Caracterização da autoincompatibilidade em populações segregantes de macieira via marcadores de DNA para alelos S

rbf Revista Brasileira de Fruticultura Rev. Bras. Frutic. 0100-2945 1806-9967 Sociedade Brasileira de Fruticultura Resumo O objetivo deste trabalho foi caracterizar genitores e respectivas populações de macieiras quanto aos alelos S para confirmar sua genealogia e avaliar a eficiência dos marcadores moleculares utilizados. Conjuntos específicos de iniciadores foram utilizados para a identificação dos alelos S via PCR. Foram avaliadas duas populações segregantes do Programa de Melhoramento Genético de Macieira da Epagri resultantes dos cruzamentos entre ‘Fred Hough’ × ‘Monalisa’ e ‘M-11/00’ × ‘M-13/91’. As segregações esperadas são 1:1:1:1 para compatibilidade total e 1:1 para semi-compatibilidade, que pode ser confirmada pelo teste X2. O cruzamento ‘Fred Hough’ (S5S19) × ‘Monalisa’ (S2S10) foi identificado como totalmente compatível, e foram identificados dois triploides entre os híbridos. O cruzamento entre ‘M-11/00’ (S3S19) × ‘M-13/91’ (S3S5) se mostrou semi-compatível baseado nos marcadores moleculares e a segregação dos alelos S nos híbridos foi de 1:1 como esperado. A segregação dos marcadores de DNA para S2, S3, S5, S10 e S19 ocorreu juntamente com seus respectivos alelos S. Dessa forma, a caracterização dos alelos S, além de permitir identificar a compatibilidade entre os genitores, serviu para identificar contaminações em populações segregantes. Introduction Gametophytic self-incompatibility is present in the reproductive process of several Malus species, including Malus × domestica Borkh. (BROOTHAERTS, 2003). Control of self-incompatibility is performed by the multiallelic locus ‘S’, located in the terminal portion of chromosome 17 (YAMAMOTO et al., 2002; DE FRANCESCHI et al., 2011, 2016). Each allele is responsible for production of a protein with co-dominance behavior, which acts within the pistil (BATLLE et al. 1995; LI et al., 2012; RAMALHO, 2012). The mechanism of self-incompatibility is as efficient as dioicia in the requirement of cross-fertilization between plants, helping to increase genetic variability (ALLARD, 1971). Currently, DNA markers are used for identification of S-alleles (MA et al., 2016; MIR et al., 2016; DE FRANCESCHI et al., 2018). However, allele-specific markers and microsatellite markers must be linked to the respective S-alleles they identify to be considered efficient (FERREIRA; GRATTAPAGLIA, 1995). When the primer is adapted to a region distant from the gene of interest, distortions may occur in the expected frequency of alleles in the segregating populations, resulting in false negative or positive results. In crosses between fully-compatible plants (all different S-alleles), the segregation pattern in the progenies is expected to follow the proportion 1:1:1:1 (RAMALHO, 2012; AGAPITO-TENFEN et al., 2015). In contrast, in crosses of semi-compatible plants, the expected pattern of S-allele segregation is expected to follow the ratio 1:1 (CHOI et al. 2002; DE FRANCESCHI et al., 2016). This is because the pollen grain bearing the S-allele, common to the diploid pistil tissue, is aborted when in contact with the pistil S-RNases (MATSUMOTO, 2014). However, in crosses between incompatible plants, in which the pair of S-alleles in each parent are the same, abortion of all pollen grains occurs, not allowing the formation of viable seeds. Therefore, the objective of this study was to characterize the parents and their respective descendants regarding identification of S-alleles by DNA markers to confirm their genealogy and to evaluate the efficiency of the markers used to identify the S-alleles. Materials and methods For characterization of self-incompatibility by identification of the S-alleles, two segregating populations of apple trees belonging to the Apple Breeding Program of the Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina - Epagri (Agricultural Research and Rural Extension Company of Santa Catarina) were evaluated. The experimental orchard is in the Experimental Station in the municipality of Caçador, in the midwestern region of the state of Santa Catarina (26°49’5’’ S and 50°59’12’’ W at 940 m AMSL). One population originated from crossing the apple varieties ‘Fred Hough’ × ‘Monalisa’ (54 plants) and the other from crossing the selections ‘M-11/00’ × ‘M-13/91’ (120 plants). These crosses were made in 2007, following routine crosses of the Apple Breeding Program for the generation of segregating populations. Young healthy leaves were collected from each of the two segregating populations and respective parents and were kept deep-frozen at -20°C in plastic bags until DNA extraction, which was performed according to Revers et al. (2005) using 0.1 g of ground plant tissue. Each polymerase chain reaction (PCR) was performed in a final volume of 15 μL, containing 1 U of Taq DNA polymerase, 1x enzyme buffer, 2.00 mM MgCl2, 0.2 mM dNTPs, 1 μM of each primer (forward and reverse), and 50 ng of genomic DNA. Primers developed by Kitahara and Matsumoto (2002) and Broothaerts (2003) were used to identify 16 S-alleles of apple trees (Table 1). PCR was performed with a T100™ thermocycler (BioRad® California, USA) programmed for 3 min at 94°C, followed by 30 denaturation cycles at 94°C for 1 min, annealing depending on the primer characteristics (see Table 1) for 1 min, and extension at 72°C for 1 min, followed by 7 min at 72°C. For the S10 primer, the final extension step was at 72°C for 10 min. Table 1 Primer sequences and temperature conditions for allele-specific PCR to identify the S-alleles of apple tree (Malus × domestica Borkh.) and restriction enzyme digestion. S-Allele Primers Sequence (5' → 3') Annealing temperature (ºC) / restriction enzymes Amplified size (bp) S1 FTC168 ATATTGTAAGGCACCGCCATATCAT 60 530 FTC169 GGTTCTGTATTGGGGAAGACGCACAA S2 OWB122 GTTCAAACGTGACTTATGCG 60 449 OWB123 GGTTTGGTTCCTTACCATGG S3 FTC177 CAAACGATAACAAATCTTAC 55 500 FTC226 TATATGGAAATCACCATTCG S4 FTC5 TCCCACAATACAGAACGAGA 60 / TaqI 274 (194+77) OWB249 CAATCTATGAAATGTGCTCTG S5 FTC10 CAAACATGGCACCTGTGGGTCTCC 59 346 FTC11 TAATAATGGATATCATTGGTAGG S6 FTC141 ATCAGCCGGCTGTCTGCCACTC 58 (1) 850 FTC142 AGCCGTGCTCTTAATACTGAATAC S7 FTC143 ACTCGAATGGACATGACCCAGT 60 302 FTC144 TGTCGTTCATTATTGTGGGATGTC S9 OWB154 CAGCCGGCTGTCTGCCACTT 62 343 OWB155 CGGTTCGATCGAGTACGTTG S10 (2) AACAAATCTTAAAGCCCAGC 60, NarI 282 (185+97) GGTTTCTTATAGTCGATACTTTG S16 FTC5 TCCCACAATACAGAACGAGA 60 / TaqI 274 (243+41) OWB249 CAATCTATGAAATGTGCTCTG S19 FTC229 TCTGGGAAAGAGAGTGGCTC 60 304 FTC230 TTTATGAACTTCGTTAAGTCTC S20 FTC141 ATCAGCCGGCTGTCTGCCACTC 60 (1) / NarI 920 (800+120) FTC142 AGCCGTGCTCTTAATACTGAATAC S22 FTC5 TCCCACAATACAGAACGAGA 60 / TaqI 274 (199+44+31) OWB249 CAATCTATGAAATGTGCTCTG S23 FTC222 CAATCGAACCAATCATTTGGT 60 237 FTC224 GGTGTCATATTGTTGGTACTAATG S24 FTC231 AAATATTGCAACGCACAGCA 60 580 FTC232 TTGAGAGGATTTCAGAGATG S26 FTC14 GAAGATGCCATACGCAATGG 54 194 FTC9 TTTAATACCGAATATTGGCG * Values in parentheses refer to the fragment size generated after digestion with the respective restriction enzymes. (1) Cycle extension time of 45 sec. (2) Primer proposed by Kitahara e Matsumoto (2002). FTC and OWB primers were developed by Broothaerts (2003). For discrimination of the S4, S16, and S22 alleles, part of the PCR-amplified product (10 μL) was digested by the restriction enzyme TaqI (for 1 h in a 65°C water bath). Likewise, for discrimination of the S20 and S10 alleles, 10 μL of the amplification product was digested by the restriction enzyme NarI (for 4 h in a 37°C water bath). The following cultivars, previously characterized for the respective S-allele, were used as positive controls for the presence of each S-allele: Fuji (S1 and S9; SASSA et al., 1996), Golden Delicious (S2 and S3; BROOTHAERTS et al., 1995), Gloster (S4; VAN NERUM et al., 2001), Gala (S5; JANSSENS et al., 1995), Marubakaido (S6 and S26; AGAPITO-TENFEN et al., 2015), Idared (S7; JANSSENS et al., 1995), McIntosh (S10; RICHMAN et al., 1997), Delicious (S19; MATSUMOTO and KITAHARA, 2000), Alkmene (S22; VAN NERUM et al., 2001), Mutsu (S20; MATSUMOTO et al., 1999), Granny Smith (S23; SCHNEIDER et al., 2001), and Braeburn (S24; KITAHARA et al., 2000). The only exception was allele S16, since no genotype with this pre-identified allele is maintained in Epagri. In addition, the same cultivars were used for primer optimization. The amplification products were analyzed by 3% agarose gel electrophoresis using a 50 bp DNA marker. The gels were stained with GelRed® fluorescence intercalation. The profiles of the amplified fragments were analyzed by images captured with a Kodak Gel Logic 212 Pro Imaging System. The S-allele amplifications whose size coincided with the positive control were identified as present. The segregation of the S-alleles was assessed using the X2 test, considering the S-alleles that were identified and the expected segregation (complete compatibility = 1:1:1:1 and semi-compatibility = 1:1). In addition, if there were S-alleles common to the parents, field crosses were carried out to obtain the fertilization rate. Self-fertilization of the parents and reciprocal cross-pollination were performed. At 40 days after pollination, the fertilization index (number of fruit with more than 20 mm formed after pollination) of each cross (fruit set) was evaluated. Results and discussion The genotypes of the parents ‘Fred Hough’ (S5S19), ‘Monalisa’ (S2S10), ‘M-11/00’ (S3S19), and ‘M-13/91’ (S3S5) identified by Brancher et al. (2020) were confirmed (Figure 1). Figure 1 Characterization of the S-alleles of the parents of the segregating populations (‘F.H.’ Fred Hough – S5S19, ‘M.’ Monalisa – S2S10, ‘11/00’ M-11/00' – S3S19, and ‘13/91’ M-13/91' – S3S5) comparing the size of the PCR fragments identified on agarose gel (3% and 50 bp ladder) with the sizes available in the literature (S2: 449 bp; S3: 500 bp; S5: 346 bp; S10: 282 [185 + 97] bp; S19: 304 bp). In the segregating population resulting from ‘Fred Hough’ (S5S19) × ‘Monalisa’ (S2S10), 49 apple trees exhibited one of the genotypes expected from this fullycompatible cross (S2S5, S5S10, S2S19, and S10S19). Distribution of the plants among the possible S-allele genotypes (Table 2) followed the expected proportion for crossing of fully-compatible genotypes: 1:1:1:1 (p > 0.05). Thus, the DNA markers used were effective in identification of the respective alleles, and confirmed that these plants were the result of the ‘Fred Hough’ × ‘Monalisa’ cross. Table 2 S-allele genotype and number of plants identified with each genotype in apple tree populations. 'Fred Hough' (S5S19) × 'Monalisa' (S2S10) S-alleles identified in the *********** population Number of plants observed Number expected X2 S5S2 13 13.5 1.85ns S5S10 15 13.5 S2S19 9 13.5 S10S19 12 13.5 S2S5S19 1 0 S5S10S19 1 0 S5S9 1 0 S9S19 1 0 S19S? 1 0 Total 54 'M-11/00' (S3S19) × 'M-13/91' (S3S5) S-alleles identified in *** segregating population Number of plants observed Number expected X2 S3S5 66 60 2.62ns S5S19 49 60 S5S10 2 0 S5S? 3 0 Total 120 * S?: unidentified S-allele. In addition, two plants were identified as having three alleles each: one with S2S5S19 and the other with S5S10S19 (Table 2). The occurrence of three S-alleles suggests the triploidy of these two plants, since the S-alleles identified were common to the S-alleles present in the parents. Triploid plants can occur naturally in the Malus genus, both in interspecific crosses and among diploid crosses (BROWN, 2012). Two of the three alleles of these plants were inherited from the female parent ‘Fred Hough’ (S5 and S19). This result coincides with that found by Janssens et al. (1995) and Sakurai et al. (2000), who identified the maternal parent as the donor of the gamete 2n (gamete not reduced) in different crosses between diploid parents, from which triploid descendants can originate. Three plants of the first cross had a S-allele different from the alleles expected for this population. The plants were genotyped as S5S9, S5S19, and S19S?, according to Table 2. These plants may have resulted from some exchange during the seedling development process or contamination by pollen that did not correspond to the cross-breeding parent. In the cross ‘M-11/00’ (S3S19) × ‘M-13/91’ (S3S5), four different genotypes were identified in the population (Table 2). There is one S-allele in common between the parents, characterizing semi-compatibility because of the gametophytic self-incompatibility mechanism (BATLLE et al., 1995; RAMALHO, 2012; MATSUMOTO, 2014; DE FRANCESCHI et al., 2016; PRATAS et al., 2018). The distribution of the plants among the possible S-allele genotypes (S5S19 and S3S5) followed the expected proportion for the crossing of semi-compatible genotypes: 1:1 (p > 0.05). In addition, there are five plants with alleles that likely are a result of contamination during formation of the population (S5S10 and S5S?). Because the S3 allele is in common, the cross ‘M-11/00’ x ‘M-13/91’, the reciprocal cross, and selffertilization of both parents were performed again to determine the fruit set of this cross. The results of the four crosses are shown in Table 3. Self-fertilization of ‘M-13/91’ did not produce fruit; the cross ‘M-13/91’ (♀) × ‘M-11/00’ (♂) exhibited 15.4% fruit set; and the cross ‘M-11/00’ (♀) × ‘M-13/91’ (♂) exhibited 35.8% fruit set. It was notable that self-fertilization of ‘M-11/00’ produces 7.5% fruit set. A hypothesis for this fruit set is that ‘M-11/00’ has some degree of self-fertility, through which some self-fertilization could naturally occur (LI et al., 2016). The formation of viable seeds in the fruit will indicate if the egg(s) was (were) fertilized or not, and then parthenocarpy may be dismissed, which is characterized by the formation of fruit without fertilization of the eggs, resulting in the absence of seeds or the presence of sterile seeds (HEGEDÜS, 2006). This fruit set obtained in both crosses diverges from the results of the original cross made in the year 2007, which had 87% fruit set (data not shown), probably because of some environmental effect or the germination capacity of the pollen currently used compared to the past. After harvest, seeds from the fruit from crosses between ‘M-11/00’ and ‘M-13/91’ and from the ‘M-11/00’ self-pollinations (if there are seeds in the self-fertilized fruit) will be genotyped to check for S-alleles. Table 3 Cross between the selections ‘M-13/91’ and ‘M-11/00’, the reciprocal cross, and self-fertilization of the parents regarding number of pollinated flowers, number of apples formed, and fruit set (%) at 40 days after pollination. Parents Number of pollinated flowers ****** of apples formed Fruit set (%) Female (♀) **** (♂) M-11/00(S3S19) M-11/00(S3S19) 133 10 7.5 M-11/00(S3S19) M-13/91(S3S5) 123 44 35.8 M-13/91(S3S5) M-13/91(S3S5) 198 0 0 M-13/91(S3S5) M-11/00(S3S19) 156 24 15.4 Conclusion The cross ‘Fred Hough’ (S5S19) × ‘Monalisa’ (S2S10) is characterized as fully-compatible, with corresponding segregation of the S-alleles (1:1:1:1). The results obtained from the segregating population of ‘M-11/00’ x ‘M-13/91’ indicate semi-compatibility and a segregation ratio of 1:1. DNA markers for the S2, S3, S5, S10, and S19 alleles co-segregated with the respective S-alleles, which was effective for characterization of genotypes. Acknowledgments The authors would like to thank Capes, CNPq (Funding Code: 404475.2016-7), Udesc, and Epagri for funding this study. AGAPITO-TENFEN, S.Z.; DANTAS, A.C.M.; DENARDI, F.; NODARI, R.O. Identification of the Er1 resistence gene and RNase S-alleles in Malus prunifolia var. ringo rootstock. Scientia Agricola, Piracicaba, v.72, n.1, p.62–68, 2015. ( AGAPITO-TENFEN S.Z. DANTAS A.C.M. DENARDI F. Identification of the Er1 resistence gene and RNase S-alleles in Malus prunifolia var. ringo rootstock. Scientia Agricola Piracicaba 72 1 2015 62 68 ALLARD, R. W. Sistemas de controle da polinização em plantas cultivadas. In: Princípios do melhoramento genético das plantas. São Paulo: Edgard Blücher, 1971. p. 189–203. ALLARD R. W. Sistemas de controle da polinização em plantas cultivadas. São Paulo Edgard Blücher 1971 p. 189–203 BATLLE, I.; ALSTON, F.H.; EVANS, K.M. The use of the isoenzymic marker gene Got-1 in the recognition of incompatibility S alleles in apple . Theoretical and Applied Genetics, Berlin, v.90, n.2, p.303–306, 1995. BATLLE I. ALSTON F.H. EVANS K.M. The use of the isoenzymic marker gene Got-1 in the recognition of incompatibility S alleles in apple . Theoretical and Applied Genetics Berlin 90 2 1995 303 306 BRANCHER, T.L.; HAWERROTH, M.C.; KVITSCHAL, M.V.; MANENTI, D.C.; GUIDOLIN, A.F. Self-incompatibility alleles in important genotypes for apple breeding in Brazil. Crop Breeding and Applied Biotechnology, Londrina, v.20, n.4, p.e28652041, 2020. BRANCHER T.L. HAWERROTH M.C. KVITSCHAL M.V. Self-incompatibility alleles in important genotypes for apple breeding in Brazil. Crop Breeding and Applied Biotechnology Londrina 20 4 2020 28652041 BROOTHAERTS, W. et al. cDNA cloning and molecular analysis of two self-incompatibility alleles from apple. Plant Molecular Biology, v. 27, n. 3, p. 499–511, fev. 1995. BROOTHAERTS W. DNA cloning and molecular analysis of two self-incompatibility alleles from apple. Plant Molecular Biology 27 3 1995 499 551 BROOTHAERTS, W. New findings in apple S-genotype analysis resolve previous confusion and request the re-numbering of some S-alleles. Theoretical and Applied Genetics, Berlin, v.106, n.4, p.703–714, 2003. BROOTHAERTS W. New findings in apple S-genotype analysis resolve previous confusion and request the re-numbering of some S-alleles. Theoretical and Applied Genetics Berlin 106 4 2003 703 714 BROWN, S. Apple. In: BADENES, M.L.; BYRNE, D.H. (ed.). Fruit breeding. Boston: Springer, 2012. p.329–367. BROWN S. Apple. Boston Springer 2012 p.329–367 CHOI, C.; TAO, R.; ANDERSEN, R.L. Identification of self-incompatibility alleles and pollen incompatibility groups in sweet cherry by PCR based s-allele typing and controlled pollination. Euphytica, Dordrecht, v.123, p.9–20, 2002. CHOI C. TAO R. ANDERSEN R.L. Identification of self-incompatibility alleles and pollen incompatibility groups in sweet cherry by PCR based s-allele typing and controlled pollination. Euphytica Dordrecht 123 2002 9 20 DE FRANCESCHI, P.; BIANCO, L.; CESTARO, A.; DONDINI, L.; VELASCO, R. Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars. Plant Molecuar Biology, The Hague, v.97, n.3, p.279–296, 2018. DE FRANCESCHI P. BIANCO L. CESTARO A. Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars. Plant Molecuar Biology The Hague 97 3 2018 279 296 DE FRANCESCHI, P.et al; COVA, V.; TARTARINI, S.; DONDINI, L. Characterization of a new apple S-RNase allele and its linkage with the Rvi5 gene for scab resistance. Molecular Breeding, Dordrecht, v.36, n.1, p.1–11, 2016. DE FRANCESCHI P. Characterization of a new apple S-RNase allele and its linkage with the Rvi5 gene for scab resistance. Molecular Breeding Dordrecht 36 1 2016 1 11 DE FRANCESCHI, P.; PIERANTONI, L.; DONDINI, L.; GRANDI, M.; SANSAVINI, S.; SANZOL, J. Evaluation of candidate F-box genes for the pollen S of gametophytic self-incompatibility in the Pyrinae (Rosaceae) on the basis of their phylogenomic context. Tree Genetics and Genomes, New York, v.7, n.4, p.663–683, 2011. DE FRANCESCHI P. PIERANTONI L. DONDINI L. Evaluation of candidate F-box genes for the pollen S of gametophytic self-incompatibility in the Pyrinae (Rosaceae) on the basis of their phylogenomic context. Tree Genetics and Genomes New York 7 4 2011 663 683 FERREIRA, M.E.; GRATTAPAGLIA, D. Introdução ao uso de marcadores moleculares em análise genética 2.ed. Brasília, (DF): Embrapa-Cenargen, 1995. FERREIRA M.E. GRATTAPAGLIA D. Introdução ao uso de marcadores moleculares em análise genética. 2.ed Brasília Embrapa-Cenargen 1995 HEGEDÜS, A. Review of the self-incompatibility in apple (Malus × domestica Borkh., syn.: Malus pumila Mill.). Internaon Joural of Hoticultural Science, Budapest, v.12, n.2, p.31–36, 2006. HEGEDÜS A. Review of the self-incompatibility in apple (Malus × domestica Borkh., syn.: Malus pumila Mill.). Internaon Joural of Hoticultural Science Budapest 12 2 2006 31 36 JANSSENS, G.A.; GODERIS, I.J.; BROEKAERT, W.F.; BROOTHAERTS, W. A molecular method for S-allele identification in apple based on allele-specific PCR. Theoretical and Applied Genetics, Berlin, v.91, n.4, p.691–698, 1995 JANSSENS G.A. GODERIS I.J. BROEKAERT W.F. A molecular method for S-allele identification in apple based on allele-specific PCR. Theoretical and Applied Genetics Berlin 91 4 1995 691 698 KITAHARA, K.; SOEJIMA, J.; KOMATSU, H.; FUKUI, H.; MATSUMOTO, S. Complete sequences of the S-genes, Sd- and Sh-RNase cDNA in apple. HortScience, Alexandria, v.35, n.4, p.712–715, 2000. KITAHARA K. SOEJIMA J. KOMATSU H. Complete sequences of the S-genes, Sd- and Sh-RNase cDNA in apple. HortScience Alexandria 35 4 2000 712 715 KITAHARA, K.; MATSUMOTO, S. Sequence of the S10 cDNA from ‘McIntosh’ Apple and a PCR-digestion Identification Method. HortScience, v. 37, p.187–190, 2002. KITAHARA K. MATSUMOTO S. Sequence of the S10 cDNA from ‘McIntosh’ Apple and a PCR-digestion Identification Method. HortScience 37 2002 187 190 LI, T.; LONG, S.; LI, M.; BAI S.; ZHANG, W. Determination S-genotypes and identification of five novel S-RNase alleles in wild Malus species. Plant Molecular Biology Reporter, Dordrecht, v.30, n.2, p.453–461, 2012. LI T. LONG S. LI M. Determination S-genotypes and identification of five novel S-RNase alleles in wild Malus species. Plant Molecular Biology Reporter Dordrecht 30 2 2012 453 461 LI, W.; YANG, Q.; GU,Z.; WU, C; MENG. D.; YU, J.; CHEN Q.; LI, Y.; YUAN,H.; WANG, D.; LI, T. Molecular and genetic characterization of a self-compatible apple cultivar, ‘CAU-1’. Plant Science, Shannon, v.252, p.162–175, 2016. LI W. YANG Q. GU Z. Molecular and genetic characterization of a self-compatible apple cultivar, ‘CAU-1’. Plant Science Shannon 252 2016 162 175 MA, Y.; XUE, H.; ZHANG, L.; ZHANG, F.; OU, C.; WANG, F.; ZHANG, Z. Involvement of auxin and brassinosteroid in dwarfism of autotetraploid apple (Malus × domestica). Scientific Reports, London, v.6, e.26719, 2016. MA Y. XUE H. ZHANG L. Involvement of auxin and brassinosteroid in dwarfism of autotetraploid apple (Malus × domestica). Scientific Reports London 6 2016 26719 MATSUMOTO, S. Apple pollination biology for stable and novel fruit production: Search system for apple cultivar combination showing incompatibility, semicompatibility, and full-compatibility based on the S-RNase allele database. International Journal of Agronomy, London, v.2014, p.1–9, 2014. MATSUMOTO S. Apple pollination biology for stable and novel fruit production: Search system for apple cultivar combination showing incompatibility, semicompatibility, and full-compatibility based on the S-RNase allele database. International Journal of Agronomy London 2014 2014 1 9 MATSUMOTO, S.; KITAHARA, K.; KOMORI, S.; SOEJIMA, J. A new S-allele in apple, “Sg”, and its similarity to the “Sf” allele from “Fuji”. HortScience, Alexandria, v.34, n.4, p.708–710, 1999. MATSUMOTO S. KITAHARA K. KOMORI S. A new S-allele in apple, “Sg”, and its similarity to the “Sf” allele from “Fuji”. HortScience Alexandria 34 4 1999 708 710 MATSUMOTO, S.; KITAHARA, K. Discovery of a new self-incompatibility allele in apple. HortScience, Alexandria, v.35, n.7, p.1329–1332, 2000. MATSUMOTO S. KITAHARA K. Discovery of a new self-incompatibility allele in apple. HortScience Alexandria 35 7 2000 1329 1332 MIR, J.; AHMED, N.; SINGH, D.B.; MALIK,G.; HAMID, A.; ZAFFER,S.; SHAFI, W. Molecular identification of S-alleles associated with self-incompatibility in apple (Malus spp.) genotypes. Indian Journal of Agricultural Sciences, New Delhi, v.86, n.1, p.78–81, 2016. MIR J. AHMED N. SINGH D.B. Molecular identification of S-alleles associated with self-incompatibility in apple (Malus spp.) genotypes. Indian Journal of Agricultural Sciences New Delhi 86 1 2016 78 81 PRATAS, M.I.; AGUIAR, B.; VIEIRA, J.; NUNES, V.; TEIXEIRA, V.; FONSECA, N.A.; IEZZONI, A.; NOCKER, S.; VIEIRA, C.P. Inferences on specificity recognition at the Malus×domestica gametophytic self-incompatibility system. Scientific Reports, London, v.8, n.1, p.1717, 2018. PRATAS M.I. AGUIAR B. VIEIRA J. Inferences on specificity recognition at the Malus×domestica gametophytic self-incompatibility system. Scientific Reports London 8 1 2018 1717 RAMALHO, M.A.P. Genética na agropecuária. 5.ed. Lavras: UFLA, 2012. RAMALHO M.A.P. Genética na agropecuária. 5.ed Lavras UFLA 2012 REVERS, L.F.; LAMPE, V.S.; OLIVEIRA, P.R.D.; CAMARGO, U.A.; LIMA, J.C. Uso prático de marcadores moleculares para seleção assistida no melhoramento de uvas de mesa apirênicas. Revista Brasileira de Fruticultura, Jaboticabal, v.28, n.1, p.104–108, 2005. REVERS L.F. LAMPE V.S. OLIVEIRA P.R.D. Uso prático de marcadores moleculares para seleção assistida no melhoramento de uvas de mesa apirênicas. Revista Brasileira de Fruticultura Jaboticabal 28 1 2005 104 108 RICHMAN, A.D.; BROOTHAERTS, W.; KOHN, J.R. Self-incompatibility RNases from three plant families: homology or convergence? American Journal of Botany, New York, v.84, n.8, p.912–917, 1997. RICHMAN A.D. BROOTHAERTS W. KOHN J.R. Self-incompatibility RNases from three plant families: homology or convergence? American Journal of Botany New York 84 8 1997 912 917 SAKURAI, K.; BROWN, S.K.; WEEDEN, N. Self-incompatibility alleles of apple cultivars and advanced selections. HortScience, Alexandria, v.35, n.1, p.116–119, 2000. SAKURAI K. BROWN S.K. WEEDEN N. Self-incompatibility alleles of apple cultivars and advanced selections. HortScience Alexandria 35 1 2000 116 119 SASSA, H.; NISHIO, T.; KOWYAMA, Y.; HIRANO, H.; KOBA, T.; IKEHASHI, H. Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily. MGG Molecular e General Genetics, v.250, p.547–557, 1996. SASSA H. NISHIO T. KOWYAMA Y. Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily. MGG Molecular e General Genetics 250 1996 547 557 SCHNEIDER, D.et al.Analysis of S-alleles by PCR for determination of compatibility in the ‘Red Delicious’ apple orchard.The Journal of Horticultural Science and Biotechnology, Berlin, v.76, n.5, p.596–600, 2001. SCHNEIDER D. Analysis of S-alleles by PCR for determination of compatibility in the ‘Red Delicious’ apple orchard. The Journal of Horticultural Science and Biotechnology Berlin 76 5 2001 596 600 VAN NERUM, I.; GEERTS, M.; VAN HAUTE, A.; KEULEMANS, J.; BROOTHAERTS, W. Re-examination of the self-incompatibility genotype of apple cultivars containing putative ’new’ S-alleles. TAG Theoretical and Applied Genetics, Berlin, v.103, n.4, p.584–591, 2001. VAN NERUM I. GEERTS M. VAN HAUTE A. Re-examination of the self-incompatibility genotype of apple cultivars containing putative ’new’ S-alleles. TAG Theoretical and Applied Genetics Berlin 103 4 2001 584 591 YAMAMOTO, T.; KIMURA, T.; SHODA, M.; IMAI, T.; SAITO, Y.; SAWAMURA, Y.; KOTOBUKI, K.; HAYASHI, T.; MATSUTA, N. Genetic linkage maps constructed by using an interspecific cross between Japanese and European pears. Theoretical and Applied Genetics, Berlin, v.106, n.1, p.9–18, 2002. YAMAMOTO T. KIMURA T. SHODA M. Genetic linkage maps constructed by using an interspecific cross between Japanese and European pears. Theoretical and Applied Genetics Berlin 106 1 2002 9 18