Abstract: Knowledge of the genotypes for the self-incompatibility locus (S-locus) in apple varieties and in genotypes being used as parents is critical for breeding and commercial production. We present a high-throughput set of molecular markers for the identification of 13 commonS-RNase alleles (S₁,S₂,S₃,S₅,S₇,S₈,S₉,S₁₀,S₂₀,S₂₃,S₂₄,S₂₅ andS₂₈). This set is composed of seven allele-specific quantitative PCR-based High-Resolution Melting assays and four multi-allelic SSR markers. Validation of these markers was performed using 86 apple accessions, including cultivars with knownS-genotypes and recent commercial varieties arising from the Plant & Food Research (PFR) cultivar breeding programme. We also characterized theS-genotypes of 183 genotypes representing some of the most valuable parents within PFR’s cultivar breeding programme. The results of this work demonstrate the practical usefulness of this marker set to provide accurate cross-compatibility information to optimise choice of pollenisers in commercial apple orchard design, and to identify compatible parents and guide parental selection when executing apple breeding programmes, to optimise fruit crop yield and quality.

Key words: 
• S-allele" type="keywords.keywordEn" href='#"S-RNase" type="keywords.keywordEn" href='#"Allele-specific PCR" type="keywords.keywordEn" href='#"S-genotyping" type="keywords.keywordEn" href='#"s01">

  • Apple (Malus domestica Borkh.) fruit are the final products of the fusion between two gametic nuclei: the male pollen grain and the female egg-cell. After the pollen grain is deposited onto the flower's stigma, it germinates and grows a pollen tube down the style to fertilize the egg-cell. The pollen can come from the same plant or from a different one. However, a broadly distributed, pre-zygotic and genetic mechanism called gametophytic self-incompatibility (GSI) prevents the self-fertilization of closely related individuals, promoting out-crossing and thereby maintaining genetic diversity. GSI is found in many angiosperms, including theSolanaceae,Rosaceae, andPlantaginaceae families^[1,2].

    Apple has a homomorphic GSI mechanism where inhibition of self-fertilization occurs through genetic or biochemical mechanisms that function regardless of flower morphology^[3] operating in a reproductive system, which has two different and tightly linked components (S-genes). One is located in the pistil and the other is specific to the pollen^[1,4]. The pistil component is an extracellular ribonuclease (S-RNase) that inhibits self-pollen tube growth^[5]. The pollen-specific component is controlled by multiple genes calledSFBBs (i.e.S-locus F-box brothers) that interact with theS-RNase in an allele-specific way^[6]. Both components work in a collaborative manner to control the single, multi-genic and multi-allelicS-locus. EachSFBB interacts withS-RNases, such that non-self-RNases are degraded allowing pollen tube growth^[7] (Fig. 1). The selective pressures underlying this collaborative recognition mechanism generate a lower diversity of theS-pollen genes than is found on theS-pistil locus, which shows a higher degree of allelic polymorphism^[8]. However, these multi-genicS-haplotypes are inherited as single segregating units keeping their functionality across generations^[9,10].

    

    Figure 1.  Genetic control of gametophytic self-incompatibility (GSI) inMalus. TheS-locus is composed of two tightly linked components, found in the pollen and pistil respectively. In GSI, the pollen self-incompatibility phenotype is controlled gametophytically, i.e., the genotype of the haploid pollen itself (gametophyte) determines its incompatibility type. For example, the pollen composition of a certain pollen donor plant is phenotypically halfS₁ and halfS₂. In the female parent, two alleles are co-dominant and both are expressed in the pistil. Pollen inhibition occurs when there is a match between the donor pollenS-haplotype and either of the two haplotypes present in the pistil, producing an incompatible reaction that inhibits the growth of the 'self' pollen tube growth. Three types of reactions can occur during a cross: (a) incompatible; neither of the two gametes will germinate, (b) semi-compatible; half the donor pollen will be inhibited and the other half will germinate and grow normally, (c) compatible; all pollen will germinate and grow normally.

    Apple breeding relies on compatible and productive cross-pollination. A breeder needs information about the self-incompatibility genotypes (S-genotypes) of both parents to execute successful crosses and facilitate the selection of individuals carrying a combination of desirable traits. Traditionally, incompatibility was determined using time-consuming cross-pollination experiments, where successful fruit set was measured over many combinations of parents. Recently, less time consuming and more cost-effective molecular markers have been implemented to replace such field experiments, using either allele-specific markers amplifying a singleS-RNase allele^[11−18] or markers based on restriction enzyme digestion of polymerase chain reaction (PCR) products (cleaved amplified polymorphic sequences CAPS or PCR-RFLP). Simple Sequence Repeat (SSR) markers have also been used for screeningS-alleles^[14,15,19]. CAPS and SSR markers have helped determine most of theS-genotypes for common commercial apple varieties. However, most of the existing assays involve the use of restriction enzymes after PCR reactions and the visualization of the products on agarose gels, making them very laborious and time-consuming when handling large numbers of samples. This can be problematic evidenced when new seedlings/selections, that are potential parents in a breeding programme, need to be checked for compatibility each season.

    The objective of this study was to develop and validate a high-throughput and practical method to identify 13 different appleS-RNase alleles (S₁,S₂,S₃,S₅,S₇,S₈,S₉,S₁₀,S₂₀,S₂₃,S₂₄,S₂₅ andS₂₈). Our method is composed of seven allele-specific High-Resolution Melting (HRM) and four multi-allelic SSR markers, which do not require post-PCR restriction enzyme digestions or agarose gels for allele-scoring analysis. We used as controls 70 out of a total of 86 commercial apple cultivars for whichS-genotypes had already been reported, to validate the accuracy of our markers for identifying the correctS-alleles. We then demonstrate the usefulness of these assays by genotyping 183 genotypes representing some of the most valuable parents within the PFR breeding program.

  A novel, rapid and high-throughput protocol forS-RNase allele genotyping

  • The four developed SSR markers Myb110a1_PFR, Myb110a2_PFR, Myb110b_PFR and GSI_SSR_PFR amplify polymorphic PCR products associated with 13 differentS-alleles (S₁,S₂,S₃,S₅,S₇,S₈,S₉,S₁₀,S₂₀,S₂₃,S₂₄,S₂₅ andS₂₈) (Table 1). All four primer pairs amplify PCR products linked to theS₁,S₃,S₅,S₇,S₂₄ andS₂₅S-alleles. Both Myb110b_PFR and GSI_SSR_PFR can be used to distinguish theS₁₀S-allele. Myb110b_PFR exclusively identifiesS₂ andS₉, whilst GSI_SSR_PFR amplifies a PCR product linked to theS₂₈S-allele. All four primer pairs, except Myb110b_PFR, amplify PCR products linked toS₂₀ and all four but Myb110a1_PFR can identify theS₂₃S-allele.

    Table 1.  Primer sequences of quantitative real-time PCR and SSR-based markers.

    Marker name	Type		Primer sequences '5 - 3'	Physical location	Genbank locus	S-Rnase alleles and product sizes (bp)
    S1_apple_PFR	HRM	Forward	ACAGGCCACTGGTGGA	not found in reference genome	MG598487.1:1981−1996	S1,S20,S24 (38)
    		Reverse	ATTGCGTATGGCATTTTCAAT	Chr17:30844510−30844530	MG598487.1:1998−2018
    S2_apple_PFR	HRM	Forward	TTGAACAAATATTATTCAATGGGGA	Chr17:31240988−31240964	MG598488.1:860−884	S2 (54)
    		Reverse	CATCGTAACTATATATACCATCCGCGTA	Chr17:31240964−31240943	MG598488.1:886−913
    S5_apple_PFR	HRM	Forward	AATTTATAAAACACGTGATCA	not found in reference genome	MG598491.1:326−346	S5 (43)
    		Reverse	GCTCCTATTGATCGATCAT	not found in reference genome	MG598491.1:350−370
    S8_apple_PFR	HRM	Forward	TTCGATTATTTTCAATTTACGCTT	Chr17:31240889−31240871	MG598494.1 :1159−1182	S8 (162)
    		Reverse	ATTTAAGGTTGTTTCTTTGCAATAC	not found in reference genome	MG598494.1 :1296−1320
    S9_apple_PFR	HRM	Forward	GCTCAGGAAATGACCCAATATAC	not found in reference genome	MG598495.1:1284−1306	S9 (61)
    		Reverse	AATATTACCTTAGTAGAATTCATGGTTGT	not found in reference genome	MG598495.1:1315−1344
    S23_apple_PFR	HRM	Forward	TTTATGGCCTTCAAACTGGAA	not found in reference genome	MG598501.1:1065−1085	S23 (42)
    		Reverse	CAGAAGATTGGGTCGGGT	not found in reference genome	MG598501.1:1089−1106
    S28_apple_PFR	HRM	Forward	TGCCTCGCTCTTGAACAAA	not found in reference genome	MG598505.1:782−800	S28 (47)
    		Reverse	CCCCGTAATTCCCATTGAATAATA	not found in reference genome	MG598505.1:805−828
    Myb110a1_PFR	SSR	Forward	TCTCCCTCATCCCAGAACA	Chr17:32151473−32151491		S1 (166),S3 (184),S5 (180),S7 (170),S20 (158),S24 (176),S25 (188)
    		Reverse	CGAGCCAAACAAAATTGGA	Chr17:32151642−32151624
    Myb110a2_PFR	SSR	Forward	CTCTCCCTCATCCCAGAACA	Chr17:32151472−32151491		S1 (325),S3 (343),S5 (339),S7 (314),S20 (317),S23 (309),S24 (320),S25 (347)
    		Reverse	TCCTACTCGGCTCGACAATC	Chr17:32151800−32151781
    Myb110b_PFR	SSR	Forward	CTTCGGGCTTATTTGGGTTT	Chr17:32187809−32187790		S1 (202),S2 (233),S3 (214),S5 (209),S7 (191),S9 (247),S10 (216),S23 (239),S24 (217),S25 (238)
    		Reverse	TTTGCCCCTTCAAAGATCAG	Chr17:32187616−32187635
    GSI_SSR_PFR	SSR	Forward	GCCCCTTACATTCCTTTTCTTT	Chr17:31704109−31704130		S1 (314),S3 (338),S5 (335),S7 (324),S10 (328),S20 (317),S23 (322),S24 (217),S25 (329),S28 (352)
    		Reverse	CAATCTTGAGTTGTCGTTGGAG	Chr17:31704430−31704409

    Additionally, we developed seven quantitative-PCR allele-specific markers. Six of these markers (S₁,S₂,S₅,S₉,S₂₃ andS₂₈_apple_PFR) amplify specific single nucleotide polymorphisms (SNPs) identified by a pairwise alignment of the coding sequences of 25S-RNase alleles, previously published by De Franceschi et al.^[20]. The seventh marker forS₈ was adapted to work with our HRM methodology, by modifying the forward primer from the previously published primers pairs (Larsen et al.^[19]) (Fig. 2). When used on their own, without the SSR markers, theseS-allele-specific qPCR markers can identify eightS-alleles in total. TheS1_apple PFR marker can resolve theS_20,S₂₄ andS₁S-alleles as separate melting curves.

    

    Figure 2.  High resolution melting (HRM) curve profiles of sevenS-allele-specific markers. Amplification curves of real-time PCR marker assays (left panels), HRM difference plots, where the derivative fluorescence signal (dF/dT) is plotted as a function of temperature (right panels). Each colour represents a specificS-genotype as shown by the legends. Light grey represents samples that were not amplified in the real-time PCR.

    Validation of the S-RNase allele genotyping method on a large set of apple commercial cultivars

  • TheS-allele genotyping of 86 apple accessions (81 different cultivars, counting a red mutant sport of 'Fuji' and four 'Gala' mutant sports) – including 70 traditional varieties with previously reportedS-alleles, five varieties with unknownS-alleles and ten newer cultivars arose from the PFR breeding programme – was undertaken using the new set of molecular markers. TheS-genotypes of 59 of the 70 traditional apple varieties were in complete agreement with previous reports and theS-genotypes of their respective parents. For ten accessions, our results for one of the twoS-alleles disagreed with those published previously (Supplemental Table S1): 'Abbondanza' had been reported as (S₃S₅)^[20], while (S₃S₇)S-alleles were detected in the present study. 'Antonovka' was reported (S₈S₃₂)^[20], while we detectedS₈ and other allele sizes that could equally be linked toS₃,S₇ orS₂₀. 'Priscilla' was reported as (S₃S₉)^[21] or (S₉S₂₀)^[22] or (S₇S₁₀)^[20], while we detected (S₇S₂₈) alleles. 'Ingrid Marie' was reported as (S₅S₄₃)^[23], while our most likely observedS-genotype was (S₃S₅), 'James Grieve' was reported as (S₅S₈)^[23], while we detected (S₅S₂₀). 'Ben Davis' was reported as (S₅S₂₃) while we detected (S₇S₂₃); 'Jonathan' was reported previously as (S₇S₉)^[24−28]; however, we detected (S₉S₂₃). 'Early Cortland' has been reported as (S₅S₂₈)^[29,30]; however, we report it here as (S₁S₂₈)^[29]. 'Yellow Transparent' has been reported as (S₁S₅)^[31,32], while we identified (S₁S₇S₉S₂₄). Finally, 'McIntosh' was reported as (S₁₀S₂₅)^[11,23], while we found (S₂S₂₅).

    The new markers also resolved bothS-alleles for three out of four traditional varieties with previously undeterminedS-genotypes: 'Red Dougherty' (S₁S₇), 'Pinkie' (S₂S₃) and 'Merton Russet' (S₅S₂₄). We identified only theS₂₅ allele for 'Paulared'. The ten cultivars that have recently arisen from the PFR breeding programme were successfully typed for theirS-alleles: 'Scired' (S₂S₉), 'PremA093' (S₂S₉), 'Scifresh' (S₂S₂₄), 'PremA153' (S₂S₂₄), 'PremA34' (S₂S₃), 'Scilate' (S₅S₉), 'PremA96' (S₅S₉), 'PremA17' (S₅S₂₄), 'PremA280' (S₅S₂₄) and 'PremA129' (S₉S₂₄). For the Canadian variety 'Sunrise' we identifiedS-alleles (S₃S₂₄).

    For a remaining set of five cultivars, just oneS-allele could be determined: 'Hetlina' and 'Geheimrat Dr Oldenburg' were reported as (S₁S_16b)^[12] and (S₃S₂₈)^[20], respectively. The respectiveS₁ andS₃S-alleles were detected in our study; however, we did not identify allele sizes that could be linked to eitherS_16b orS₂₈ using the new markers. For 'Benoni', reported as (S₅S₁₁)^[33], we detected theS₅ allele, as well as other marker alleles not linked to the expectedS₁₁. 'Regent' was reported as (S₃S₁₀)^[34]; we identified theS₁₀ allele, but noS₃-linked alleles were observed. Instead, an allele linked toS₂₅ was detected. Finally, 'Panenské České' was reported as (S₇S₁₀)^[20], but only theS₇ allele was detected using three SSR markers. Marker alleles linked toS₁₀ were not found, rather the Myb110b marker detected alleles linked toS₃ andS₂₄.

    Diversity ofS-RNase alleles in the PFR apple cultivar breeding programme

  • When the new markers were screened over 183 genotypes, including some of the most valuable parents within the PFR apple cultivar breeding programme, we were able to determine theirS-genotypes. These genotypes are the seedlings of 76 biparental families (Supplemental Table S2). For 32 of these families (from a total of 132 selections), theS-genotypes of their parental pedigrees could be verified. The frequency ofS-RNases alleles found among this pool of genotypes is shown inFig. 3. Among the 183 genotypes screened,S₂ was the most commonS-allele, present in 21.3% of the samples, followed byS₃ (19.9%),S₂₄ (18.6%),S₅ (17.5%),S₂₃ (13.7%),S₉ (5.7%) andS₇ (0.8%). RareS-alleles wereS₂₈ (0.5%),S₂₅,S₁ andS₂₀ (0.3% each). Only 2.2% of theS-alleles could not be assigned. The most prevalent genotypes were: (S₃S₂₄), (S₂S₂₄), (S₂S₅), (S₂S₂₃), (S₃S₂₃), (S₃S₅) and (S₅S₂₃) observed at frequencies of: 7.1, 7.1, 6.3, 5.2, 4.4, 4.1 and 3%, respectively. Other less prevalent genotypes were: (S₅S₂₄), (S₅S₉), (S₃S₉), (S₂S₃), (S₉S₂₄), (S₂S₉) and (S₂₃S₂₄), which were observed at frequencies of 2.2, 1.6, 1.4, 1.4, 1.4, 1.1 and 0.8%, respectively. The following rare genotypes were each found at a frequency of 0.3%: (S₁S₃), (S₂S₇), (S₅S₇), (S₇S₉), (S₂₀S₂₈) and (S₃S₂₈). For 1.1% of the selections, just one allele was identified (S₃?) in 0.8% and (S₂₃?) in 0.3%).

    

    Figure 3.  Frequency ofS-alleles andS-genotypes of the 183 apple advanced selections of the PFR’s breeding programme. Inner plot shows the percentage frequency distribution ofS-alleles from the total 366 alleles observed among the 183 genotypes tested. Outer plot represents the absolute frequency of eachS-genotype. All outer slices not showing a percentage value in the figure represent 0.3% respectively.

  • A high-throughput method to identify theS-genotypes of apples was developed and validated in this study. This will help to inform the selection of compatible parental combinations when designing a crossing programme. We present a new high-throughput marker set based on four multi-allelic SSR and seven allele-specific qPCR markers. The four SSR markers can identify 13S-RNase-alleles (S₁,S₂,S₃,S₅,S₇,S₈,S₉,S₁₀,S₂₀,S₂₃,S₂₄,S₂₅ andS₂₈) and the seven HRM markers allow the identification of eightS-RNase-alleles (S₁,S₂,S₅,S₈,S₉,S₂₀,S₂₃ andS₂₈). The identification of the 13S-alleles can be achieved economically and efficiently by employing three PCR reactions, using two (Myb110b_PFR and GSI_SSR_PFR) of the four SSR markers and theS8_apple_PFR marker (with the addition of an M13-tail on the 3'-end). These three markers can be multiplexed by using different fluorescent labels that can be simultaneously separated and scored on a capillary electrophoresis instrument.

    Alternatively, for laboratories with access to a real-time qPCR system, as well as a capillary electrophoresis instrument, the seven qPCR allele-specific markers and one of the SSR markers (either Myb110b_PFR or GSI_SSR_PFR) will be sufficient to identify and resolve the whole set of 13S-alleles.

    The usefulness of the new markers was validated in over 59 of 70 well-established apple cultivars with knownS-genotypes. Ten of the discrepancies with previous assays are supported by our results from parental pedigree allele analysis, acknowledging that some might be mistakes with labelling, or incorrect germplasm harvest: 'Priscilla' has been reported as (S₃S₉)^[21], (S₉S₂₀)^[22] or (S₇S₁₀)^[20], while we detected (S₇S₂₈), withS₂₈ as probably coming from 'Starking Delicious', which is reported as (S₉S₂₈)^[29]. 'Ingrid Marie' was reported as (S₅S₄₃)^[23], while our most likely observedS-genotype was (S₃S₅), whereS₅ is derived from 'Cox's Orange Pippin' (S₅S₉). However, neitherS₄₃ norS₃ has been reported in 'Cox's Pomona'S-genotype (S₁S₃₄)^[19]. 'Early Cortland' has been reported as (S₅S₂₈)^[29,30], which is consistent with its parentage: 'Cortland' (S₅S₂₅)^[29] and 'Lodi' (S₁S₂₈)^[30]; however, we reported it here as (S₁S₂₈)^[29]. 'Abbondanza' was reported as (S₃S₅)^[20], while (S₃S₇)S-alleles were detected here. 'Antonovka' was reported (S₈S₃₂)^[20], while we detected alleleS₈; however, we observed different allele sizes for our SSR markers that we could link to eitherS₃,S₇ orS₂₀. There are different 'Antonovka' accessions^[35], so it is probable that they have differentS-genotypes. 'James Grieve' was reported as (S₅S₈)^[23], while we detected (S₅S₂₀), although the Myb110b_PFR marker showed an additional 202 bp allele, which is linked toS₁S-RNase, but did not exhibit any other allele sizes linked toS₁ in any of the other SSR or qPCR markers. Then, 'Ben Davis' was reported as (S₅S₂₃), while we detected (S₇S₂₃), with the same allele sizes found and expected for 'Lady Williams' (S₇S₂₃). 'Jonathan' was reported previously as (S₇S₉)^[24−28]; however, we characterised it as (S₉S₂₃), but note that we do have molecular and phenotypic indicators suggesting this could be an incorrectly identified accession in the PFR germplasm (Vincent Bus, pers. comm.). Finally, 'Yellow Transparent' is reported as (S₁S₅)^[31,32], while we identified (S₁S₇S₉S₂₄), which is consistent with this cultivar being a tetraploid sport mutant^[36].

    We demonstrated the usefulness of the markers by determining theS-genotypes of ten newer cultivars arising from the PFR breeding programme ('Scired', 'PremA093', 'Scifresh', 'PremA153', 'PremA34', 'Scilate', 'PremA96', 'PremA17', 'PremA280' and 'PremA129'). TheS-genotype information for such new cultivars is valuable information for growers, enabling them to plant compatible pollenisers in commercial orchards. Despite the high diversity ofS-RNase alleles that have been characterized inMalus (at least 35 differentS-alleles were found among cultivars in Matsumoto's database^[29]), the common worldwide practise of using a relatively small pool of cultivars that combine premium fruit quality as well as resistance to pests and environmental stresses in breeding programmes leads to new cultivars with restricted allelic combinations. Among the 183 PFR apple genotypes tested here, a pool of only 11S-alleles was found (S₁,S₂,S₃,S₅,S₇,S₉,S₂₀,S₂₃,S₂₄,S₂₅, andS₂₈). This is not surprising given that all theS-alleles of the main founders of PFR's cultivar breeding programme [namely 'Splendour' (S₂S₉), 'Cox's Orange Pippin' (S₅S₉), 'Red Delicious' (S₉S₂₈), 'Golden Delicious' (S₂S₃), 'Red Dougherty' (S₁S₇), 'Worcester Pearmain' (S₂S₂₄), 'Jonathan' (S₂₃S₉), 'Fuji' (S₁S₉), 'Braeburn' (S₉S₁₄), 'Granny Smith' (S₃S₂₃), 'James Grieve' (S₅S₂₀), 'Wagener' (S₃?), 'Cripp's Pink' (S₂S₂₃) and 'Akane' (S₇S₂₄)^[34]] have ten of these 11S-alleles. However, it is possible that the wider parental pool also has undetectedS-alleles beyond these 11, as some breeding parents, not represented in the 183 genotypes tested here, are also derived from minor founders.

    The most frequently observed allele wasS₂ (21.3%), which is one of the two alleles carried by 'Royal Gala' (S₂S₅), a parent or grandparent in pedigrees of most of the PFR genotypes. For instance, 'Scired', 'Sciros', 'Scilate', and 'Sciray' were used as the pollen parents for many crosses and they all are progeny of a cross between 'Gala' (S₂S₅) and 'Splendour' (S₂S₉). 'Gala's' parentage is 'Golden Delicious' (S₂S₃) and 'Kidd's Orange Red' (S₅S₉), so 'Golden Delicious' is the source of this allele in 'Gala' or 'Royal Gala' crosses.

    S₃ was the second most abundant allele (19.9%), being present in crosses of genotypes with 'Pinkie' in their parentage. 'Pinkie' likely inherited the allele from 'Granny Smith' (S₃S₂₃), although we do not know theS-alleles carried by its other parent A679-2. Also, crosses produced using 'Fiesta' (S₃S₅), have inherited theS₃ allele from 'Idared' (S₃S₇), which has 'Wagener' (S₃?) as a parent.

    AlleleS₂₄ was observed in 18.6% of the genotypes, those arising from crosses with 'Braeburn' (S₉S₂₄) as one of the parents: 'Scifresh' is a progeny of the cross 'Braeburn' 'Royal Gala'; while 'PremA153' is derived from a 'Gala' × 'Braeburn' cross. 'PremA129' has 'Braeburn' as a grandparent, being a progeny of 'PremA280' × 'Scired', with 'PremA280' having 'Gala' and 'Braeburn' as parents. 'PremA17' (S₅S₂₄) also has this allele, presumably from 'Braeburn': this cultivar was derived from a cross between genotypes A045R13T007 × A020R02T167, which unfortunately are no longer available in the orchard. Another source of this allele is 'Akane' (S₇S₂₄)^[34], which inherited it from 'Worcester Pearmain' (S₂S₂₄).

    The origin ofS₅ (17.5%) in our breeding programme is 'Cox's Orange Pippin' (S₅S₉). Crosses that involve 'Fiesta' or 'James Grieve' have 'Cox's Orange Pippin' as a grandparent. It is also a great-grandparent in crosses that have 'Gala' as a parent and a great-great-grandparent in crosses that include cultivars such as 'Sweetie', 'PremA17' and 'PremA96' as parents.

    TheS₂₃ allele is present (13.7%) in seedlings derived from our A068 family; however, we still need to confirm the source of the allele as we do not know theS-genotypes of grand-parents.S₉ is found in 5.7% of seedlings derived from crosses where one of the parents was 'Scired', or alternatively with 'Splendour' as one of the grandparents or great-grandparents. Other important sources of theS₉ allele are 'Cox Orange Pippin' (S₅S₉) and 'Braeburn' (S₉S₂₄).

    At the other end of the scale, theS₇ allele was only found in 4.8% progeny of crosses with 'Red Free' (S₃S₇) as a parent or grandparent and in some crosses using 'Akane' (S₇S₂₄)^[34], which has 'Jonathan' (S₇S₉) as the likely parental source of this allele. The allelesS₂₅ andS₂₈ occur rarely in the PFR breeding programme. They probably come from 'McIntosh' (S₁₀S₂₅) and 'Delicious' (S₉S₂₈), respectively; however, the pedigree of the few selections with these alleles is not complete: further information is needed to confirm this hypothesis.

    According to Sheick et al.^[18], 11S-alleles (S₁,S₂,S₃,S₅,S₉,S₁₀,S₂₀,S₂₃,S₂₄,S₂₅, andS₂₈) are represented among the U.S. industry's most produced apples. These are predominantly coming from 'Red Delicious' (S₉S₂₈), 'Gala' (S₂S₅), 'Granny Smith' (S₃S₂₃), 'Fuji' (S₁S₉), 'Golden Delicious' (S₂S₃), 'Honeycrisp' (S₂S₂₄), 'McIntosh' (S₁₀S₂₅), 'Rome' (S₂₀S₂₄), 'Cripps Pink' (S₂S₂₃), and 'Empire' (S₁₀S₂₈). These same 11S-alleles butS₁₀ are represented in the New Zealand PFR breeding programme. Instead ofS_10, we haveS₇ included in our pool ofS-alleles, which is represented in crosses having 'Jonathan' (S₇S₉) and 'Red Free' (S₃S₇) in their pedigree. Another recent study by Lays Brancher et al.^[37] identified 11S-alleles (S₁,S₂,S₃,S₅,S₇,S₉,S₁₀,S₁₉,S₂₀,S₂₃, andS₂₄) among 42 apple genotypes, including cultivars, advanced selections and accessions of the Apple Germplasm Bank of Epagri (Caçador, Santa Catarina, Brazil). TheS₃ andS₅ alleles were most frequent (30.2% and 18.6%, respectively). The higher frequency of these alleles can be explained as 26 of the 42 accessions tested were direct or indirect descendants from the cultivars Imperatriz (S₃S₅), Golden Delicious (S₂S₃) and/or Gala (S₂S₅), which have served as the basis for the crosses of the Epagri Apple Breeding Program.

    A Danish study by Larsen et al.^[19] found 25S-alleles (S₁,S₂,S₃,S₄,S₅,S₆,S₇,S₈,S₉,S₁₀,S₁₁,S_16b,S_16c,S₂₀,S₂₁,S₂₃,S₂₄,S₂₅,S₂₆,S₂₈,S₃₁,S₃₃,S₃₄,S₃₆ andS₄₀) in 432Malus accessions including a selection ofM. domestica cultivars of mainly Danish origin (402 accessions), as well as a selection of otherMalus species (30 accessions). Among the 402 Danish accessions the alleleS₃ (28 %) was the most common followed byS₁ andS₇ (both 27 %). Previous studies^[16,38] using cultivars from Northern Europe and the Carpathian basin found similar results whereS₃ andS₇ were the two most commonS-alleles.

    Although selections used as parents in breeding programmes around the world are different due to consumer preferences, climate conditions, resistances to pest and diseases, etc., there is a common set of cultivars among worldwide breeding programmes^[39]. These are 'Golden Delicious' (S₂S₃), 'Braeburn' (S₉ S₂₄), 'Fuji' (S₁S₉), 'Gala' (S₂S₅), 'Granny Smith' (S₃S₂₃), 'Idared' (S₃S₇), 'Jonathan' (S₇S₉) and 'Red Delicious' (S₉S₂₈). TheS-alleles carried by these cultivars are also the most common among the total 183 advanced selections tested within PFR's breeding programme. TheS₃ allele is the most commonS-allele worldwide as seen in the previous studies mentioned here and among other older studies including European, American and Japanese cultivars^[12,23,40]. This low diversity ofS-RNase alleles highlights the need of introducing breeding cultivars with some of the less commonS-alleles into breeding programmes to increase mate compatibility among parental selections.

  Plant material

  • Leaves from 86 apple cultivars were collected at PFR, Havelock North, New Zealand, and Washington State University, Pullman, WA, USA. Total genomic DNA was extracted using the cetyltrimethyl ammonium bromide (CTAB) method^[41]. This DNA was used as a set for evaluating the new markers (Supplemental Table S1). Additionally, leaves from 183 apple genotypes, from 76 biparental families, were collected from trees in PFR's elite parental apple collections, to identify theirS-genotypes.

    SSR-basedS-allele markers

  • Three new primer pairs were designed around two single sequence repeats (SSR) linked to theMyb110a andMyb110b genes^[42] and named Myb110a1_PFR, Myb110a2_PFR and Myb110b_PFR, which are closely linked to theS-locus on apple chromosome 17. A fourth primer pair was designed for a SSR located within theS-locus (GSI_SSR_PFR) (Table 1). Design of the primer pairs was based on the GDDH13v1.1^[43] apple genome as a reference and employed using the Krait software^[44]. The M13 sequence TGTAAAACGACGGCCAGT was added to the 5′ end of the forward primer to enable the use of Schuelke's^[45] approach to fluorescent labelling. PCR was performed in a 15 µL reaction mixture containing 1.5 mM MgCl_2, 200 uM dNTPs, 13 nM of forward primer, 200 nM of reverse primer, 8.33 µL DNA-free water and 1× PCR Buffer (-MgCl₂) and 0.5 U of Platinum™ Taq DNA polymerase (Thermo Fisher Scientific, 10966034). The conditions of the touchdown PCR included an initial denaturing at 94 °C for 2 min, then five cycles (94 °C for 55 s, 65 °C for 55 s (decreased by 1 °C each cycle), 72 °C for 1 min and 39 s), then 35 cycles (94 °C for 55 s, 55 °C for 55 s and 72 °C for 1 min and 39 s) and a final extension at 72 °C for 10 min. The final amplicons were subjected to capillary electrophoresis using an ABI 3500 DNA sequence analyser (Applied Biosystems, Foster City, USA) and sized using GenScan™ 500 LIZ Size Standard (Applied Biosystems). SSR allele profiles were analysed using GeneMarker™^[46] version 2.20 (SoftGenetics LLC®, State College, PA, USA, www.softgenetics.com).

    Quantitative real-time PCR-based markers

  • SNPs were identified by performing a multiple sequencing progressive pairwise alignment^[47] of the coding sequences of 25S-RNase alleles previously published by De Franceschi et al.^[20] in Geneious version 10.0.9 (https://www.geneious.com), with the following parameters: global alignment with free end gaps algorithm, 70% similarity cost matrix, gap open penalty of 11.9, gap extension penalty of 2 and 2 refinement iterations.

    SevenS-RNase allele specific primer pairs namedSx_apple_PFR (x being: 1, 2, 5, 8, 9, 23 or 28 alleles) were designed to amplify a single product of 250 bp or less. These primer pairs can be used on a conventional PCR machine or by employing the High Resolution Melting (HRM) methodology^[48] on a quantitative PCR instrument. The primer pairs forS8_apple_PFR marker were modified from the ones previously published by Larsen et al.^[19].

    Conventional PCR reactions were carried out in 15 µL volume containing 1× PCR buffer mix (Invitrogen), 200 μM of each dNTP, 1.5 mM MgCl₂, 3 μM each primer, 0.1 U Platinum™ DNA polymerase (Thermo Fisher Scientific, 10966034) and 20 ng template DNA. Amplifications were carried out on a MasterCycler ProS thermocycler (Eppendorf). The conditions of the touchdown PCR included an initial denaturing at 95 °C for 5 min, then ten cycles (94 °C for 30 s, 60 °C for 30 s (decreasing 1 degree in each cycle) and 72 °C for 45 s), then forty cycles (94 °C for 30 s, 50 °C (forS5_apple_PFR primer pair) or 55 °C (forS1, 2, 9, 23, 28_apple_PFR primer pairs) for 30 s and 72 °C for 45 s) and a final extension at 72 °C for 5 min. PCR products were then visualized on a 2% agarose gel stained with RedSafe™ 20000x (ChemBio, UK) after 1 h of electrophoresis at 100 V.

    Quantitative PCR reactions were performed in a total volume of 10 µL containing 20 ng of template DNA, 2.5 mM MgCl₂, 200 nM forward and reverse primers and 1× HRM master mix (Roche Applied Science). PCR and HRM were performed on a LightCycler® 480 (Roche Diagnostics). The PCR parameters were an initial denaturation step of 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 20 s. Following amplification, the samples were heated to 95 °C for 1 min and then cooled to 40 °C for 1 min. Melting curves were generated with continuous fluorescence acquisition during a final ramp from 65 °C to 95 °C at 4.8 °C/s, followed by a final cooling step of 40 °C for 30 s. The resultant fluorescence data were processed using the LightCycler® 480 software (version 1.5; Roche Applied Science). Primer sequences, fragment sizes and their respective associatedS-RNase alleles are shown inTable 1.

    S-genotypingMalus cultivars and breeding seedlings

  • The four SSR-based markers were initially screened using a DNA set from 70 out of a total of 86 apple cultivars with knownS-genotypes, based on previously reported CAPS or PCR-RFLP detection methods (as referenced inSupplemental Table S1). Following this, the seven HRM assays were screened over the same cultivars to validate the allele-specificity of each primer pair (Table 1).

    Following the screening of the first DNA set, all 11 markers forS-genotype were further validated using 183 apple genotypes from the PFR breeding programmes. TheS-alleles were confirmed by verifying theS-genotype composition within each family and by examining their pedigree composition up to the grandparent level. For the PFR breeding populations, a summary of theS-genotype composition of the tested seedlings within families was made.

    Data availability

  • Raw data and R script for statistical analysis are available at linkhttps://github.com/hrpelg/Rnotebook_Self-incompatibility

  • We demonstrated the efficiency of a set of markers for theS-locus in aMalusdomestica germplasm set with knownS-genotypes and we determined theS-genotypes of uncharacterized cultivars, with an emphasis on new commercial releases. We showed theS-genotyping efficacy of this method on a large sample of advanced apple genotypes from the PFR breeding programme, whereS-genotypes were concordant with their parental pedigree.

    This robust, reproducible, simple and cost-efficientS-RNase-genotyping method is an alternative to the present molecular approaches. The existing molecular methods employ single allele specific markers per every singleS-allele or use marker based restriction enzyme digestions of PCR products to distinguish among fewS-alleles needing to be visualized on agarose gels. The flexibility of our method permits to know 13 differentS-alleles by employing just three different PCR reactions in a laboratory provided with a capillary electrophoresis instrument. These three PCR reactions can be multiplexed in a single electrophoresis run by using three different fluorescent colours. Alternatively, if a qPCR instrument is also available, this can be done using seven different HRM-markers and a single SSR marker. The use of a qPCR instrument allows the analysis of 384 samples per run or the multiplexing of four markers per PCR for every 96 samples.

    This method is provided to scientists, breeders and growers to select compatible pollenisers and to develop new cultivars. The benefits of knowing theS-alleles that each parental selection carries are: pollination success between compatible parental pollen and pistil, higher yields of orchards planted with compatible varieties and possible parentage identificationof unknown seedlings' descent due to undesired open pollination.

  • We would like to gratefully thank Stijn Vanderzande for providing the US plant material and Susan E. Gardiner for helpful advice and review of the manuscript. This work was funded by Prevar Limited (https://prevar.co.nz).

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1List of 86 commercial apple cultivars used as a validation set in this study, allele sizes (bp) per marker, observed and expected (by previous published assays)S-genotypes and parental pedigrees.^aS-genotypes in disagreement with previous reports,*S-genotypes not reported prior this study and (?) UnknownS-allele or unconfirmed pedigree. Each differentS-allele and its associated allele sizes are highlighted in a different colour.
  • Supplemental Table S2List of 183 apple genotypes from the PFR cultivar breeding programme grouped by family, allele sizes (bp) per marker and observedS-genotypes.

  • Copyright: © 2021 by the author(s). Exclusive Licensee Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visithttps://creativecommons.org/licenses/by/4.0/.

  Figure(3)  Table(1)References(48)

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  López-Girona E, Bowatte DR, Smart MEM, Alvares S, Brancher TL, et al. 2021. A high-throughput S-RNase genotyping method for apple. Fruit Research 1: 10 doi: 10.48130/FruRes-2021-0010

  López-Girona E, Bowatte DR, Smart MEM, Alvares S, Brancher TL, et al. 2021. A high-throughputS-RNase genotyping method for apple.Fruit Research 1: 10doi:10.48130/FruRes-2021-0010