/10.1111/pbr.12399
  » https://doi.org/10.1111/pbr.12399
• LIU, Y.; LI, B.; WANG, C.; LIU, C.; KONG, X.; ZHU, J.; DAI, H. Genetics and molecular marker identification of a resistance to glomerella leaf spot in apple. Horticultural Plant Journal, Amserdam, v.2, n.3, p.121-25, 2016. https://doi.org/10.1016/j.hpj.2016.06.002
  » https://doi.org/10.1016/j.hpj.2016.06.002
• VALDEBENITO-SANHUEZA, R. M.; BECKER, W.; BONETI, J. I. S.; KATSURAYAMA, Y.; CZERMAINSKI, A. B. C. Manejo das doenças de verão na produção integrada de maçã Bento Gonçalves: Embrapa Uva e Vinho, 2022. (Circular técnica, 36).

Edited by

Alexandre Pio Viana

Publication Dates

• Publication in this collection
  04 Nov 2024
• Date of issue
  2024

History

• Published
  14 Oct 2024
• Received
  08 Apr 2024
• Accepted
  18 July 2024