Inheritance of S-genotypes in Paviot × Kabaasi apricot F₁ progenies

ABSTRACT

Self-incompatibility plays an important role in the fertilization of fruit species such as apricot. Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility, which is controlled by a multi-allelic S-locus. In this study, S-alleles of 77 F₁ progenies derived from Paviot, which is one of the French local cultivars, and Kabaasi, one of the most important Turkish dried apricot cultivars, parents were identified by S-RNase intron regions polymerase chain reaction (PCR) amplification and DNA sequencing. The results from the S-allele PCR analysis revealed that the Paviot female parent had an S_cS₂ genotype and the Kabaasi male parent had S₁S₉ alleles. Forty-three of the F₁ progenies showed self-compatibility allele (S_c) by having either S_cS₉ or S_cS₁ alleles. Thirty-four of the F₁ progenies were self-incompatible by having either S₂S₁ or S₂S₉ alleles. The distributions of detected alleles in F₁ progenies were determined as follows: S_cS₁ 31.2%, S₁S₂ 27.3%, S_cS₉ 24.7% and S₂S₉ 16.8%. The results from the study are relevant for the data obtained in apricot breeding programmes in the selection of crossing combinations and in the establishment of commercial orchards.

KEYWORDS:

• Prunus armeniaca
• Paviot
• Kabaasi
• F₁ progenies
• self-incompatibility
• S-genotypes

Introduction

Apricot is one of the most important fruit species due to its commercial importance. Turkey ranks first in the world in apricot production with approximately 676.000 metric tons annually, with the apricot production fluctuating from year to year due to spring frosts.[1] In recent years, molecular techniques are widely used to characterize fruit tree germplasm resources. DNA molecular markers are highly effective and informative in the assessment of the genetic diversity and genetic relationships of fruit trees.[2–6]

Self-incompatibility in Prunus species, including apricot, is controlled by a simple multi-allelic S-locus that rejects its own pollen and has a homomorphic, gametophytic self-incompatibility system.[7] Self-incompatibility alleles allow pollen tube growth in the pistil and access to the ovaries. In cases where the same allele disputes exist on haploid pollen and diploid pistil, the growth of pollen tubes is blocked and a dispute arises due to the S-allele-specific S-RNase gene ribosomal RNA degradation system.[8–11] The effects of the self-incompatibility mechanism force pollination with foreign pollens to occur; and thus the emergence of genetic diversity and an increase in heterozygosity.[12]

Many European apricot cultivars are self-compatible, whereas those in Central Asia and Iran and the Caucasus, including Turkey, are self-incompatible.[13] Prominent apricot cultivars grown in Turkey are located within the Iran–Caucasian eco-geographical group. The apricot cultivars of economic importance in Turkey are found to be, generally, self-incompatible.[2,14–16]

Self-incompatibility plays an important role in the fertilization of fruit species such as apricot. Self-incompatible apricot cultivars need suitable pollinators for fructification.[17,18] A breeding target is to breed self-compatible apricot cultivars having high fruit quality. Therefore, it is imperative to determine the S-alleles of apricot cultivars.[19]

The self-incompatibility mechanism can be determined using conventional methods such as pollination tests and pollen tube growth tests. However, these tests are labour-intensive and time-consuming, and also influenced by environmental factors.[20,21] The use of molecular techniques to reveal S-alleles in genotypes has recently become the most commonly used method that gives the best results, as it allows early selection.[18]

In this study, we report S-alleles of Paviot, which is one of the French local cultivars, and Kabaasi, one of the most important Turkish dried apricot cultivars, by S-RNase intron region polymerase chain reaction (PCR) amplification and DNA sequencing. In addition, the segregation of S-alleles in Paviot × Kabaasi F₁ populations is also reported.

Materials and methods

Plant material

The plant materials were provided by the Apricot Research and Application Central Directorate of Agricultural Center of İnönü University. In this study, 77 F₁ progenies and their parents (Paviot and Kabaasi) were used. F₁ progenies were produced in a project supported by TUBITAK [project no: TOGTAG-3099] in the period 2003–2005. Leaf samples from each plant were stored at 4 °C after lyophilization.

DNA extraction

DNA isolation from leaf samples was performed using the CTAB (cetyl trimethyl ammonium bromide) protocol according to Doyle and Doyle [22] with minor modifications.[23] The concentration of isolated DNA was determined by comparison with λ-DNAs quantified by agarose gel electrophoresis (Thermo Electron Corporation EC135-90, Madison, WI, USA).

S-allele PCR amplification

Primer pairs developed by Romero et al. [24] and Vilanova et al. [25] were used as listed in Table 1 in order to determine the S-alleles by PCR analysis. Each PCR reaction of 25 µL contained 75 mmol/L of Tris-HCl (pH 8.8), 20 mmol/L of (NH₄)₂SO₄, 2 mmol/L of MgCl₂, 0.1% Tween 20, 100 µmol/L of each deoxyribonucleoside triphosphate, 0.2 µmol/L of each primer, 1.0 units of Taq DNA polymerase (Thermo, Waltham, MA, USA) and 50 ng of DNA. For PCR amplification, the samples were pre-denatured at 94 °C for 3 min, followed by 35 cycles in which the samples were denatured for 45 s at 94 °C, annealed for 45 s at 54 °C and extended for 60 s at 72 °C. For the final extension step, the samples were kept at 72 °C for 10 min. The PCR products were separated by electrophoresis in a 3% agarose gel with 0.5X TBE (Tris-borate-ethylenediaminetetraacetic acid) buffer based on band sizes and were visualized under ultraviolet light (Vilber Lourmat Infinity 1100, Collégien, France) by staining with ethidium bromide.

Table 1. The primers used to determine S-alleles of apricots.

Primers	Primer sequence	Reference
SRc-R	5'-GGC CAT TGT TGC ACA AAT TG-3'	Vilanova et al.[25]
SRc-F	5'-CTC GCT TTC CTT GTT CTT GC-3'	Romero et al.[24]

DNA sequencing

DNA sequence analysis of PCR products was performed by Sanger sequencing at Medsantek Company (İstanbul, Turkey). The S-alleles of the parents were determined by comparing the sequences using BLAST (basic local alignment search tool) with those available in the NCBI (National Center for Biotechnology Information) databases.

Data analysis

All results were analysed using the SPSS (version 15) statistical analysis package and the data are mean values with standard deviation (±SD) from three replications. Data were analysed by analysis of variance and significant differences between the groups were determined by the multiple comparison procedure according to Duncan.[26] Differences were considered statistically significant at (P < 0.05).

Results and discussion

PCR amplifications using the SrcF–SrcR primer combination detected an allele of 353 bp in Paviot genotype, which corresponds to the self-compatibility S_c allele reported by Vilanova et al.[25] According to Tao et al.,[27] all genotypes without S_c alleles did not fructify, and they were self-incompatible. In addition to the S_c allele, three other alleles were identified: two in Paviot (328 and 267 bp) and one in Kabaasi (373 bp) (Figure 1).

Figure 1. Electrophoregram of S-alleles of parents and F₁ progenies amplified with the SrcF and SrcR primers for the first intron region. P: Paviot, K: Kabaaşı. DNA molecular size marker: Thermo Generuler 50 bp DNA ladder (Waltham, MA, USA).

The Paviot and Kabaasi S-allele genotypes showed homology with the allele sequences from Prunus armeniaca with S_c (353 bp), S₂ (328 bp), S₁ (373 bp) and S₉ (267 bp) available in the NCBI database.[23,28] The Paviot genotype was found to have S_cS₂ alleles and to be self-compatible due to the presence of S_c alleles. It was reported that the Kabaasi genotype has the S₉ allele, and it is the most common allele found in the local Turkish apricot varieties such as Adilcevaz 5, Akcadag Gunay, Cataloglu, Cekirge 52, Cologlu, Dortyol 2, Haci Haliloglu, Hasanbey, Ismailaga, Kadıoglu, Kamelya, Kurukabuk, No 2 Zerdali, Ozal, Seftalioglu, Soganci and X3 Zerdali.[2]

Previous selfing studies carried out under specific conditions showed that the Kabaasi cultivar is self-compatible,[16] whereas others have found it to be self-incompatible.[14,15] In this study, the Kabaasi cultivar had S₁S₉ alleles and was determined to be self-incompatible. Halasz et al. [2] reported that the Kabaasi cultivar had S₉S₁₃ alleles, and it was self-incompatible. In that study, the alleles carried by individuals were identified based on the band sizes in electrophoresis gel images. The band sizes were estimated by separating the PCR products by agarose gel electrophoresis; however, it was sometimes very difficult to estimate such bands when the sizes of the alleles were very close to each other. In this study, to overcome this problem, the bands in Kabaasi and Paviot cultivars were bidirectionally sequenced, BLAST searched in NCBI GenBank and in them, the S-alleles were determined as S₁S₉.

Apricots belonging to the Iran–Caucasian eco-geographical group have been reported to be mostly self-incompatible, those in the European eco-geographical group, mostly self-compatible and 60% of the varieties in Turkey have been found to be self-incompatible.[16,29–31] In this study, the Kabaasi cultivar, which is in the Iran–Caucasian eco-geographical group, was found to be self-incompatible, whereas the Paviot cultivar, which belongs to the European eco-geographical group, was found self-compatible.

Of the 77 F₁ progenies tested, 34 samples did not have S_c alleles and were identified to be self-incompatible (Table 2). According to Halasz et al.,[2] there was absence of an S_c allele in 44 of 51 apricot genotypes grown in Turkey and they were found to be self-incompatible. Halasz et al. [31] also found that 60% of the varieties grown in Turkey are self-incompatible. In this study, 43 of the F₁ progenies had either S_cS₁ or S_cS₉ alleles, and they were self-compatible. On the other hand, 34 F₁ progenies were found to have S₁S₂ and S₂S₉ incompatibility alleles (Table 3). Burgos et al. [10] reported that the self-compatibility allele was dominant over the self-incompatibility allele. Similarly, in their study conducted on the self-incompatibility status of apricot F₁ populations in, Chen et al. [32] found that the self-compatibility allele was dominant over the self-incompatibility allele; and the S-genotype in Katy, the main individual, was heterozygous (S_cS₈).

Table 2. S-genotypes of parents and F₁ progenies in this study.

Progenies	Allele	Progenies	Allele	Progenies	Allele	Progenies	Allele
Paviot	ScS2	PK 19	S1S2	PK 39	S2S9	PK 59	ScS1
Kabaasi	S1S9	PK 20	ScS1	PK 40	S1S2	PK 60	ScS1
PK 1	S1S2	PK 21	ScS1	PK 41	ScS1	PK 61	ScS1
PK 2	ScS1	PK 22	S1S2	PK 42	ScS1	PK 62	S2S9
PK 3	ScS1	PK 23	S1S2	PK 43	ScS1	PK 63	S1S2
PK 4	ScS1	PK 24	ScS9	PK 44	S2S9	PK 64	S2S9
PK 5	S2S9	PK 25	S2S9	PK 45	ScS1	PK 65	ScS9
PK 6	S1S2	PK 26	S2S9	PK 46	ScS1	PK 66	ScS1
PK 7	S1S2	PK 27	S2S9	PK 47	S2S9	PK 67	ScS1
PK 8	S1S2	PK 28	S1S2	PK 48	ScS9	PK 68	S1S2
PK 9	S2S9	PK 29	ScS9	PK 49	ScS9	PK 69	S2S9
PK 10	ScS1	PK 30	S1S2	PK 50	ScS9	PK 70	ScS1
PK 11	ScS1	PK 31	S1S2	PK 51	S1S2	PK 71	ScS9
PK 12	S2S9	PK 32	ScS9	PK 52	ScS9	PK 72	ScS1
PK 13	ScS9	PK 33	S1S2	PK 53	ScS9	PK 73	S1S2
PK 14	S1S2	PK 34	ScS1	PK 54	S1S2	PK 74	ScS9
PK 15	ScS1	PK 35	ScS9	PK 55	ScS9	PK 75	ScS9
PK 16	ScS9	PK 36	ScS9	PK 56	ScS9	PK 76	S1S2
PK 17	ScS1	PK 37	ScS1	PK 57	ScS9	PK 77	S1S2
PK 18	S2S9	PK 38	S1S2	PK 58	ScS1

Note: P, Paviot; K, Kabaasi; PK, Paviot × Kabaasi.

Table 3. Fruit characteristics of some F₁ progenies.

Progenies	Fruit weight (g)	Kernel weight (g)	Brix (%)	Fruit shape	Peel colour	Flesh colour
PK 10	32.1 ± 1.2^c	3.1 ± 0.01^b	23.8 ± 1.1^b	Oval	Yellow	Yellow
PK 12*	28.0 ± 0.2^d	3.4 ± 0.06^b	21.0 ± 1.6^c	Oval	Green	Yellow
PK 13	42.3 ± 2.3^ab	3.6 ± 0.06^ab	26.5 ± 1.2^a	Oblong	Orange	Orange
PK 14	35.2 ± 0.8^c	3.6 ± 0.05^ab	23.0 ± 1.7^b	Oval	Orange	Orange
PK 17**	35.7 ± 0.6^c	2.8 ± 0.05^c	23.5 ± 1.3^b	Round	Orange	Orange
PK 20**	39.8 ± 0.5^b	3.6 ± 0.01^ab	25.0 ± 1.3^a	Elliptic	Yellow	Yellow
PK 33	23.7 ± 0.2^e	2.6 ± 0.03^c	23.8 ± 1.2^b	Oval	Yellow	Cream
PK 38	37.7 ± 1.1^b	2.8 ± 0.02^c	21.5 ± 1.7^c	Round	Yellow	Yellow
PK 41	39.3 ± 0.4^b	3.4 ± 0.02^b	21.0 ± 1.8^c	Elliptic	Yellow	Cream
PK 46	48.2 ± 0.5^a	4.0 ± 0.09^a	21.6 ± 1.5^c	Oval	Yellow	Yellow
PK 52	38.6 ± 0.2^b	3.4 ± 0.02^b	26.2 ± 1.6^a	Oval	Yellow	Yellow
PK 58**	45.3 ± 0.7^a	3.3 ± 0.07^b	26.8 ± 1.4^a	Round	Yellow	Cream
PK 59	44.5 ± 0.9^a	3.2 ± 0.07^b	23.3 ± 1.1^b	Elliptic	Orange	Orange
PK 63	35.2 ± 0.1^c	3.2 ± 0.07^b	21.5 ± 1.4^c	Round	Yellow	Yellow
PK 65	28.5 ± 0.2^d	2.8 ± 0.02^c	22.0 ± 1.9^b	Oval	Yellow	Yellow
PK 67**	40.4 ± 1.3^ab	3.1 ± 0.05^b	17.1 ± 1.7^d	Elliptic	Green	Cream
PK 70	46.5 ± 0.4^a	3.5 ± 0.04^ab	22.8 ± 1.4^b	Oval	Yellow	Yellow
PK 72**	43.8 ± 1.9^ab	3.8 ± 0.06^a	24.5 ± 0.05^b	Elliptic	Yellow	Yellow
PK 74	33.2 ± 1.2^c	3.4 ± 0.08^b	26.6 ± 1.2^a	Oval	Orange	Orange
PK 77	41.8 ± 1.1^ab	3.7 ± 0.03^a	20.5 ± 1.0^c	Round	Yellow	Yellow

Note: Values are means (±SD) of three replications. Data followed by different letters are significantly different from each other (P < 0.05) according to Duncan's test [26].

*High yield. **Very high yield.

The distribution of alleles in the F₁ progenies was observed to be as follows: S_cS₁ in 31.2%, S₁S₂ in 27.3%, S_cS₉ in 24.7%, and S₂S₉ in 16.8%. Two alleles were identified in the Paviot genotype with sizes of 353 and 328 bp, and two other alleles were found in the Kabaasi genotype with sizes of 267 and 373 bp. Comparison of the DNA sequences of all the four different alleles with those in GenBank showed that the S_c and S₂ alleles were found in the Paviot genotype, whereas S₁ and S₉ were identified in the Kabaasi genotype. In total, 43 F₁ progenies were found to have an S_c allele and thus, to be self-compatible, whereas 34 F₁ progenies were self-incompatible due to the absence of an S_c allele.

Table 3 shows the pomological features of some F₁ progenies that had high quality in 2011–2013. F₁ progenies Nos. 17, 20, 58, 67 and 72 were found to have high yield and high quality fruit in pomological studies. At the same time, they were determined to be self-compatible, too (Table 2). These progenies can be used as parents in breeding programmes or as cultivar candidates in commercial apricot orchards.

Conclusions

In this study, S-allele specific PCR was used to identify the S-alleles of 77 F₁ progenies and their parents. The DNA sequences of four alleles were obtained and compared in the NCBI GenBank database. S_c and S₂ alleles were found in the Paviot genotype, whereas S₁ and S₉ alleles were identified in the Kabaasi genotype. It was determined that 43 F₁ progenies had the S_c allele; and thus they were self-compatible. On the other hand, 34 F₁ progenies had no S_c allele; therefore, they were self-incompatible. The distributions of the detected alleles in the F₁ progenies in Paviot (S_cS₂) and Kabaasi (S₁S₉) parents were determined as follows: S_cS₁ 31.2%, S₁S₂ 27.3%, S_cS₉ 24.7% and S₂S₉ 16.8%. In breeding studies, the development of new genotypes with the desired characteristics and with known compatibility pattern of parents is of crucial importance in terms of both the required time and the cost of manpower. Therefore, the results from this study are relevant based on the data obtained in apricot breeding programmes in the selection of crossing combinations and in the establishment of commercial orchards.

Acknowledgments

The authors thank Lorenzo Burgos, Nuria Alburquerque and Lydia Bremaud from Centro de Edafologia y Biologia Aplicada del Segura (CEBAS-CSIC), Murcia, Spain, for providing valuable help, support and advice.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was supported by The Council of Higher Education of Turkey.