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. 2018 Apr 27;9:527. doi: 10.3389/fpls.2018.00527

Optimizing Production in the New Generation of Apricot Cultivars: Self-incompatibility, S-RNase Allele Identification, and Incompatibility Group Assignment

Sara Herrera 1,†, Jorge Lora 2,†, José I Hormaza 2, Maria Herrero 3, Javier Rodrigo 1,*
PMCID: PMC5935046  PMID: 29755489

Abstract

Apricot (Prunus armeniaca L.) is a species of the Rosaceae that was originated in Central Asia, from where it entered Europe through Armenia. The release of an increasing number of new cultivars from different breeding programs is resulting in an important renewal of plant material worldwide. Although most traditional apricot cultivars in Europe are self-compatible, the use of self-incompatible cultivars as parental genotypes for breeding purposes is leading to the introduction of a number of new cultivars that behave as self-incompatible. As a consequence, there is an increasing need to interplant those new cultivars with cross-compatible cultivars to ensure fruit set in commercial orchards. However, the pollination requirements of many of these new cultivars are unknown. In this work, we analyze the pollination requirements of a group of 92 apricot cultivars, including traditional and newly-released cultivars from different breeding programs and countries. Self-compatibility was established by the observation of pollen tube behavior in self-pollinated flowers under the microscope. Incompatibility relationships between cultivars were established by the identification of S-alleles by PCR analysis. The self-(in)compatibility of 68 cultivars and the S-RNase genotype of 74 cultivars are reported herein for the first time. Approximately half of the cultivars (47) behaved as self-compatible and the other 45 as self-incompatible. Identification of S-alleles in self-incompatible cultivars allowed allocating them in 11 incompatibility groups, six of them reported here for the first time. The determination of pollination requirements and the incompatibility relationships between cultivars is highly valuable for the appropriate selection of apricot cultivars in commercial orchards and of parental genotypes in breeding programs. The approach described can be transferred to other woody perennial crops with similar problems.

Keywords: Prunus armeniaca, self-incompatibility, S-alleles, S-genotype, ovary, pollen tube, pollination, style

Introduction

Apricot (Prunus armeniaca L.) is considered as one of the most delicious temperate fruits (Faust et al., 1998). Apricot is a species of the Rosaceae, one of the most economically important plant families in temperate regions worldwide (Dirlewanger et al., 2004). Although the Latin name of apricot (armeniaca) and later its scientific name (P. armeniaca) could wrongly suggest an origin from Armenia, apricot was indeed originated in Central Asia, where the first orchards of apricot were described 406-250 BC (Janick, 2005), whereas Armenia was the route by which apricot first entered Europe. Apricot was already mentioned as Mela armeniaca by Roman authors around 50 A.D., which could indicate its introduction in the Roman empire during the first century (Faust et al., 1998). The English name apricot (apricock in the old spelling) derives from the Arabic and Greek term al-praecox that means “early fruit” (Faust et al., 1998; Janick, 2005). Traditionally, apricot cultivars have been classified in six main groups depending on the geographical origin: Dzhungar-Zailij, East Chinese, European Iranian-Caucasian, Middle-Asian, and North Chinese (Layne et al., 1996).

Apricot cultivars from the Central Asian, the Dzhungar-Zailij, and the Iranian-Caucasian groups are mostly self-incompatible. However, cultivars from the European group, which is the least variable and the most recent, are mainly self-compatible and include most of the commercial cultivars (Mehlenbacher et al., 1991; Hormaza et al., 2007). In the Rosaceae, the incompatibility mechanism to reduce self-fertilization and promote outcrossing is based on cell-cell recognition that is determined genetically by a gametophytic self-incompatibility System (GSI). This mechanism acts through the inhibition of pollen tube growth in the style (de Nettancourt, 2001) and is controlled by a multiallelic locus named S, encoding two linked genes that determine the pistil and pollen genotypes (Charlesworth et al., 2005). A ribonuclease (S-RNase), which is a glycoprotein secreted in the style mucilage, determines the allele specificity of the style (Tao et al., 1997) whereas an F-box protein (SFB) specifically expressed in pollen determines pollen allele specificity (Ushijima et al., 2003).

The introduction of an increasing number of new apricot cultivars from different breeding programs is resulting in an important renewal of plant material worldwide (Zhebentyayeva et al., 2012). Thus, the initial classification of six ecogeographical groups is becoming increasingly complex, since many of the new cultivars are derived from crosses between genotypes of different ecogeographical groups (Faust et al., 1998; Halász et al., 2007). Moreover, although most traditional apricot cultivars in Europe are self-compatible (Burgos et al., 1997), the use of self-incompatible cultivars developed in North America as parental genotypes in several breeding programs, with the objective of incorporating resistance to sharka (Hormaza et al., 2007; Zhebentyayeva et al., 2012), is leading to the introduction of new self-incompatible cultivars. As a consequence, there is an increasing need to interplant those new cultivars with cross-compatible cultivars to ensure fruit set in commercial orchards. However, the pollination requirements of many of these new cultivars are unknown.

The pollination requirements of a cultivar can be established by carrying out controlled pollinations in the field and recording the percentage of fruit set. Final fruit set in apricot is usually established during the first 4 weeks following pollination (Rodrigo et al., 2009; Julian et al., 2010). However, incompatibility can be determined more accurately under a fluorescence microscope by the observation of pollen tube growth through the style in self- and cross-pollinated flowers in squash preparations of pistils after staining with aniline blue (Burgos et al., 1993; Rodrigo and Herrero, 1996, 2002; Julian et al., 2010). In self-incompatible genotypes and incompatible crosses, pollen tube growth is arrested in the style and, therefore, fertilization of the ovules is prevented since no pollen tubes reach the ovary. However, in self-compatible genotypes and compatible crosses, pollen tubes can grow along the style and reach the ovary, where fertilization of some of the two ovules can take place. This histochemical approach allows the identification of pollination failure from diverse environmental factors that can affect fruit set under field conditions (Guerra and Rodrigo, 2015).

In addition, advances in the study of the molecular determinants of self-incompatibility have allowed developing tools to analyze the allelic composition of the self-incompatibility locus. Thus, the identification of the S-RNase gene in apricot (Romero et al., 2004; Sutherland et al., 2004) allowed developing an S-allele genotyping PCR strategy, similar to those developed for cherry or almond (Sutherland et al., 2004). To date, 33 S-alleles (S1 to S20, S22 to S30, S52, S53, Sv, and Sx), including one allele for self-compatibility (Sc), have been identified in apricot (Halász et al., 2005; Vilanova et al., 2005; Zhang et al., 2008; Muñoz-Sanz et al., 2017; Murathan et al., 2017), although additional alleles have been included in the NCBI database and not yet published. These studies allowed the determination of different apricot S-genotypes from different countries (Halász et al., 2010; Kodad et al., 2013a,b; Muñoz-Sanz et al., 2017) that are included in, up to now, 17 incompatibility groups (Szabó and Nyéki, 1991; Egea and Burgos, 1996; Halász et al., 2010; Lachkar et al., 2013).

Due to the increasing release of a high number of apricot cultivars in the last years with unknown self-incompatibility genotypes, in this work we analyze the pollination requirements of a group of 92 apricot cultivars, including traditional and new cultivars released from different breeding programs. Self-compatibility was established by the observation of pollen tube behavior under the microscope following self-pollination. Incompatibility relationships between cultivars were established by the identification of S-alleles by PCR analysis. The results obtained allowed assigning each cultivar to its corresponding incompatibility group.

Materials and methods

Plant material

Leaf and flower samples from 92 apricot cultivars, including traditional cultivars from different origins and new cultivars from different breeding programs (Table 1), were collected from diverse collections for pollination experiments and S-RNase genotyping.

Table 1.

Country of origin, number of pistils examined, percentage of pistils with pollen tubes halfway the style, at the base of the style, and reaching the ovule, percentage of style traveled by the longest pollen tube, mean number of pollen tubes at the base of the style, and self-incompatibility (SI) or self-compatibility (SC) of 92 apricot cultivars analyzed in this work.

Cultivar Country of origin Number of pistils examined Pistils (%) with pollen tubes Percentage of style traveled by the longest pollen tube Mean number of pollen tubes at the base of the style SI/SC
Halfway the style At the base of the style Reaching the ovule
AC1 USA 13 100 0 0 82 0 SI
ASF0401 France 17 94 0 0 65 0 SI
ASF0402 France 24 100 0 0 65 0 SI
Avirine (Bergarouge) France 13 100 0 0 62 0 SI
CA-26 (Almater) Spain 20 100 5 5 70 0 SI
Colorado Spain 30 90 0 0 64 0 SI
Cooper Cot USA 10 100 0 0 65 0 SI
Durobar (Almadulce) Spain 23 100 0 0 67 0 SI
Farely France 10 100 0 0 63 0 SI
Feria Cot France 10 100 0 0 78 0 SI
Flash Cot USA 10 70 0 0 54 0 SI
Flodea Spain 11 100 0 0 71 0 SI
Goldbar USA 20 100 0 0 62 0 SI
Goldrich USA 72 94 3 3 69 0 SI
Goldstrike 01a USA 40 100 0 0 71 0 SI
Goldstrike 02a USA 20 100 0 0 72 0 SI
Harcot Canada 44 95 0 0 62 0 SI
Hargrand Canada 49 100 14 14 77 0 SI
Henderson USA 47 91 15 9 75 0 SI
Holly Cot France 20 100 0 0 61 0 SI
JNP Spain 20 100 5 5 75 0 SI
Lilly Cot USA 47 96 2 0 67 0 SI
Magic Cot USA 30 100 0 0 65 0 SI
Maya Cot France 10 100 0 0 66 0 SI
Medaga France 10 100 0 0 71 0 SI
Megatea Spain 10 100 0 0 62 0 SI
Moniqui Spain 18 100 6 0 79 0 SI
Monster Cot USA 10 100 0 0 70 0 SI
Muñoz Spain 21 100 0 0 72 0 SI
Orangered USA 10 90 0 0 64 0 SI
Pandora Greece 23 100 4 0 75 0 SI
Peñaflor 01a Spain 29 100 7 7 71 0 SI
Perle Cot USA 28 93 4 0 72 0 SI
Pinkcot France 34 97 9 0 83 0 SI
Priabel France 10 90 10 0 81 0 SI
Robada USA 25 96 0 0 63 0 SI
Spring Blush France 40 83 3 3 55 0 SI
Stark Early Orange USA 51 98 33 16 87 0 SI
Stella USA 13 100 23 15 85 0 SI
Sun Glo USA 64 100 2 0 71 0 SI
Sunny Cot USA 10 100 0 0 65 0 SI
Sweet Cot USA 20 95 0 0 66 0 SI
Vanilla Cot USA 20 100 0 0 79 0 SI
Veecot Canada 29 100 3 3 74 0 SI
Wonder Cot USA 37 100 0 0 69 0 SI
AC2 USA 10 100 100 100 100 2.2 SC
Aprix 20 Spain 15 100 73 53 100 1.4 SC
Aprix 33 Spain 10 100 100 100 100 1.1 SC
Aprix 9 Spain 14 100 86 64 100 2.1 SC
ASF0404 (Apriqueen) France 22 100 91 91 100 3 SC
Berdejo Spain 10 100 100 100 100 1.9 SC
Bergecot France 20 100 95 95 100 2.5 SC
Canino Spain 29 100 100 83 100 2.0 SC
Charisma South Africa 23 100 100 100 100 3 SC
Corbato Spain 60 100 98 93 100 3.2 SC
Delice Cot France 15 100 87 53 100 1.1 SC
Faralia France 11 100 100 100 100 2 SC
Farbaly France 22 100 86 77 100 2.0 SC
Farbela France 6 100 100 100 100 1.8 SC
Farclo France 9 100 100 89 100 1.4 SC
Fardao France 9 100 100 100 100 4.1 SC
Farfia France 10 100 100 100 100 3 SC
Farhial France 10 100 100 100 100 3 SC
Farius France 12 100 100 100 100 2 SC
Farlis France 22 100 100 100 100 2 SC
Fartoli France 10 100 100 100 100 3 SC
Flopria Spain 10 100 100 100 100 2 SC
Golden Sweet USA 21 100 95 95 100 2 SC
Lady Cot France 26 100 77 77 100 2.3 SC
Lorna USA 17 100 100 100 100 3 SC
Luizet France 10 100 90 80 100 1 SC
Medflo France 8 100 100 100 100 1.9 SC
Mediabel France 12 100 100 100 100 1.2 SC
Mediva France 9 100 89 89 100 2.3 SC
Mirlo Anaranjado Spain 10 100 100 100 100 2.1 SC
Mirlo Blanco Spain 10 100 100 100 100 2 SC
Mitger Spain 50 100 100 100 100 2.4 SC
Palsteyn South Africa 30 100 100 100 100 3 SC
Paviot France 12 100 91 91 100 1.2 SC
Peñaflor 02a Spain 6 100 83.3 66.6 100 1.4 SC
Pepito del Rubio Spain 12 100 100 90 100 2.2 SC
Playa Cot France 10 100 100 70 100 1.7 SC
Pricia France 9 100 100 100 100 2 SC
Primidi France 9 89 78 78 100 2 SC
Rouge Cot France 10 100 90 70 100 1.55 SC
Rubista France 19 95 89 89 100 1.7 SC
Sandy Cot France 10 100 100 100 100 2.3 SC
Soledane France 21 100 100 100 100 3 SC
Swired Switzerland 9 100 100 90 100 1.8 SC
Tadeo Spain 36 100 97 97 100 2.4 SC
Tom Cot USA 10 100 100 100 100 3 SC
Victor 1 14 100 93 93 100 2.1 SC
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a

Diverse origin.

Pollination experiments

To explore self-(in)compatibility, self-pollinations of the 92 apricot cultivars were carried out in the laboratory. Pollen tube growth was observed in self-pollinated flowers under the microscope (Table 1). As control, a group of flowers of each cultivar were cross-pollinated with pollen from “Canino” or “Katy,” which are considered as universal pollinizers for apricot (Zuriaga et al., 2013).

Pollen was extracted from flowers collected at the balloon stage. For this purpose, the anthers were removed and dried at laboratory temperature during 24 h. After that, pollen grains were sieved by using a fine mesh (0.26 mm) and used immediately or frozen at −20°C until further use. Pistils were obtained from flowers collected 1 day before anthesis, at balloon stage. After the removing of petals, sepals and stamens, the pistils were maintained on wet florist foam at laboratory temperature (Rodrigo and Herrero, 1996). For each self- and cross-pollination, a group of 20–25 flowers were hand pollinated with the help of a paintbrush 24 h after emasculation. After 72 h, they were fixed in ethanol (95%)/acetic acid (3:1, v/v) during 24 h, and conserved at 4°C in 75% ethanol (Williams et al., 1999). When observations of pollen tube growth were not clear, each cross was repeated every year during the flowering period up to 4 years. In order to evaluate pollen viability, after hand pollination, pollen from each cultivar was scattered on a solidified pollen germination medium (Hormaza et al., 1996). After 24 h, preparations were observed under the microscope. Pollen grains were considered viable when the length of the growing pollen tubes was higher than the pollen grain diameter.

For histochemical preparations, the pistils were washed three times for 1 h with distilled water and left in 5% sodium sulphite at 4°C for 24 h. Then, to soften the tissues, they were autoclaved at 1 kg/cm2 during 10 min in sodium sulphite (Jefferies and Belcher, 1974). To stain callose, the softened pistils, were stained with 0.1% (v/v) aniline blue in 0.1 N K3PO4 (Linskens and Esser, 1957). The observation of pollen tube behavior along the style was performed by a Leica DM2500 microscope (Cambridge, UK) with UV epifluorescence using 340–380 bandpass and 425 longpass filters. The percentage of style traveled by the longest pollen tube and the mean number of pollen tubes at the base of the style were recorded on at least 10 pistils in each cross. Cultivars were considered as self-incompatible when pollen tube growth was arrested in the style in most pistils from self-pollinated flowers, and as self-compatible when more than half of the pistils displayed at least one pollen tube reaching the base of the style.

DNA extraction

For the identification of S-alleles, young leaves were collected in spring. Genomic DNA from 92 cultivars (Table 2) was isolated following the protocol described by Hormaza (2002) and using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). NanoDrop™ ND-1000 spectrophotometer (Bio-Science, Budapest, Hungary) was used to measure DNA concentrations to analyze the quantity and quality of DNA.

Table 2.

Incompatibility group (I.G.) and S-RNase genotype of 92 apricot cultivars analyzed in this study and 30 additional cultivars analyzed in previous studies.

I.G. S-RNase genotype Cultivars analyzed in this study Cultivars analyzed in previous studies References
I S1S2 AC1x
Hargrand 2, 5, 14
Katy 13, 14
Goldrich 1, 2, 3, 5, 6, 10, 13, 14
Castleton 14
Farmingdale 9
Giovanniello 9
Lambertin-1 2, 14
II S8S9 Pinkcotx
Perle Cotx
Ceglédi óriás 5, 8
Cologlu 12
Kadioglu 12
Ligeti óriás 5, 8
Seftalioglu 12
Szegedi M. 14
III S2S6 ASF0401x
Avirine (Bergarouge)x
Moniqui 2, 6, 14
Iri Bitirgen 12
V S2S8 Holly Cotx
Sweet Cotx
Alyanak 12
Ziraat Okulu 12
VIII S6S9 Orangeredw 14
ASF0402x
Wonder Cotx
Stark Early Orangew 14
Feria Cotx
Sunny Cotx
JNPx
Cataloglu 12
Ozal 12
Soganci 12
XVIIIz S1S3 Cooper Cotx
Perfection 1
XIXz S2S3 Mayacotx
Sun Glo 2, 3, 4, 6
XXz S2S9 Magic Cotx
Goldstrike 02v,x
Hasanbey 12
XXIz S3S8 Spring Blushx
Lilly Cotx
Kayseri P.A 12
XXIIz S3S9 Durobar (Almadulce)x
Hendersonw 14
Flodeax
Akcadag Günay 12
XXIIIz S7S9 Goldbarx
Kurukabuk 12
S2 Pandorax
Veecot
Muñozx
Peñaflor 01v, x
Hardgrand
Goldrich
Búlida 9
Lornay 9
Perla 9
S3 Coloradoy
Ninfa 9
S4 Harcot
S6 Stella
S8 Vanilla Cotx
Robadax
Katy 7
Krimskyi Medunec 5
S9 Flash Cotx
Goldstrike 01v,x
CA-26 (Almater) x
Farelyx
Medagax
Megateax
Monster Cotx
Priabelx
Self-compatible cultivars S2Sc Berdejox
Canino 3, 10, 13
Paviotx
Pepito del Rubio 2, 3
Peñaflor 02v,x
Bergecotx
Medivax
Primidix
Sandy Cotx
S3Sc Priciax
Rubistax
S6Sc Aprix 20x
Aprix 9x
Faraliax
Farlisx
Medflox
Mediabelx
S7Sc Charismax
S9Sc AC2x
Flopriax
Tom Cotx
Sc Soledanex
ASF0404 (Apriqueen)x
Mirlo Blanco 11
Mitger
Tadeo
Corbatoy
Aprix 33x
Delice Cotx
Farbalyx
Farbelax
Farclox
Fardaox
Farfiax
Farhialx
Fariusx
Fartolix
Lady Cotx
Mirlo Anaranjado
Luizetx
Playa Cotx
Swiredx
Rouge Cotx
S1S2 Lornax,y
Palsteynx,y
S2S9 Victor 1x
S3 Golden Sweetx
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(1) Egea and Burgos (1996); (2) Burgos et al. (1998); (3) Alburquerque et al. (2002); (4) Sutherland et al. (2004); (5) Halász et al. (2005); (6) Vilanova et al. (2005); (7) Chen et al. (2006); (8) Halász et al. (2007); (9) Donoso et al. (2009); (10) Raz et al. (2009); (11) Egea et al. (2010); (12) Halász et al. (2010); (13) Zuriaga et al. (2013); (14) Muñoz-Sanz et al. (2017).

v

Diverse origin.

w

S9 and S17 have been considered the same allele.

x

S-RNase genotypes first reported in this study.

y

Cultivars in which S-RNase genotype reported herein differs from that reported in other studies.

z

Incompatibility groups first reported in this study.

S-RNase allele identification by PCR analysis

Amplification reactions for the first intron region of the S-RNase gene were carried out with the combination of the fluorescently labeled forward primer SRc-F (5′-CTCGCTTTCCTTGTTCTTGC-3′) with the reverse primer SRc-R (5′-GGCCATTGTTGCACAAATTG-3′; Romero et al., 2004; Vilanova et al., 2005). PCR amplifications were carried out in 15 μl reaction volumes, containing 10x NH4 Reaction Buffer, 25 mM Cl2Mg, 2.5 mM of each dNTP, 10 μM of each primer, 100 ng of genomic DNA and 0.5 U of BioTaq™ DNA polymerase (Bioline, London, UK). The temperature profile used had an initial step of 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 55°C and 3 min at 72°C, and a final step of 5 min at 72°C.

The sizes of the products obtained by PCR were analyzed in a CEQ™ 8000 capillary electrophoresis DNA analysis system (Beckman Coulter, Fullerton, CA, USA) and compared and classified according to Vilanova et al. (2005) and Kodad et al. (2013b). Primers Pru-C2 (5′-CTTTGGCCAAGTAATTATTCAAACC-3′) and Pru-C4R (5′-GGATGTGGTACGATTGAAGCG-3′) were used for the amplification of the second intron region as recommended by Vilanova et al. (2005), but with the addition of 10 cycles and using 55°C of annealing temperature as indicated by Sonneveld et al. (2003). Amplified fragments of the second intron were separated on 1% (w/v) agarose gels and DNA bands were visualized using the nucleic acid stain SYBR Green (Thermo).

Sequencing of genomic PCR products

Two PCR fragments of 420 and 430 bp obtained by the automatic sequencer were isolated using the NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). Cloning was performed using CloneJET PCR Cloning Kit (Thermo) and by electroporation in E. coli Single-Use JM109 Competent Cells (Promega). The search for similarities in the sequences of the NCBI database was performed with BLAST (http://www.ncbi.nlm.nih.gov/BLAST, version 2.2.10). The 420 bp fragment resulted in a fragment of 414 bp after one sequencing reaction whereas the initial 430 bp fragment resulted in a fragment of 421 bp after two sequencing reactions.

Results

Pollination experiments

Self-compatibility of 92 apricot cultivars was established by the observation of pollen tube behavior in pistils under the microscope after self-pollinations (Table 1). Germinated pollen grains were observed in the stigma (Figure 1A) in all the pollinations performed. The establishment of self-incompatibility or self-compatibility could be carried out for all the cultivars. Approximately half of the cultivars (47) behaved as self-compatible, displaying most pistils with pollen tubes growing along the style (Figure 1B) and at least one pollen tube reaching the base of the style (Figure 1C). On the other hand, in 45 cultivars pollen tubes arrested their growth in the style (Figure 1D) and no pollen tubes reached the base of the style in most of the pistils. Consequently, these cultivars were considered as self-incompatible. As expected, in all cross-pollinations pistils displayed pollen tubes at the base of the style. Between one and four pollen tubes at the base of the style were observed in self-compatible cultivars.

Figure 1.

Figure 1

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Pollen tube growth in self-pollinated apricot flowers. (A) Pollen grains germinating at the stigma surface. (B) Pollen tubes growing along the style. (C) Pollen tubes reaching the base of the style. (D) Pollen tube arrested in the style. Scale bars, 100 μm.

S-RNase allele PCR analysis

To confirm the results obtained in the pollination experiments, PCR analyses using specific primers from conserved regions of the apricot S-RNase locus were used to identify the S-RNase alleles of 92 apricot cultivars (Table 2). The information reported herein has been compiled with the S-RNase genotype of 30 additional cultivars previously determined showing the compatibility relationships among all the cultivars whose S-RNase genotype is known (Table 2). Cultivars have been allocated according their S-RNase alleles in 11 incompatibility groups, six of them reported here for the first time. Some cultivars with previously reported S-genotypes were initially used to confirm the size of S-RNase alleles previously identified using the primer pairs SRc-F and SRc-R (Vilanova et al., 2005) that amplify the first intron of the apricot S-RNase and allowed identifying the S-RNase alleles of the rest of the cultivars analyzed (Figure 2).

Figure 2.

Figure 2

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Gene structure of the P. armeniaca RNase gene. Genomic sequence of the S4 allele showing the exons in green square, the primers used for the identification of S-alleles and the five conserved regions (C1, C2, C2, C3, RC4, and C5), and one hypervariable region (RHV) in blue square.

Two alleles, S1 and S7, could not be distinguished with the primers SRc-F/SRc-R, since these alleles showed similar fragment sizes in the first intron (Table 3). Thus, the PruC2/PruC4R primer combination designed from P. avium S-RNase-cDNA sequences (Tao et al., 1999) was additionally used to amplify the second intron. The alleles Sc and S8 had also similar fragment sizes in the first intron, and, in this case, the self-compatibility or self-incompatibility observed in the pollination experiments was used to distinguish between both alleles in each cultivar. A fragment of 420 or 430 bp was detected in some of the cultivars. These band sizes are close to the S6 allele, which has been reported as 424 bp (Kodad et al., 2013b) or 423 bp (Halász et al., 2010). To elucidate if the 430 bp and 420 bp bands obtained by an automatic sequencer correspond to new or pre-existing alleles, both fragments were cloned and sequenced, resulting in fragments of 421 and 414 bp, respectively.

Table 3.

S-alleles identified or/and sequenced in Prunus armeniaca.

Alleles Gen bank accession Sequence Fragment 1st intron Fragment 2nd intron References
Sc EF491872/DQ386735 Partial CDS, 1st intron 355a,e 2800c Halász et al., 2007
S1 AY587561 CDS, 1st and 2nd intron 408a,e 2260b,e Romero et al., 2004
S2 AY587562 CDS, 1st and 2nd intron 334a,e 990b Romero et al., 2004
S3 274a,e ~450b,e Vilanova et al., 2005
S4 AY587564 CDS, 1st and 2nd intron 249a,e 247b,e Romero et al., 2004
S5 375a 1400b Vilanova et al., 2005
S6/S52 KF951503 (S52) CDS, 1st and 2nd intron 421a,e 1386b,e Unpublished
S7 408a,e 900b,e Vilanova et al., 2005
S8 AY884212 Partial CDS, 2nd Intron 355a,e 691b Feng et al., 2006; Halász et al., 2007
S9 AY864826 Partial CDS 414a,e 749b,e Feng et al., 2006
S10 AY846872 Partial CDS, 2nd Intron 583d Feng et al., 2006
S11 DQ868316 Partial CDS, 2nd Intron 464d Zhang et al., 2008
S12 DQ870628 Partial CDS, 2nd Intron 360d Zhang et al., 2008
S13 DQ870629 Partial CDS, 2nd Intron 401d Zhang et al., 2008
S14 DQ870630 Partial CDS, 2nd Intron 492d Zhang et al., 2008
S15 DQ870631 Partial CDS, 2nd Intron 469d Zhang et al., 2008
S16 DQ870632 Partial CDS, 2nd Intron 481d Zhang et al., 2008
S17 DQ870633 Partial CDS, 2nd Intron 487d Zhang et al., 2008
S18 DQ870634 Partial CDS, 2nd Intron 1337d Zhang et al., 2008
S19 EF133689 Partial CDS, 2nd Intron 546d Zhang et al., 2008
S20 EF160078 Partial CDS, 2nd Intron 1934d Zhang et al., 2008
S22 HM053569 Partial CDS, 2nd Intron 550d Unpublished
S23 EU037262 Partial CDS, 2nd Intron 505d Wu et al., 2009
S24 EU037263 Partial CDS, 2nd Intron 168d Wu et al., 2009
S25 EU037264 Partial CDS, 2nd Intron 583d Wu et al., 2009
S26 EU037265 Partial CDS, 2nd Intron 289d Wu et al., 2009
S27 EU836683 Partial CDS, 2nd Intron 230d Wu et al., 2009
S28 EU836684 Partial CDS, 2nd Intron 948d Wu et al., 2009
S29 EF185300 Partial CDS, 2nd Intron 285d Wu et al., 2009
S30 EF185301 Partial CDS, 2nd Intron 956d Wu et al., 2009
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a

Amplified using SRc-(F/R).

b

Amplified using Pru-C2 and Pru-C4R.

c

Amplified using Pru-C2 and Pru-C3R.

d

Amplified using other primers.

e

Our results.

The 421 bp fragment showed a 99% identity with the S52 present in the NCBI database. This allele was initially included in the NCBI database and unpublished but, recently, it has been reported in some Turkish apricot cultivars (Murathan et al., 2017). Since the S6 allele had not been previously sequenced, the S52 allele could indeed correspond to the S6 allele. The S6 allele could also be identified with the primers Pru-C2/Pru-C4, showing a PCR-fragment of around 1400 bp (1386 bp) that included the second intron; a 1386 bp fragment was also amplified in the S52 allele with the same primer combination strongly suggesting that S6 and S52 could be the same allele. Thus, in this work, the 421 bp fragment was assigned to the S6 allele.

The sequence of the 414 bp fragment showed high sequence similarity to S-alleles from other Prunus species, but not to any S-allele of Prunus armeniaca present in NCBI databases. The second intron of this allele was amplified with the primers Pru-C2/Pru-C4, showing a PCR-fragment of around 700 bp. Its cloning, sequencing and alignment revealed a 99% identity with the S9 allele [AY853594 (Feng et al., 2006)] and, consequently, the 414 bp fragment was assigned to the S9 allele.

The 45 self-incompatible cultivars were grouped in incompatibility groups according to their S genotypes following the numbering proposed by Halász et al. (2010) and Lachkar et al. (2013). While 26 of the cultivars analyzed were assigned to 11 different incompatibility groups, 19 cultivars were not assigned since only one S-RNase allele was detected (Table 2). S2 was the most frequent allele, appearing in 22 cultivars, followed by Sc (21), S9 (19), S6 (16), S3 (10), S8 (6), and S1 (4), while S7 was the least frequent allele found in only two cultivars.

Discussion

Self-pollination of the 92 apricot cultivars analyzed in this work and observation of pollen tubes under the microscope showed that 47 behaved as self-compatible and 45 as self-incompatible. The self-(in)compatibility of 68 cultivars is reported herein for the first time. The results in the remaining 23 cultivars have been compared with previous reports in which self-(in)compatibility was determined by the evaluation of the percentage of fruit set after self-pollinations in the field (Egea and Burgos, 1996; Rodrigo and Herrero, 1996; Burgos et al., 1997; Egea et al., 2010; Muñoz-Sanz et al., 2017) or by the observation of pollen tube growth in pistils after self- and cross-pollinations (Egea and Burgos, 1996; Rodrigo and Herrero, 1996; Egea et al., 2010; Milatovic et al., 2013a,b). Thus, results herein agree with previous reports for “Canino,” “Corbato,” “Luizet,” “Mirlo Blanco,” “Mirlo Anaranjado,” “Mitger,” “Palsteyn,” “Paviot,” “Pepito del Rubio,” “Tadeo,” and “Tom Cot” as self-compatible, and also for “Bergarouge,” “Goldrich,” “Goldstrike,” “Harcot,” “Hargrand,” “Moniqui,” “Orangered,” “Pinkcot,” “Robada,” “Stark Early Orange,” “Stella,” “Sun Glo,” and “Veecot” as self-incompatible.

Approximately half of the cultivars analyzed (49%) were self-incompatible, a very high percentage compared to the situation some years ago when most European cultivars were self-compatible (Mehlenbacher et al., 1991), including the most traditional cultivars (Burgos et al., 1997). The other half (51%) were self-compatible. Due to this dramatic increase in the number of self-incompatible apricot cultivars, knowing their pollination requirements is necessary in order to choose compatible pollinizers in designing new commercial orchards as well as in selecting parental genotypes in apricot breeding programs.

The first case of cross-incompatibility of apricot cultivars in the European group was reported in the early 1990s in the Spanish cultivar Moniqui (Egea et al., 1991). The first incompatibility group in apricot, which consisted of three North American cultivars, was established several years later based on microscopic observations (Egea and Burgos, 1996), and, later, a more extensive study with 123 apricot cultivars, reported self-incompatibility in 42 cultivars (Burgos et al., 1997). Afterward the S-RNase proteins of seven S-alleles (S1–S7) and one allele associated with self-compatibility (Sc) were identified; the proteins were separated by non-equilibrium pH gradient electrofocusing (NEpHGE) in a gel that was later stained for ribonuclease activity (Alburquerque et al., 2002). The identification of the gene involved in GSI allowed S-allele identification by PCR analysis (Romero et al., 2004). In this first study, three S-alleles were sequenced (S1, S2, and S4) (Romero et al., 2004). In a later study, S-allele genotyping using the SRc-F and SRc-R primers, which have also been used in our study and amplify the first intron, identified four self-incompatibility alleles (S3, S5, S6, and S7) and one allele for self-compatibility (Sc) (Vilanova et al., 2005). Nine additional S-alleles (S8–S16) were identified by Halász et al. (2005) in 23 apricot accessions, mostly from Hungary. These last 13 alleles (S3, S5–S16) were only identified by PCR analysis. From then on, several studies have identified additional S-alleles by sequencing [S9 and S10 (Feng et al., 2006); S11–S20 (Zhang et al., 2008); S23–S30 (Wu et al., 2009)], but some of them have only been included in the NCBI database and not yet published. Some unpublished alleles such as S52 (Murathan et al., 2017)/S6 (our results) and S22 (Muñoz-Sanz et al., 2017), have already been associated to several cultivars. Most of these studies have been performed in apricot cultivars from different origins like China, Turkey, Hungary, or Spain and, in some cases, the use of different primers or just the inaccuracy of band identification in a gel or even analyzed by an automatic fragment analyzing system can result in misidentification and the appearance of numerous homologies (Muñoz-Sanz et al., 2017). For example, differences in reported fragment size can be found in the S2 [334 bp herein; 327 bp in Vilanova et al. (2005); 332 bp in Kodad et al. (2013b)] or Sc [358 bp herein; 353 bp in Vilanova et al. (2005); 355 bp in Kodad et al. (2013b)] alleles in addition to the S6 allele mentioned above. Moreover, many of the S-alleles, such as S10–S30 alleles, in which the first intron is still unknown, have only been partially sequenced.

From the 92 cultivars analyzed herein, 74 have been reported for the first time. Two alleles could be identified in most cultivars. However, a single allele was identified in 19 self-incompatible cultivars that may be due to inefficient PCR amplification of the S-RNase allele, in which the PCR primers may have a preferential amplification of the detected allele or caused by mismatching of PCR primers. Alternatively, the similar size of two alleles that show overlapping PCR fragments could make their identification difficult. Thus, the identification of additional S-RNase alleles in these genotypes requires more work focused on characterizing the S-locus. In 22 self-compatible cultivars, the identification of a unique allele could also be due to homozygosis of the self-compatible allele (Sc). Higher S-allele frequencies in the cultivars analyzed correspond to alleles S2, Sc, and S9 (19–22%). The allele Sc is associated with self-compatibility, and the alleles S2 and S9 are present in different cultivars resistant to sharka from USA (“Goldrich,” “Henderson,” “Orangered,” and “Sun Glo”) and Canada (“Hargrand” and “Veecot”), which have been used as parental genotypes in different breeding programs (Hormaza et al., 2007; Zhebentyayeva et al., 2012). On the other hand, the allele S7 is the least frequent and is present in only two cultivars: “Charisma” from South Africa, and “Goldbar” from USA.

Results herein, while confirmed the S-RNase genotype of 16 cultivars reported in previous studies, showed differences in the S-RNase genotype of “Corbato,” “Colorado,” “Lorna,” and “Palsteyn” reported previously (Burgos et al., 1998; Alburquerque et al., 2002; Vilanova et al., 2005; Donoso et al., 2009; Raz et al., 2009; Muñoz-Sanz et al., 2017). To clarify their S-genotype, it would be necessary to identify their S-alleles in additional samples of the same cultivars. In this sense, sequencing can reveal the differences between a band fragment of an initial size, such as in our case, in which the initial fragment of 420 and 430 bp resulted in a S-allele of 414 bp (S9) and 421 bp (S6) respectively, after sequencing. Moreover, the sequence of the S6 (fragment of 421 bp) could reveal its similarity to S52 that has been recently assigned to Turkish apricot cultivars (Murathan et al., 2017).

The self-incompatibility identification has allowed allocating the cultivars to their corresponding incompatibility groups. This information, compiled with those reported in previous studies, has allowed describing six new incompatibility groups (XVIII–XXIII). Thus, self-incompatible cultivars within the same incompatibility group have the same S-genotype and are genetically incompatible with each other. On the other hand, cultivars with at least one different S-allele are placed in different incompatibility groups and are inter-compatible.

Although self-incompatibility was observed in nearly half of the cultivars analyzed herein, self-compatibility was still found in a good number of apricot cultivars. Self-compatibility has been related to particular S-alleles in different self-incompatible Prunus species, as almond (Fernández i Martí et al., 2014; Company et al., 2015), Japanese apricot (Ushijima et al., 2003), peach (Tao et al., 2006; Hanada et al., 2014), sour cherry (Prunus cerasus L.) (Yamane et al., 2003), and sweet cherry (Wunsch and Hormaza, 2004; Marchese et al., 2007; Cachi and Wünsch, 2014). The breakdown of the incompatibility system has been reported in these Prunus species affecting the function of S-locus in the stylar S-determinant (S-RNase) and in the pollen S-determinant (F-box protein, SFB). It has also been related to mutations outside the S-locus (Hegedus et al., 2012; Company et al., 2015). In apricot, self-compatibility has been related with the insertion of a 358-bp fragment in the S-haplotype-specific F-box (SFB) gene. This insertion has been reported in self-compatible Spanish (Vilanova et al., 2006) and Hungarian (Halász et al., 2007) cultivars and, since they only differ in two nucleotides in the intron region, a common origin for them has been suggested (Halász et al., 2007). Moreover, a detailed study on the S8 allele, a very common allele in Hungarian cultivars, suggests that Sc could derive from the S8 allele (Halász et al., 2007). Thus, the insertion of 358-bp in the SFB gene is only present in the Sc haplotype and both alleles can only be distinguished by primers designed based on the SFB sequence such as the degenerate primers AprSFB-F1/R (Halász et al., 2007) or, as in our case, by pollination experiments.

The microscopy observations herein revealed self-compatibility in “Lorna” and “Palsteyn,” cultivars with the S1S2 genotype, “Victor 1” with the genotype S2S9, and “Golden Sweet” with the allele S3, but the Sc allele was not reported. A different mutation that is not linked to the S-locus has also been related to self-compatibility with a loss of pollen S-activity (Vilanova et al., 2006) and it has recently been associated with the M-locus (Zuriaga et al., 2013; Muñoz-Sanz et al., 2017). Due to its limited distribution, it has been suggested that the M-locus would be in a very early stage of dispersion (Muñoz-Sanz et al., 2017). Interestingly, the Sc allele is generally found in cultivars with the m-haplotype suggesting that this could be the result of a relaxed constrain for self-compatible selection (Muñoz-Sanz et al., 2017). A similar S-genotype is also found in self-compatible “Katy,” in which the compatibility was associated to the M-locus (Zuriaga et al., 2013). A further analysis could reveal if self-compatibility in “Lorna,” “Palsteyn,” “Victor 1,” and “Golden Sweet” is also related to the M-locus.

Studies on S-allele identification can also provide information on the genetic diversity of the species. Thus, Chinese cultivars are mostly self-incompatible with a higher number of S-alleles (Zhang et al., 2008) compared with the limited number of S-alleles found in Western countries (Halász et al., 2007). A study on genetic diversity using AFLP markers showed a decreasing genetic diversity of apricot cultivars from the former USSR to Southern Europe (Hagen et al., 2002; Halász et al., 2007), which is coherent with the Asian origin of the species. However, although most traditional European cultivars are self-compatible (Burgos et al., 1997), due to breeding and crosses with Asian cultivars, the number of self-incompatibility cultivars is recently increasing in Western countries (Muñoz-Sanz et al., 2017; Murathan et al., 2017) and our study confirms this trend. Moreover, our results also show six new incompatibility groups in addition to the initial 17 incompatibility groups described so far.

Thus, the results obtained in this work using pollen tube growth observation in self-pollinated pistils, has allowed establishing the self-compatibility or self-incompatibility of 92 cultivars, including traditional and the main current cultivars as well as new cultivars from different breeding programs. S-RNase allele identification has allowed the allocation of a number of cultivars to their corresponding incompatibility groups, determining the incompatibility relationships between cultivars. The combination of these two complementary approaches results in valuable information for the appropriate selection of cultivars in commercial orchards and for the selection of parental genotypes in apricot breeding programs and a similar approach could be used in other woody perennial crops.

Author contributions

JR, JH, and MH: conceived the study; SH, JL, JR, JH, and MH: designed the experiments and wrote the paper; SH and JL performed the microscope observations, the PCR analysis, and analyzed the data; SH and JL contributed equally to this work.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Erica Fadón, Reyes López, and Yolanda Verdún for technical assistance. We gratefully acknowledge AFRUCCAS for providing plant material used in this study.

Footnotes

Funding. This work was supported by Ministerio de Economía, Industria y Competitividad (MEIC) - European Regional Development Fund, European Union (AGL2012-40239, AGL2013-43732-R, AGL2016-77267-R, and AGL2015-74071-JIN); Instituto Nacional de Investigación Agraria (RFP2015-00015-00, RTA2014-00085-00; RTA2017-00003-00); Gobierno de Aragón - European Social Fund, European Union (Grupo Consolidado A12_17R) and Agroseguro S.A.

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