S-allele Constitution and Flowering Time Synchronization Preconditions for Effective Fertilization in Sweet Cherry (Prunus avium L.

Romanian Biotechnological Letters Vol. 20, No. 6, 2015 Copyright 2015 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER S-allele Constitution and Flowering Time Synchronization Preconditions for Effective Fertilization in Sweet Cherry (Prunus avium L.) Orchards Received for publication, September 9, 2014 Accepted, February 10, 2015 SANJA RADIČEVIĆ, SLAĐANA MARIĆ, RADOSAV CEROVIĆ Department of Pomology and Fruit Breeding, Fruit Research Institute, Čačak, Serbia Address correspondence to: Fruit Research Institute, Kralja Petra I/9, 32000 Čačak, Republic of Serbia Tel.: +381 32 221 375; Fax: +381 32 221 391; Email: saniaradicevic@gmail.com Abstract Although sweet cherry (Prunus avium L.) shows gametophytic self-incompatibility which is controlled by the multiallelic locus S, overlap in phenophase of full flowering is another crucial factor for the most effective pollination and fertilization between compatible cultivars. This study was carried out to determine the S-RNase genotype using the PCR method with consensus and allele-specific primers, and to investigate flowering time and abundance of 13 introduced sweet cherry cultivars over the six-year period, under the agro-environmental conditions of West Serbia. According to flowering time, the assessed sweet cherry cultivars were classified into three groups: mid-early [ Burlat (S 3 S 9 ), Early Lory (S 1 S 9 ), Lapins (S 1 S 4 '), Vera (S 1 S 3 ) and Celeste (S 1 S 4 ')], mid-late [ Merchant (S 4 S 9 ), Kordia (S 3 S 6 ), Summit (S 1 S 2 ) and Karina (S 3 S 4 )] and late flowering [ Starkrimson (S 3 S 4 '), Germersdorfer (S 3 S 12 ), Carmen (S 4 S 5 ) and Regina (S 1 S 3 )]. Based on the S-genotype and sufficient overlapping in full bloom, we offered a recommendation for effective pollination and fertilization of economically important sweet cherry cultivars, which could be very important for both breeders and growers, as well as for the further investigation in this field of research. Keywords: Prunus avium L., cultivars, S-allele genotyping, (in)compatibility groups, flowering time 1. Introduction Sweet cherry (Prunus avium L.) flowers express a self-incompatibility system which is controlled by the polymorphic S-locus with gametophytic action. The interaction between the components in the style (S-allele specific ribonuclease product of the S-RNases gene) (1) and in the pollen (S specific F-box protein, product of the SFB gene) (2), determines the outcome of fertilization. Fertilization will be prevented if a pollen expresses the same S-allele as one of the two S-alleles present in the style (3). Different S-alleles have been cloned and sequenced in sweet cherry; consensus and allele-specific primers have been designed to amplify across each of the two introns (4, 5). Both introns, especially the second one, allow most of the alleles to be distinguished according to the size of polymerase chain reaction (PCR) products. S-allele genotyping in sweet cherry led to the rapid confirmation of S-alleles and incompatibility groups. 18 S-alleles in 47 incompatibility groups in sweet cherry have been reported (6). The cause of self-compatibility in sweet cheery is attributed to the artificial pollen-part ' ' ' mutation of S 3 and S 4 (7), the natural pollen-part mutation of S 5 (3), and the mutation of a Romanian Biotechnological Letters, Vol. 20, No. 6, 2015 10997

SANJA RADIČEVIĆ, SLAĐANA MARIĆ, RADOSAV CEROVIĆ non S-locus EMPaS02 which also leads to a loss of pollen function (8). Despite the availability of self-compatible cultivars, most widely grown cultivars in the region of Serbia are self-incompatible. Thus, in commercial orchards, cross-compatible cultivars with different S-genotypes that flower simultaneously are planted together to ensure successful crosspollination and satisfactory fruit yield. If we add to this the other important aspects of the reproductive process characteristics of flowering and stable overlap in the full bloom of the main cultivars and pollenizers from year to year, pollen quality and ensuring of its transfer, stigma receptivity, ovule longevity etc. then it is clear to what extent these factors influence the fertilization efficiency, which is manifested as an optimal fruit set and yield. In certain years, the interaction of these complex relationships may cause the yield of commercially important cultivars, even in cases of fully- and semi-compatible combination of cultivars flowering about the same time, supported by an optimal arrangement of pollenizers and with an adequate pollen transfer (9). An appropriate choice of the main cultivars based on fruit quality and ripening time, as well as the choice of pollenizers providing high and stable yield, but also having a significant commercial value, is a basic requirement in modern cherry production. This study aimed to find out the best possible choice of newly introduced sweet cherry cultivars for planning of commercial orchards and breeding parent combinations by determining their self-incompatibility genotypes and average flowering period overlap based on six-year study of their flowering time in West Serbia. 2. Materials and Methods 2.1. Plant material and experimental design Thirteen introduced sweet cherry (Prunus avium L.) cultivars were used in this study viz., Biggareau Burlat ( Burlat ), Carmen, Celeste, Early Lory, Germersdorfer, Karina, Kordia, Lapins, Merchant, Regina, Starkrimson, Summit and Vera. The trial was performed at Preljinsko brdo facility (43 53' N; 20 21' E; 350 m above the sea) of the Fruit Research Institute Čačak, West Serbia, which is a typical area for fruit cultivation. Cultivars were grafted on Gisela 5 rootstock. The experimental orchard was established in 2005, with ten trees of each cultivar, at 4.0 1.5 m distance. Zahn Spindle was the applied training system. A few of the assessed cultivars, i.e. Burlat and Germersdorfer are well known and widely grown in Serbia, while the others are currently gaining in popularity. In addition, literature provides data on the studies of pomotechnological and other traits of some analyzed cultivars in region of Serbia, i.e. Celeste, Early Lory, Karina, Kordia, Lapins, Regina, Starkrimson and Summit (10, 11, 12). 2.2. DNA extraction and determination of S-genotype Young leaves of the studied cultivars were frozen in liquid nitrogen and stored at 80 C until use for the extraction. Frozen leaf samples were ground with four ball-bearings in Mixer Mill MM 400 (Retsch GmbH, Haan, Germany). Genomic DNA was than extracted using the CTAB method (13), with addition of 1% -mercaptoethanol and 2% polyvinylpyrrolidone (PVP 40) in the buffer. DNA samples in TE buffer were stored at 20 C until use. To identify the S-RNase alleles in the assessed cultivars, polymerase chain reactions were performed with the consensus primer pairs specific for the first and the second intron (PaConsI-F + -R and PaConsII-F + -R, respectively) and primers specific for alleles S 1 to S 5 (4, 5). PCR products were separated by electrophoresis in a 1.5% agarose gel (70 V for 3 4 h). Fragments were visualised by ethidium bromide staining and sized by comparison with a 1 Kb plus DNA ladder (Invitrogen, Groningen, the Netherlands). 10998 Romanian Biotechnological Letters, Vol. 20, No. 6, 2015

S-allele Constitution and Flowering Time Synchronization Preconditions for Effective Fertilization in Sweet Cherry (Prunus avium L.) Orchards 2.3. Flowering phenophase investigation Flowering phenophase investigated by monitoring and taking notes on flowering onset, full flowering and end of flowering 10% of flowers open, 80% of flowers open and 90% of petals fallen off, respectively (14). Abundance of flowering graded as follows: excellent 5, very good 4, good 3, poor 2, bad 1, and without flowers 0. Results of flowering time and abundance investigation presented as average data obtained over the six-year period (2009 2014). According to flowering time, the studied sweet cherry cultivars were classified into three groups: mid-early, mid-late and late flowering. Classification was based on the finding that flowering time of Burlat is estimated as mid-early (15, 16, 17). On the basis of the average overlap in full bloom of 5 8 days which ensures effective pollination and fertilization in sweet cherry (18), as well as on the basis of S-allele genotyping and classification of cultivars into incompatibility groups, a recommendation was given for their growing in orchards. 3. Results and Discussions 3.1. Identification of S-RNase allele constitutions in assessed sweet cherry cultivars The fact that self-incompatibility and cross-(in)compatibility in sweet cherry is attributed to a multi-allelic S-locus with gametophytic action, as well as that the S-allelic constitution of certain cultivars was corrected several time, impose the necessity for S-genotyping. The genotyping of cultivars provides useful information for breeders using it in crossing programmes, and indeed for nurserymen and growers in order to achieve an effective pollination.the use of the primers specific for the first (figure 1) and the second intron (figure 2), and allele-specific primers (figure 3) allowed the clear determination of the S-RNase genotypes in the assessed cultivars (table 1). The following S-RNase allelic constitutions were confirmed (table 2): S 3 S 9 ( Burlat ), S 4 S 5 ( Carmen ), S 1 S 4 ' ( Celeste and Lapins ), S 1 S 9 ( Early Lory ), S 3 S 12 ( Germersdorfer ), S 3 S 4 ( Karina ), S 3 S 6 ( Kordia ), S 4 S 9 ( Merchant ), S 1 S 3 ( Vera and Regina ), S 3 S 4 ' ( Starkrimson ) and S 1 S 2 ( Summit ). The S-RNase genotypes for the cultivars Carmen, Celeste, Early Lory, Karina, Kordia, Lapins, Merchant, Regina, Starkrimson, Summit and Vera were in agreement with the previously reported results (6). However, for Burlat (S 3 S 9 ; Group XVI), our results were consistent with previously reported (19, 6), but not with those who reported S 4 S 5 and S 3 S 5 genotypes (Group VII), respectively (20, 21). Namely, S 4 S 5 genotype of Group VII was corrected to S 3 S 5 (22), and it was shown that Burlat does not belong to Group VII and does not have an S 5 allele (19). Among the cultivars assessed in this study, for Germersdorfer two allelic constitutions have been reported so far (S 3 S 4 and S 3 S 12 ). Germersdorfer is incompatible with Bing and Napoleon (15), which belong to Group III (6), whereas S 3 S 4 genotype was confirmed for Germersdorfer collected from the Fruit Collection of Fruit Research Institute Čačak, Serbia (23). Our results for Germersdorfer (S 3 S 12 genotype, Group XXII) are in agreement with the previously reported (6, 24). Different sweet cherry genotypes have the same name, and the same genotypes can have various names (6). As an example of this, the author mentioned cultivars from Group XXII (S 3 S 12 ), which includes well known cultivars like Germersdorfi, Nasenkirsche, Noire de Meched, Nordwunder, Schneiders Spate Knorpelkirsche, Ziraat 0900 and many others. It is obvious that further genotyping of sweet cherry cultivars, i.e. with set of highly polymorphic SSRs spanning the whole genome, can enable correction of the problematic genotypes. Romanian Biotechnological Letters, Vol. 20, No. 6, 2015 10999

SANJA RADIČEVIĆ, SLAĐANA MARIĆ, RADOSAV CEROVIĆ Figure 1. PCR products of the S-RNase amplified fragment obtained with consensus primers for the first intron of assessed sweet cherry cultivars: 1 Burlat ; 2 Carmen ; 3 Celeste ; 4 Early Lory ; 5 Germersdorfer ; 6 Karina ; 7 Kordia ; 8 Lapins ; 9 Merchant ; 10 Regina ; 11 Starkrimson ; 12 Summit ; 13 Vera ; 1Kb plus DNA ladder (M) Figure 2. PCR products of the S-RNase amplified fragment obtained with consensus primers for the second intron of assessed sweet cherry cultivars: 1 Burlat ; 2 Carmen ; 3 Celeste ; 4 Early Lory ; 5 Germersdorfer ; 6 Karina ; 7 Kordia ; 8 Lapins ; 9 Merchant ; 10 Regina ; 11 Starkrimson ; 12 Summit ; 13 Vera ; 1Kb plus DNA ladder (M) Figure 3. PCR products of the S-RNase amplified fragment obtained with primers specific for alleles S 1 (a), S 2 (b), S 3 (c), S 4 (d) and S 5 (e) in assessed sweet cherry cultivars: 1 Burlat ; 2 Carmen ; 3 Celeste ; 4 Early Lory ; 5 Germersdorfer ; 6 Karina ; 7 Kordia ; 8 Lapins ; 9 Merchant ; 10 Regina ; 11 Starkrimson ; 12 Summit ; 13 Vera ; 1Kb plus DNA ladder (M) 11000 Romanian Biotechnological Letters, Vol. 20, No. 6, 2015

S-allele Constitution and Flowering Time Synchronization Preconditions for Effective Fertilization in Sweet Cherry (Prunus avium L.) Orchards Table 1. S-RNase alleles identification of the assessed sweet cherry cultivars with consensus and allele-specific primers Results with consensus Results with consensus Amplification with primer primer Cultivar allele-specific primers for the first intron for the second intron Allele 1 Allele 2 Allele 1 Allele 2 S 1 S 2 S 3 S 4 S 5 Burlat S 3 S 9 S 3 S 9 + Carmen S 4 S 5 S 4 S 5 + + Celeste S 1 S 4 S 1 S 4 + + Early Lory S 1 S 9 S 1 S 9 + Germersdorfer S 3 S 12 S 3 S 12 + Karina S 3 S 4 S 3 S 4 + + Kordia S 3 S 6 S 3 S 6 + Lapins S 1 S 4 S 1 S 4 + + Merchant S 4 S 9 S 4 S 9 + Regina S 1 S 3 Difficult to Difficult to distinguish distinguish + + Starkrimson S 3 S 4 S 3 S 4 + + Summit S 1 S 2 S 1 S 2 + + Vera S 1 S 3 Difficult to Difficult to distinguish distinguish + + S 6, S 9 and S 12 alleles were confirmed with consensus primers for the first and the second intron Table 2. S-genotype and incompatibility group of the assessed sweet cherry cultivars Cultivar S-genotype Incompatibility group Burlat S 3 S 9 XVI Carmen S 4 S 5 V Celeste S 1 S 4 ' Self-compatible Early Lory S 1 S 9 XVIII Germersdorfer S 3 S 12 XXII Karina S 3 S 4 III Kordia S 3 S 6 VI Lapins S 1 S 4 ' Self-compatible Merchant S 4 S 9 XXI Regina S 1 S 3 II Starkrimson S 3 S 4 ' Self-compatible Summit S 1 S 2 I Vera S 1 S 3 II a. Flowering phenophase investigation The earliest and the latest average flowering onset were observed in Burlat and Regina, respectively (figure 4). Flowering onset of cultivars within a species depends on the origin and genotypic characteristics, but it may also vary by location and year, making it impossible to speculate on a precise order in terms of flowering onset, but rather on the order which can be expected with the highest probability (25). The flowering onset span of the cultivars investigated in this paper was 11 days; the earliest and the latest flowering sweet cherry cultivars covered the span of three to nine days (26). Sweet and sour cherries are stone fruit species with a short period between the flowering onset and full flowering, which lasts up to three days (25). As result of this study, full flowering occurred two to four days after flowering onset. Five sweet cherry cultivars showed excellent flowering abundance, i.e. Lapins, Merchant, Kordia, Karina and Regina ; in Germersdorfer it was the lowest (grade 3.4; figure 4). Romanian Biotechnological Letters, Vol. 20, No. 6, 2015 11001

SANJA RADIČEVIĆ, SLAĐANA MARIĆ, RADOSAV CEROVIĆ The flowering period in the assessed sweet cherry cultivars lasted between 8 ( Summit ) and 14 days ( Burlat ). The flowering period in seasons with earlier and later beginning of flowering lasts 15 and 6 days, respectively (25). Previous studies reported that flowering period in different sweet cherry cultivars lasts 8 10 days (26), 11 18 days (27), or 15 days (10). The investigated cultivars were classified into three groups in terms of flowering onset (table 3). The results concerning the sequence of flowering of some cultivars and their belonging to the specific flowering-time group, are mainly in agreement with those reported by other authors. If there are different data on the time of flowering of certain cultivars, these are mostly the neighboring groups of flowering time. For example, Kordia has classified in group of late flowering cultivars (17), whereas the flowering time of Lapins was described as mid-late (28). This was expected, given the differences in the locality, the length of tested period, rootstock and training system. Contradictory data exist only on the flowering time of Early Lory (29). 11002 Figure 4. Flowering phenophase of introduced sweet cherry cultivars (2009-2014) Table 3. Classification of introduced sweet cherry cultivars, according to the flowering onset Cultivars Mid-early flowering Mid-late flowering Late-flowering Burlat Merchant Starkrimson (-) Early Lory Kordia Germersdorfer Lapins Summit Carmen Vera (+) Karina (+) Regina Celeste (+) b. Cultivars composition according to the S-allele constitution and flowering time Knowledge of the S-allele constitution and flowering time of sweet cherry cultivars is very useful when advising farmers in choosing the appropriate pollenizers to ensure production in commercial orchards, as well as for breeders in making proper choice of parental genotypes in breeding programmes and for enhancing genetic research related to this field. Two or three cross-compatible cultivars must be planted in cherry orchards in order to achieve effective cross-pollination (30). In modern plantations, where a larger number of cultivars is grown due to different ripening times, it is necessary to take into account their mutual (in)compatibility. Our results suggested that Vera and Regina are not only incompatible, but also have inadequate flowering synchronicity (figure 5). On the other hand, the potential combinations in Romanian Biotechnological Letters, Vol. 20, No. 6, 2015

S-allele Constitution and Flowering Time Synchronization Preconditions for Effective Fertilization in Sweet Cherry (Prunus avium L.) Orchards our research were mostly semi-compatible. Although in semi-compatible crosses a half of pollen tubes are arrested in the upper third of the style, in the expression of pollen-pistil interaction during progamic phase, inferiority of semi-compatible compared to full-compatible combinations in terms of fertilization efficacy, has not been determined (31). If pollenizer has a good flowering time synchronicity with the main cultivar, it is irrelevant whether they make a semi- or fully-compatible cross. In both cases, a sufficient number of pollen tubes will be able to reach the ovary, and successfully complete the fertilization within ovules of good vitality. In addition to this, the importance of pollinizer cultivars for self-compatible sweet cherry cultivars was reported (9, 32), showing that satisfactory crop of Stella was largely a result of outcrossing rather than selfing, as well as that Sunburst showed no evidence of selffertilization. However, the use of self-compatible cultivars (with pollen part mutation S 4 ') as a male parent in pollination which normally would have been incompatible and semi-compatible, resulted in semi-compatible and fully compatible crosses, respectively (i.e. Karina (S 3 S 4 ) Starkrimson (S 3 S 4 ') and Carmen (S 4 S 5 ) Starkrimson (S 3 S 4 '); figure 5). Cultivar Burlat Early Lory Lapins Vera Celeste Merchant Kordia Summit Karina Starkrimson Germersdorfer Carmen Regina Burlat Early Lory Lapins Vera Celeste Merchant Kordia Summit Karina Starkrimson Germersdorfer Carmen Regina Male genotype; Female genotype; Compatible; Semi-compatible; Incompatible; Flowering time overlap; Insufficient overlap in certain seasons; Insufficient overlap. Figure 5. Flowering time and (in)compatibility in the assessed sweet cherry cultivars It is for this reason that the practical problem is not only incompatibility, but also choosing the main cultivars and pollenizers with sufficient overlapping in full bloom as a precondition for potential pollination, i.e. good flowering synchronicity. For commercial orchards, and reducing the production risk, overlap in flowering should be stable from year to year, especially at the beginning of full flowering (figure 5; white fields). Many years of overlapping in full flowering of sweet cherry cultivars for a period of 5 8 days is the basis for a potential pollination (18). This overlap should be accompanied by a short span at the Romanian Biotechnological Letters, Vol. 20, No. 6, 2015 11003

SANJA RADIČEVIĆ, SLAĐANA MARIĆ, RADOSAV CEROVIĆ beginning of full flowering (up to 2 3 days). It should be also kept in mind that an earlier flowering cultivar is a better pollenizer for a cultivar with one or two days later flowering, than conversely. The phenomenon can be interpreted by the maturity of the female parts, with simultaneous immaturity of the male parts of flower at the time of flower opening, which emphasizes the advantages of previously flowering over later flowering pollenizers (9). The eight-year study indicated a good overlap in the full flowering phenophase of Kordia and Summit in similar agro-ecological conditions (28). Mid-early flowering cultivars (flowering time overlap with most of the other cultivars) which are self-compatible (universal pollenizers) can be a very important element of the cultivar compositions in sweet cherry orchards (16). In this sense, the results of our work point to Lapins and Celeste, which also have a significant commercial value in certain agro-ecological conditions (11, 17, 12). Our previous study revealed that Lapins has an excellent abundance of flowering and a high pollen germination in vitro (16). In contrast to this, cultivars with big differences in terms of flowering time cannot be recommended as suitable pollenizers, even when their classification according to incompatibility groups allows cross-pollination. Insufficient overlap implies a small number of days during full flowering, which is related to the end of a phenophase (figure 5; dark grey fields). Insufficient overlap in the full flowering may be a limiting factor in successful pollination and fertilization in certain seasons (figure 5; light grey fields). These cultivars could make suitable crosses in certain seasons and localities, if S-genotype requirements are satisfied. However, the risks of insufficient overlap in full flowering and, consequently, of low fruit setting remains. This is particularly pronounced when low temperature occurs at the flowering onset of a main cultivar, delaying the onset of pollenizer. The effect could be more pronounced when female cultivar has a short ovule longevity. 4. Conclusions Sweet cherry is a fruit species grown worldwide in order to produce fruits for human consumption, wherein fruit set, initiated by pollination and subsequent fertilization, is also an important aspect of the reproductive cycle. This study has provided valuable insights into the S-allelic constitutions and overlap in phenophase of full flowering of 13 newly introduced sweet cherry cultivars grown under the agro-environmental conditions of West Serbia, as essential preconditions for effective fertilization. Furhtemore, this represents a compendium for breeders to extend the list of sweet cherry cultivars that can be used as parents and to introduce interesting new traits (e.g. self-compatibility), as well as for growers in choosing appropriate pollenizers for economically important cultivars with excellent pomological and technological traits. It is apparent that the high polymorphism of S-RNase can be used for cultivar genotyping and identification, as well as that it is necessary in particular for quick and reliable identification of introduced cultivars. Acknowledgements This work was conducted under Research Project TR31064: Development and preservation of genetic potential of temperate zone fruits, supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia. 11004 Romanian Biotechnological Letters, Vol. 20, No. 6, 2015

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