S-RNase Alleles Research Articles

NaPi/S<sub>X</sub>-RNase segregates as a functional S-RNase and is induced under phosphate deficiency in Nicotiana alata

In plants, class III T2 RNases involves two groups of structurally similar proteins, but with different biological functions: S-RNases and non-S-RNases. S-RNases have been involved in self-incompatibility whereas non-S-RNases have been implicated in stress responses. Here we report a novel class III RNase termed NaPi/Sx-RNase, which works both in self-incompatibility and in response to phosphate deficiency. The NaPi/Sx-RNase gene was identified in roots of Nicotiana alata grown in the absence of inorganic phosphate. Phylogenetic analysis showed that NaPi/Sx-RNase was included within the class III RNase T2 group. The NaPi/Sx-RNase was expressed in styles and its temporal expression increased in parallel to stylar development, with a slight decrease after anthesis. Progeny analysis showed that NaPi/Sx-RNase and S107-RNase, a functional allele of the self-incompatibility system, segregated in a 1:1 ratio. The progeny segregation of a semicompatible cross, in which NaPi/Sx-RNase was shared by the two parents, exhibited a pattern consistent with a functional S-RNase allele. Considering genetic segregation, primary structure, and physiological role, the NaPi/Sx-RNase may be either an S-RNase with diversified functions or a non-S-RNase linked to the S-locus. To our knowledge, this is the first evidence for a specific function of the S-locus other than the self-incompatibility reaction. These results support the hypothesis that the self-incompatibility and stress responses may have evolved from a common origin.

Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars.

Data obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads. The reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S 5 , S 6a , S 6b , S 8 , S 11 , S 23 , S 39 , S 46 , S 50 and S 58 ). Moreover, sequence divergence clearly suggests that alleles S 6a and S 6b , proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.

Identification of S -genotypes in 18 pear accessions and exploration of the breakdown of self-incompatibility in the pear cultivar Xinxue

Abstract Self-incompatibility is a genetic mechanism in flowering plants that promotes outcrossing and prevents inbreeding. In this study, the reported 15 S-RNase alleles were isolated from 18 pear accessions that contained 14 different S-genotypes. The S-RNase alleles were only expressed in the style, not in the root, stem, leaf, fruit, sepal, and pollen. Pollen tube grew into self-styles and self-pollinated fruit set involved the breakdown of self-incompatibility in cv. Xinxue. However, the two S-RNase alleles in cv. Xinxue had identical amino acid sequences to those self-incompatible cultivars, and S5- and S6-RNase were normally expressed in style, indicating that the loss of self-incompatibility in cv. Xinxue likely resulted from pollen-part mutation. Further, S5S5 and S6S6 genotyped individuals were identified in self-pollinated progeny, indicating that S5 and S6 genotyped pollen were compatible with the self-styles of cv. Xinxue. A genetic analysis showed that the segregation ratio of S5S5, S5S6, and S6S6 was approximately 0:2:1 (χ2 = 3.505 χ20.05, 2 = 5.99), suggesting that the breakdown of self-incompatibility could be caused by modified factor(s) located outside of the S-locus. These results are useful for parental assignment and understanding self-incompatibility reaction.

Identification of 21 novel S-RNase alleles and determination of S-genotypes in 66 loquat (Eriobotrya) accessions

As observed in other self-incompatible species in the Pyrinae subtribe, loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus, which encodes a polymorphic stylar ribonuclease (S-RNase). This allows the female reproductive organ (style) to recognize and reject the pollen from individuals with the same S-alleles, but allows the pollen from individuals with different S-alleles to effect fertilization. The S-genotype is therefore an important consideration in breeding strategies and orchard management. In an attempt to optimize the selection of parental lines in loquat production, the S-RNase alleles of 35 loquat cultivars and their 26 progeny, as well as five wild loquat species, were identified and characterized in this study. The best pollinizer cultivar combinations were also explored. A total of 28 S-alleles were detected, 21 of which constituted novel S-RNase alleles. The S-haplotypes S2 and S6 were the most frequent, followed by S 29 , S 31 , S 5 , S 24 , S 28 , S 33 , S 34 , S 32 , and S 15 , while the rare alleles S 1 , S 9 , S 14 , S 16 , S 17 , S 18 , S 19 , S 20 , S 21 , S 22 , S 23 , S 27 , and S 35 were only observed in one of the accessions tested. Moreover, the S-genotypes of five wild loquat species (E. prinoides, E. bengalensis, E. prinoides var. dadunensis, E. deflexa, and E. japonica) are reported here for the first time. The results will not only facilitate the selection of suitable pollinators for optimal orchard management, but could also encourage the crossbreeding of wild loquat species to enhance the genetic diversity of loquat cultivars.

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

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.

Identification of S genotypes in loquat (Eriobotrya japonica Lindl.) based on allele specific PCR

Loquat (Eriobotrya japonica Lindl) is an evergreen fruit tree and belongs to Maloideae in the Rosaceae, which carries the RNase-dependent gametophytic self-incompatibility system (GSI). Use of appropriate pollinator trees is essential for increasing fruit production. S-allele specific amplification system is an efficient and rapid method to identify S-genotypes. In this research, according to the sequences of S-RNase alleles, 9 specific primer pairs for amplifying S-RNase alleles (S2, S7, S6, S8, S9, S10, S11,S12 and S13) were designed, which located in the introns or in the areas with high polymorphism. The specificity of the 9 allele specific primer pairs was evaluated by eight loquat cultivars with known S-genotypes. After being verified their effectiveness, they were used to S-genotype 39 loquat cultivars from Longan and loquat nurseries in Fuzhou national fruit germplasm. The results showed that the 39 loquat cultivars were S-genotyped efficiently by allele specific PCR with the 9 primer pairs, in which 28 cultivars were identified for the first time and they were classified into 18 groups according to the genotype. The results also proved that the 9 S-allele specific primer pairs can efficiently identified S-genotype, which will be very useful for choosing pollinators or crossing parents in loquat production and breeding.

Identification of two new S-RNases and molecular S-genotyping of twenty loquat cutivars [Eriobotrya japonica (Thunb.) Lindl.

Loquat (Eriobotrya japonica Lindl.), a member of Rosaceae, exhibits gametophytic self −incompatibility (GSI) that is controlled by the multiallelic S-locus. The S-locus encodes polymorphic S-RNase for the pistil-part S determinant. Identifying the S-genotypes of loquat cultivars is important for the selection of pollen donors for fruit production and breeding of loquat cultivars. In this study, we determined 20 loquat cultivar S-genotypes by PCR-based analysis with the consensus and allele-specific primers. In the 20 loquat cultivars, 9 S-haplotypes were identified including 7 S-RNases (S2, S7, S6, S8, S9, S10, S11) previously reported and two new S-RNase alleles. The partial genomic sequences of the two new S-RNases show high similarity (>95%) with Pyrus S-RNase, inserted by an intron in hypervariable region (RHV) with the length of 151bp and 806bp respectively, and encode amino acid sequences having the common structures of S-RNases. The two S-RNase genes were deposited in GenBank with name S12-RNase (accession number: KR149297) and S13-RNase (accession numbers: KR149298). Twenty loquat cultivars were classified into 12 groups according to S-genotypes. Seventeen cultivars have two different S-haplotypes except ‘Fengyu’, ‘Ninghaibai’ and ‘Bahong’ containing one S-haplotype. This research would contribute to a more efficient breeding program and pollinator selection of loquat.

Characteristic of pollen tube that grew into self style in pear cultivar and parent assignment for cross-pollination

Pear is one of the most important fruits in the world. In recent years, many new varieties have been bred. However, little is known about these new varieties, including pollen germination, self-(in)compatibility, and the best pollinator for the varieties. In this study, by surveying the length of the pollen tubes that grew into the self styles, pollen germination and self-(in)compatibility of the 127 pear cultivars were determined. All tested pollen germinated on the self stigmas, but the lengths of pollen tube growth into the self styles were different among the pear cultivars. Of these 127 cultivars, 56, 58, and 8 cultivars presented strong, intermediate, and weak self-incompatibility, respectively. Moreover, 5 cultivars presented self-compatibility because their pollen tubes grew to the bottom of the self styles and higher fruit sets with self-pollination were verified in the field. Furthermore, to assign the best pollinator for fruit production and/or new variety breeding, the S-RNase alleles that shared almost identical sequences were integrated together and then re-designated to discern different function-specificity. As a result, the original 68 S-RNase alleles in eastern pear were integrated into 48 re-designated function-specific S-RNase alleles. This integration is promising for assigning the best pollinator for the 36 main pear cultivars in China. Unless the integrated S-RNase alleles, in addition, the S-RNase alleles, which have extremely high identities in amino acid sequences, should be excluded to avoid cross-incompatibility between two parents, leading to poor fruit production and the loss of genetic information in this chromosome

S_allele identification and genetic diversity analysis of apricot cultivars

ABSTRACTIn this study, the sexual incompatibility and S-allele diversity of 24 Turkish apricot cultivars, Paviot and Sakıt-1 as parents and 127 F1 progenies were identified by polymerase chain reaction (PCR) and sequencing techniques. Additionally, genetic diversity and relatedness among the 24 cultivars were determined using 18 simple sequence repeat (SSR) markers from the genus Prunus. PCR for S_alleles identified nine different S-RNase alleles in the 24 apricot cultivars, namely Sc, S1, S2, S8, S9, S20, S24, S52, and S53. All primers amplified only one S_allele in the cultivars Adilcevaz-1, Adilcevaz-3, Ethembey, Pasamismisi, Canakkale, and Soganci. Most of the Turkish cultivars were self-incompatible. The Sc allele was present in only three cultivars (Canakkale, Ethembey, Imrahor) that are, therefore, self-compatible. The S_alleles of cultivars Paviot and Sakıt-1 displayed homology with the Sc, S2 and S20, and S52 alleles. In the 127 F1 genotypes, the two S_alleles of Paviot were inherited by roughly half of the offspring, while about 76% of the offspring inherited the S52 allele from Sakıt-1, and less than 24% inherited S20. The amplification using all SSR 18 primers was successful and produced 128 polymorphic alleles with an average of 7.11 alleles per locus. Among the apricot cultivars studied, expected heterozygosity (He) ranged from 0.33 to 0.72, observed heterozygosity (Ho) ranged from 0.42 to 1.00, PIC values were between 0.28 and 0.89, and similarity rates were between 0.30 and 0.68. The cultivars Levent and Ozal were genetically closest (0.68) while cultivars Sakıt-3 and Sogancı were the most distinct (0.30).

Simple Sequence Repeat and S-locus Genotyping to Explore Genetic Variability in Polyploid Prunus spinosa and P. insititia.

Polyploid Prunus spinosa (2n=4×) and P. insititia (2n=6×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programmes. In Hungary, 17 cultivar candidates were selected from wild-growing populations including 10 P. spinosa, 4 P. insititia and three P. spinosa×P. domestica hybrids (2n=5×). Their taxonomic classification was based on their phenotypic characteristics. Six simple sequence repeats (SSRs) and the multiallelic S-locus genotyping were used to characterize genetic variability and reliable identification of the tested accessions. A total of 98 SSR alleles were identified, which presents 19.5 average allele number per locus, and each of the 17 genotypes could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified. The complete and partial S-genotype was determined for 8 and 9 accessions, respectively. The identification of a cross-incompatible pair of cultivar candidates and several semi-compatible combinations help maximize fruit set in commercial orchards. Our results indicate that the S-allele pools of wild-growing P. spinosa and P. insititia are overlapping in Hungary. A phylogenetic and principal component analysis confirmed the high level of diversity and genetic differentiation present within the analysed genotypes and helped clarify doubtful taxonomic identities. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species. The analysed accessions represent huge genetic potential that can be exploited in commercial cultivation.

Detection of Self Incompatibility Genotypes in Prunus africana: Characterization, Evolution and Spatial Analysis.

In flowering plants, self-incompatibility is an effective genetic mechanism that prevents self-fertilization. Most Prunus tree species exhibit a homomorphic gametophytic self-incompatibility (GSI) system, in which the pollen phenotype is encoded by its own haploid genome. To date, no identification of S-alleles had been done in Prunus africana, the only member of the genus in Africa. To identify S-RNase alleles and hence determine S-genotypes in African cherry (Prunus africana) from Mabira Forest Reserve, Uganda, primers flanking the first and second intron were designed and these amplified two bands in most individuals. PCR bands on agarose indicated 26 and 8 different S-alleles for second and first intron respectively. Partial or full sequences were obtained for all these fragments. Comparison with published S-RNase data indicated that the amplified products were S-RNase alleles with very high interspecies homology despite the high intraspecific variation. Against expectations for a locus under balancing selection, frequency and spatial distribution of the alleles in a study plot was not random. Implications of the results to breeding efforts in the species are discussed, and mating experiments are strongly suggested to finally prove the functionality of SI in P. africana.

S-Genotype Profiles of Turkish Apricot Germplasm

In flowering plants, gametophytic self-incompatibility, controlled by a single locus with several allelic variants, is one of the major problems preventing self-fertilization. Among fruit trees, apricots show to a high degree self-incompatibility, especially in Middle-Asian and Iranian-Caucasian eco-geographical groups. In the present study, self-(in)compatibility characteristics of a total of 236 apricot genotypes (218 Turkish and 18 foreign) found within the National Apricot Germplasms of Apricot Research Institute in Malatya, Turkey was studied. Analyses were carried out by using four primer pairs (SRc-F and SRc-R, EM-PC2consFD and EM-PC3consRD, AprSC8-R and PaConsI-F, AprFBC8-F and AprFBC8-R). A total of 11 S-RNase alleles (S2, S3, S6, S7, S8, S9, S11, S12, S13, S20 and Sc) were determined in the 236 apricot genotypes. As Turkish and foreign apricot genotypes are determined mostly self-incompatible, the data obtained hereby might be of good use for apricot breeding programs and more practically, for apricot new plantations; thus pollinator cultivars should be considered when self-incompatible apricot cultivars are being used.

S-Genotype Profiles of Turkish Apricot Germplasm

In flowering plants, gametophytic self-incompatibility, controlled by a single locus with several allelic variants, is one of the major problems preventing self-fertilization. Among fruit trees, apricots show to a high degree self-incompatibility, especially in Middle-Asian and Iranian-Caucasian eco-geographical groups. In the present study, self-(in)compatibility characteristics of a total of 236 apricot genotypes (218 Turkish and 18 foreign) found within the National Apricot Germplasms of Apricot Research Institute in Malatya, Turkey was studied. Analyses were carried out by using four primer pairs (SRc-F and SRc-R, EM-PC2consFD and EM-PC3consRD, AprSC8-R and PaConsI-F, AprFBC8-F and AprFBC8-R). A total of 11 S-RNase alleles (S2, S3, S6, S7, S8, S9, S11, S12, S13, S20 and Sc) were determined in the 236 apricot genotypes. As Turkish and foreign apricot genotypes are determined mostly self-incompatible, the data obtained hereby might be of good use for apricot breeding programs and more practically, for apricot new plantations; thus pollinator cultivars should be considered when self-incompatible apricot cultivars are being used.

A high-throughput method for genotyping S-RNase alleles in apple.

We present a new efficient screening tool for detection of S-alleles in apple. The protocol using general and multiplexed primers for PCR reaction and fragment detection on an automatized capillary DNA sequencer exposed a higher number of alleles than any previous studies. Analysis of alleles is made on basis of three individual fragment sizes making the allele interpretation highly accurate. The method was employed to genotype 432 Malus accessions and exposed 25 different S-alleles in a selection of Malus domestica cultivars of mainly Danish origin (402 accessions) as well as a selection of other Malus species (30 accessions). The allele S3 (28 %) was the most common among the Danish cultivars followed by S1 and S7 (both 27 %). The alleles S36 and S40 not previously reported from M. domestica were found in 6 and 17 cultivars, respectively. Complete allelic composition was found in 91 % of the 369 diploid accessions and in 86 % of the 63 triploids concerned. We further identified a relatively high frequency of S33 and S34, which has not been considered by most previous studies. The protocol presented here is easy to adopt and saves both time and work effort compared to previous methods. The robustness is illustrated by the great accuracy and a high number of S-alleles presented.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-016-0448-0) contains supplementary material, which is available to authorized users.

Characterization of a new apple S-RNase allele and its linkage with the Rvi5 gene for scab resistance

Apple (Malus × domestica Borkh.) is a species possessing S-RNase-based self-incompatibility. Knowledge of the S-genotype of cultivars is very important both for breeders and growers, in order to plan compatible crosses and cultivar combinations. Here we report a revision of the S-genotype of the cultivar Murray which is in disagreement with previous reports, and the characterization of the newly identified S-RNase allele. Moreover, Murray carries the Rvi5 gene for apple scab resistance, which was mapped at the bottom of linkage group 17, in a position close to that of the S-locus. To evaluate whether the segregation of S-alleles can influence that of Rvi5, we analyzed the linkage between the two loci in 215 progeny individuals from the cross Galaxy × Murray. Our results suggest that the S-genotype of cultivars used as parents can dramatically influence the frequency of seedlings inheriting the resistance gene, and this aspect can be exploited to increase the efficiency of breeding programs aimed at pyramiding scab resistance genes.

Identification and characterization of S-RNase genes and S-genotypes in Prunus and Malus species

Gu, C., Wang, L., Korban, S. S. and Han, Y. 2015. Identification and characterization of S-RNase genes and S-genotypes in Prunus and Malus species. Can. J. Plant Sci. 95: 213–225. Most Rosaceae fruit trees such as Prunus and Malus species exhibit gametophytic self-incompatibility that is genetically controlled by the S-locus. In turn, the S-locus contains at least two tightly-linked S-determinant genes, a pistil S-RNase and a pollen SFB. In this study, S-genotypes of 120 cultivated and wild Prunus accessions (peach) and seven wild Malus accessions (crabapple) have been characterized. Among cultivated Prunus genotypes, four S-RNase alleles, designated S 1 , S 2 , S 3 , and S 4 , have been identified, and they share typical structural features of S-RNases from all other self-incompatible Prunus species. Four S-genotypes, S 1 S 2 , S 1 S 3 , S 1 S 4 , and S 2 S 2 , were identified in peach cultivars, while only one S-genotype S 1 S 2 for wild Prunus species. The S 1 S 2 genotype is predominant in peach cultivars, accounting for 58.3% of all evaluated accessions. Similarly, four SFB alleles were identified in peach cultivars and wild accessions. However, all the four SFB alleles encode truncated proteins due to a frame-shift mutation, resulting in loss of hyper-variable and/or variable regions. For Malus species, a total of 14 S-RNase alleles are identified, and of those, two alleles encode truncated proteins. Overall, the genetic variation of both S-RNase and SFB genes in peach is significantly lower than that of S-RNase and SFB genes in self-incompatible Malus and/or Prunus species. The relationship between the genetic variation of SFB genes and the diversification of S-RNase genes in Rosaceae is also discussed.

Identification of the Er1 resistence gene and RNase S-alleles in Malus prunifolia var. ringo rootstock

Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) is a major insect pest that has significant economic impact on apple growers worldwide. Modern breeding technologies rely on several molecular tools to help breeders select genetic determinants for traits of interest. Consequently, there is a need for specific markers linked to the genes of interest. Apple scions and rootstocks have an additional barrier to the introduction of pest resistance genes due to the presence of self-incompatibility S-RNase alleles. The genetic characterization and early identification of these alleles can amplify the contribution of a breeding program to the selection of resistant genitors that are as compatible as possible. In this study, we identified the Er1 gene involved in the resistance to WAA in Malus prunifolia var. ringo, also known as ‘Maruba Kaido’ rootstock, and we analyzed the inheritance pattern of the WAA resistance Er1 gene in a segregant population derived from Malus pumila ‘M.9’ and ‘Maruba Kaido’ rootstocks. The self-incompatibility of S-RNase alleles S6S26 of ‘Maruba Kaido’ were also identified along with their inheritance pattern. We also confirmed the identification of the S1S3 alleles in the ‘M.9’ rootstock. To the best of our knowledge, this is the first study to characterize WAA resistance and RNase S-alleles in ‘Maruba Kaido’. Furthermore, we discuss the potential use of the genetic markers for these genes and their potential impact on apple breeding programs.

S-genotype identification based on allele-specific PCR in Japanese pear.

Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S1–S9 and Sk) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S1/S1–S9/S9 and S4sm/S4sm) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs.

Diversification and distinctive structural features of S-RNase alleles in the genus Solanum.

The multigenic and multiallelic S-locus in plants is responsible for the gametophytic self-incompatibility system, which is important to prevent the detrimental effects of self-fertilization and inbreeding depression. Several studies have discussed the importance of punctual mutations, recombination, and natural selection in the generation of allelic diversity in the S-locus. However, there has been no wide-ranging study correlating the molecular evolution and structural aspects of the corresponding proteins in Solanum. Therefore, we evaluated the molecular evolution of one gene in this locus and generated a statistically well-supported phylogenetic tree, as well as evidence of positive selection, helping us to understand the diversification of S alleles in Solanum. The three-dimensional structures of some of the proteins corresponding to the major clusters of the phylogenetic tree were constructed and subsequently submitted to molecular dynamics to stabilize the folding and obtain the native structure. The positively selected amino acid residues were predominantly located in the hyper variable regions and on the surface of the protein, which appears to be fundamental for allele specificity. One of the positively selected residues was identified adjacent to a conserved strand that is crucial for enzymatic catalysis. Additionally, we have shown significant differences in the electrostatic potential among the predicted molecular surfaces in S-RNases. The structural results indicate that local changes in the three-dimensional structure are present in some regions of the molecule, although the general structure seems to be conserved. No previous study has described such structural variations in S-RNases.

Genetic identification of apple cultivars bred in Korea using simple sequence repeat markers

Among the many DNA markers currently available, simple sequence repeat (SSR) markers are well developed and widely used for gene identification and cultivar discrimination by apple breeders and researchers. We developed SSR fingerprints for identification and to reveal the genetic diversity of 22 apple cultivars released from Korea. We used a total of 22 SSR markers. Among them, 15 SSR markers were developed from the complete sequence of ‘Golden Delicious’ and 7 SSR markers were chosen from the HiDRAS database. Except for somatic mutants, cultivar identification of all Korean bred apple genotypes was accomplished by using only two SSR markers (CH03d07 and KFBG-7). Parent-offspring relationships analysis was performed to investigate the real parents of three suspicious cultivars (‘Gamhong’, ‘Hongan’, and ‘Green Ball’). Based on the SSR markers experiment and reported S-RNase allele, we confirmed that ‘Fuji’ was used as paternal parent of ‘Gamhong’ and either ‘Hongro’ or ‘Natumidori’ were used as paternal parent of ‘Hongan’. Moreover, ‘Jonathan’ rather than ‘Fuji’ seems to be a paternal parent of ‘Green Ball’.