Polyacrylamide gel electrophoresis of S-RNase fragments for identification of S-genotypes of Japanese pear (Pyrus pyrifolia)

Japanese pear (Pyrus pyrifolia) exhibits gametophytic self-incompatibility (GSI) controlled by a complex and multiallelic S locus. The pistil-part product of the S locus is the polymorphic ribonuclease, S-RNase. Information on S-genotypes is important for the production and breeding of Japanese pears. Molecular analyses of S-genotypes of Japanese pear have been conducted with the CAPS (cleaved amplified polymorphic sequence) system; PCR amplification of S-RNase fragments by a common primer pair followed by digestion with restriction enzymes each of which cleaves a specific S haplotype. Here, we show that the separation of S-RNase fragments by polyacrylamide gel electrophoresis (PAGE) distinguishes four out of nine S haplotypes of Japanese pear without restriction digestion. S³-, S⁵-, S⁶- and S⁸-RNases were identified as distinct bands by PAGE. S³- and S⁵-RNases were separated by PAGE despite their identical fragment sizes. Using this system, three Japanese pear lines with unknown S-genotypes were analyzed. The newly determined S-genotypes of the lines were confirmed by CAPS analysis.     

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 S^k) 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 S^4sm/S^4sm) 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.     

Reverted glutathione S-transferase-like genes that influence flower color intensity of carnation (Dianthus caryophyllus L.) originated from excision of a transposable element

A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar ‘Daisy’ carries both defective genes, whereas a spontaneous deep-colored mutant ‘Daisy-VPR’ lost the element from DcGSTF2-dTac1. This finding confirmed that dTac1 is active and that the resulting reverted gene, DcGSTF2rev1, missing the element is responsible for this color change. Crosses between the pale-colored cultivar ‘06-LA’ and a deep-colored cultivar ‘Spectrum’ produced segregating progeny. Only the deep-colored progeny had DcGSTF2rev2 derived from the ‘Spectrum’ parent, whereas progeny with pale-colored flowers had defective forms from both parents, DcGSTF2mu and DcGSTF2-dTac1. Thus, DcGSTF2rev2 had functional activity and likely originated from excision of dTac1 since there was a footprint sequence at the vacated site of the dTac1 insertion. Characterizing the DcGSTF2 genes in several cultivars revealed that the two functional genes, DcGSTF2rev1 and DcGSTF2rev2, have been used for some time in carnation breeding with the latter in use for more than half a century.     

Identification and characterization of genomic DNA sequences of the S-ribonuclease gene associated with self-incompatibility alleles S1 to S5 in European pear

The stylar products of the S‐locus for the gametophytic self‐incompatibility (GSI) system in the Rosaceae are ribonucleases (S‐RNases). Recently, sequences for 13 pear S‐RNase alleles have been published and named following a letter–symbol nomenclature (S_a to S_d and S_h to S_p). To establish the correspondence between these sequences and the self‐incompatibility alleles we have described previously (S₁ to S₅), we have amplified genomic DNA with consensus primers from the cultivars, ‘Williams’ (S₁S₂), ‘Coscia’ (S₃S₄), ‘Butirra Precoce Morettini’ (S₁S₃), ‘Santa Maria Morettini’ (S₂S₃) and ‘Doyenne du Comice’ (S₄S₅) and identified PCR products specifically associated with each S allele. Cloning and sequencing of the amplification products has revealed that they correspond to European pear sequences already deposited in the database. This allowed us to link S‐RNase sequences with S allele phenotypes and to determine a correspondence between the symbol–letter nomenclature used to name S‐RNase sequences and the number‐based nomenclature used to name S alleles. Based on this result the prediction of new cross‐incompatibilities among pear cultivars is discussed. Finally, we propose a unified number‐based nomenclature to avoid future confusion denominating S alleles in pear.     

Analysis of pollen tube growth in situ to investigate self-incompatibility in the wild potato Solanum commersonii

Summary Pollen tube growth was investigated in a diallelic crossing design with seven genotypes of the diploid wild potato species Solanum commersonii, accession O/S UR-9, CIP 762459. Pollen tube growth in the style was recorded using a combined quantitative and qualitative evaluation scale. Clear-cut differences in pollen tube growth behavior in compatible and in partially or completely incompatible crosses were detected. Diallelic crossing of the seven randomly chosen genotypes, intercrossing within two progeny families, and backcrossing of two progeny populations to the parents revealed the existence of a one-locus gametophytic system of stylar incompatibility. The S-allele status of all genotypes investigated was determined.     

Levels of self-incompatibility in wallflower,Cheiranthus cheiri L.

Summary Approximately 1000 plants from 30 wallflower cultivars were selfpollinated in the open flower and bud stage. Only a very small number of plants failed to set seed and the averages ranged from 3.3 to 11.0 seeds per siliqua. Cultivars with mixed colours appeared to show most self-incompatibility and it is suggested that they form un-evolved populations in terms of the incompatibility system by comparison with their related single-colour cultivars.S1 progenies tended to remain at the same self-incompatibility level as their parents although some segregation occurred with approximately the same number of plants from low to high self-incompatibility and vice-versa.S1 progenies showed no apparent inbreeding depression, as is the case also for their descendants, but there was a tendency for progeny heterogeneity to be associated with high parental self-incompatibility while homogeneous inbreds were most often derived from more self-compatible parents.     

New self-incompatibility alleles in apricot (Prunus armeniaca L.) revealed by stylar ribonuclease assay and S-PCR analysis

Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility controlled by a single locus with several allelic variants. An allele for self-compatibility (S_C) and seven alleles for self-incompatibility (S₁–S₇) were described previously. Our experiments were carried out to ascertain whether the number of allelic variants of apricot S-locus was indeed so small. Twenty-seven apricot accessions were analysed for stylar ribonucleases by non-equilibrium pH gradient electrofocusing (NEpHGE) to determine their S-genotype. To validate the results of electrofocusing, the applicability of the S-gene-specific consensus PCR primers designed from sweet cherry sequences was tested. NEpHGE revealed 12 bands associated with distinct S-alleles in newly genotyped cultivars. Cherry consensus primers amplified 11 alleles out from 16 ones, which indicated that these primers could also recognize most of the S-RNase sequences in apricot, and provided an efficient tool to confirm or reject NEpHGE results. By combining the protein and DNA-based methods, complete or partial S-genotyping was achieved for 23 apricot accessions and nine putatively new alleles (provisionally labelled S₈–S₁₆) were found. Their identity needs to be confirmed by pollination tests or S-allele sequencing. This study provides evidence that similarly to other Prunus species, the S-locus of apricot is more variable than previously believed.     

A comparison of methods for hybrid seed production using self-incompatibility in swedes (Brassica napus ssp. napobrassica)

Procedures for producing seed of hybrid swedes using self-incompatibility were examined. Single-cross, double-cross and modified double-cross hybrids were compared in isolation plots using natural pollinators and in polythene tunnels using blow-flies. With good coincidence of flowering and the same flower colour, nearly 100% hybrid seed was produced by natural pollinators with the single-crosses, the double-cross and one of the two modified double-cross hybrids; the other modified double-cross hybrid produced 87%hybrid seed. With poor coincidence of flowering and different flower colours the proportion of hybrids dropped to 61%. Using different flower colours and blow-flies as pollinators in polythene tunnels, higher levels of outcrossing were produced than in isolation plots with natural pollinators; the opposite result was obtained when the same flower colour was used. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of self-incompatibility alleles in pear cultivars (Pyrus communis L.)

Self and cross-incompatibility determination by means of fruit and seed set experiments or pollen tube growth observations in the style has been frequently reported to be unclear in pear (Pyrus communis L.). Thus,in order to develop a reliable in vivo method to test pollen-pistil incompatibility in pear, pollen tube performance has been studied along the pistil following self and cross-pollinations. Results show that, while pollen tube growth in the style may be an unclear test, ovule observation at the microscope for the presence of pollen tube in the nucellus is a proper method to test incompatibility in this crop. With this analysis we could identify S-alleles of ‘Williams’ (S₁S₂) and ‘Coscia’(S₃S₄), and three of the four possible S-genotypes resulting from the ‘Williams’ × ‘Coscia’ cross, as represented by ‘Butirra Precoz Morettini’ (S₁S₃), ‘Santa Maria Morettini’ (S₂S₃)and ‘Tosca’ (S₁S₄). This result demonstrates that ‘Williams’ and ‘Coscia’ cultivars do not share any allele in common. We also established two new inter-incompatibility groups in pear. Furthermore, the presence of a common allele between ‘Williams’ and ‘Agua de Aranjuez’,and ‘Coscia’ and ‘Agua de Aranjuez’, three apparently unrelated old cultivars, may indicate a narrower genetic base than expected for European pear. This finding together with the fact that 40% of new released cultivars have direct or indirect parental relationship with the cultivars ‘Coscia’ and/or ‘Williams’, anticipates the possibility of new cases of cross-incompatibility for this crop in the future. Both the method described and the determination of the S-genotypes will facilitate the characterisation of self and cross-incompatibility relationships in this species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Overcoming the self-incompatibility of Raphanus sativus by application of plant hormones,amino acids and vitamines,and by temperature treatment of pollen

Summary Overcoming self-incompatibility by application of three kinds of plant hormones, sucrose, 3 kinds of amino acids and 2 kinds of vitamines was tested in cvs. Honbashi-taibyo Minowase (H-Mino) and Minowase (Mino) of Raphanus sativus. Effects differed between the cultivars. In H-Mino, BA (100 mg/l) and glutamic acid, folic acid and nicotinic acid (500 mg/l) resulted in higher fruit set and higher number of seeds per pollinated flower. In Mino, BA and NAA (100 mg/l) and glutamic acid and glycine (500 mg/l) induced a high number of seeds per pollinated flower. These chemicals, however, induced parthenocarpic fruit set, especially GA3. From the observation of pollen on stigmas washed with glutamic acid, it appeared that the pollen-tube penetrated into a papilla cell after 1 hour and openings of papillae and detached pollen grains and tubes were found after 2 hours as the result of successful pollentube penetration of papillae. Pollen was heated at 50°C for 30, 45 or 60 minutes, at 60°C for 15, 30 or 45 minutes and at 70°C for 10, 20 or 30 minutes prior to self-pollination. In H-Mino, 60 and 70°C were effective, and expecially 60°C for 15 or 30 minutes resulted a higher percentage fruit set and more seeds per fruit. In Mino, although 50–70°C were effective, the mean number of seeds per pollinated flower was lower than in H-Mino.     

Distorted genetic segregation of the transposon mPing at the long arm of chromosome 12 in rice

A class II transposable element, mPing exists in the rice genome ubiquitously and can transpose even in ordinary cultivation conditions. A copy of mPing was identified at the long arm of chromosome 12. In reciprocal backcrossed F₁s between a heterozygote and a homozygote without mPing, the male gametes with this mPing from heterozygotes were transmitted to the next generation at a lower frequency than those without mPing, resulting in distorted genetic segregation in self-fertilized progenies, as well as in F₁s after backcrossing. Pollens with mPing tended to germinate on stigma less than those without mPing. These results, however, could not explain the lower transmission of male gametes with mPing. In addition, no excision of mPing was observed in a homozygote. Thus, it was suggested that male gametes with mPing were eliminated partly from pollination to fertilization by negative competition against male gametes without mPing. Less formation of microspores with mPing in meiosis could also be a cause for the distorted segregation, although this could not be examined. At least two ORFs, whose functions have not been identified, are located near this mPing. It is plausible that either of these ORFs or both are necessary for the normal functioning of male gametes.     

Identification of QTLs for fruit quality traits in Japanese apples: QTLs for early ripening are tightly related to preharvest fruit drop

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars ‘Orin’ and ‘Akane’ using F₁ seedlings derived from a cross between these cultivars. The ‘Orin’ map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the ‘Akane’ map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by ‘Akane’ are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.     

Influence of timing of cross-versus self-pollination on self-incompatibility in Brussels sprout inbreds

Summary The impact of cross pollination 30 h, 4 h, and immediately prior to self-pollination, and self-pollination 2, 4, 8, 12, 24 and 30 h prior to cross-pollination was assessed for pollen tube number per style, seed number per siliqua, and proportion of self-seed (sibs) per siliqua, in two inbred lines of brussels sprout. Pollination procedure had a marked effect on the amount of sib-and hybrid seed produced. Cross pollination 30 h prior to self-pollination produced significantly greater numbers of sib progeny, as did self-pollination 8–12 h before cross pollination. Prior application of self pollen reduces the number of pollen tubes in the style. The results of this experiment suggest that self-incompatibility in brussels sprout could better be assessed by self-pollination with a subsequent cross pollination 8 h later, and the determination of amounts of sib and F₁ hybrid seed per siliqua.     

Pollen and pollination experiments. V. An empirical basis for a mentor pollen effect observed on the growth of incompatible pollen tubes in pear

Summary The self-incompatible pear cultivar Doyenne du Comice was selfed with the aid of the mentor pollen technique (self pollen mixed 1:1 with compatible pollen) and the pioneer pollen method (compatible pollen applied 14 h in advance of the self pollen). Observations on tube growth in the style showed that inviable methylated pollen was ineffective either as mentor or pioncer pollen, having no effect on the performance of the self pollen which stopped growing at about one quarter of the style from the stigma. Calculations made on the basis of the obtained data indicated that the viable untreated or irradiated pioneer and mentor pollen, the former somewhat better than the latter, aided the self pollen tubes to reach the base of the style.     

The progamic phase in Cichorium intybus L. Pollen tube growth in the style,incompatibility reaction and gametophytic competition

Summary Pollen germination and tube growth were studied following compatible, incompatible and pseudo-compatible pollinations in chicory. Pollen germination begins 3 minutes after compatible pollinations. The earliest pollen tubes reach the ovary 17 minutes later. Many of the later germinating pollen tubes are arrested and burst at the stigma papillae. In the transmitting tissue inhibitional effects due to negative interactions between pollen tubes are frequently observed. Complete self-incompatibility results in total inhibition of germination. In case of pseudo-self-compatibility, some pollen germinate but germination and stigma penetration are delayed and often result in pollen bursting. There is no self-incompatibility reaction in the transmitting tract but if the pollen tubes reaching this tissue are relatively numerous, negative interactions between them occur as after compatible pollinations. An hypothesis is presented which attributes the negative interactions between pollen tubes to the diffusion of a substance from the growing pollen tubes. This substance would also provoke pollen bursting on the stigma.     

Breakdown of self-incompatibility in perennial ryegrass at high temperature and its uses in breeding

Summary The maximum temperature (34°C) at which perennial ryegrass plants from one breeding population successfully shed pollen was determined and the seed set of ten clones taken from the same population and self-pollinated both in an unheated glasshouse and at a constant 34°C during anthesis was compared. Seed set by selfing of all ten clones was markedly increased by heat treatment, from a mean of 2.3% to 30.7%. Heat treatment of anthers, but not stigmas, greatly enhanced pollen tube growth on stigmas from the same genotype. Germination of progeny was not affected by the heat treatment. Heat treatment of ten clones from a breeding population with contrasting morphology and growth rate was not so successful. Only one clone shed pollen freely at 34°C and showed a marked improvement in subsequent seed set. The advantages to the breeder of routinely producing full-sib families of elite perennial ryegrass parents by selfing are outlined.     

Occurrence of self-compatibility, self-incompatibility and unilateral incompatibility after crossing diploid S. tuberosum (SI) with S. verrucosum (SC): I. Expression and inheritance of self-compatibility

Diploid Solanum tuberosum (tbr), 2n=2x=24,can be crossed with S. verrucosum (ver) only when the latter is used as a pistillate parent but not reciprocally. This conforms to the phenomenon of unilateral incompatibility (UI) where a self-compatible species, like ver (SC) cannot be used as a male parent to cross with a self-incompatible (SI) parent like tbr. Even if ver × tbr hybrids are made, the F1 hybrids possess cytoplasmic male sterility and thus hinder genetic analysis of crossing barriers. Exceptionally, however, some diploid genotypes of tbr (SI) can be used as pistillate parents to cross with ver, and such exceptional tbr clones are called `acceptors'. Repeated backcrossing of acceptors to ver have resulted in male fertile genotypes that possess tbr cytoplasm and ver nucleus. These genotypes were used for the genetic analysis of `acceptance' and UI in thse experiments. It was found that acceptance of ver-pollen by tbr-pistils is based on a dominant gene A that expresses only in the absence of an inhibitor I. In the F₁ hybrids, only the S-allele of tbr was expressedbut not that of ver. Concomitant with this observation, it was shown that ver does not produce style-specific S-glycoproteins that are responsible for self-incompatible reaction in diploid potato. Although the the F₁ populations were SC, they segregated into SC and SI genotypes giving skewed segregation ratios for this trait. Because of this as well as the disappearance and re-appearance of SC trait in the offspring generations, it was necessary to postulate a more complex interaction between A and I. Models are presented in order to explain acceptance, non-acceptance and the expression of UI. It is concluded that at least four different loci are involved in the expression of UI. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of self-incompatibility alleles and pollen incompatibility groups in sweet cherry by PCR based s-allele typing and controlled pollination

A total of 17 pollen incompatibility groups in sweet cherry (Prunusavium L.) were identified among 46 accessions by PCR based S-alleletyping analysis and by controlled test pollinations. Two putativeS-alleles different from S ₁ to S ₆,S _z and S _y were identified. Five S-genotypes, S ₁ S ₅, S ₁ S ₆,S ₂ S ₆, S ₄ S ₆, andS ₅ S ₆, combinations of S ₁ toS ₆ alleles that had not previously been identified from cultivars in NYSAES, were positively confirmed by PCR based S-genotyping analysis. Also, the S-genotypes of cultivars in some pollen incompatibility groups that had previously been incorrectly reported have been clarified. Several popular cultivars, which were previously used as testers for S-allele typing analysis, were found to have been inaccurately genotyped. In addition, the S-genotypes and self-incompatibility groups of some relatively recentlyintroduced cultivars were identified. The molecular typing system ofS-genotypes based on PCR is a useful and rapid method for identifying newS-alleles and incompatibility groups in sweet cherry.     

Two linked genes on rice chromosome 2 for F1 pollen sterility in a hybrid between Oryza sativa and O. glumaepatula

Hybrid incompatibility plays an important role in establishment of post-zygotic reproductive isolation. To unveil genetic basis of hybrid incompatibilities between diverged species of genus Oryza AA genome species, we conducted genetic dissection of hybrid sterility loci, S22(t), which had been identified in backcross progeny derived from Oryza sativa ssp. japonica (recurrent parent) and South American wild rice O. glumaepatula near the end of the short arm of chromosome 2. The S22(t) region was found to be composed of two loci, designated S22A and S22B, that independently induce F₁ pollen sterility. Pollen grains containing either of the sterile alleles (S22A-glum^s or S22B-glum^s) were sterile if produced on a heterozygous plant. No transmission of the S22A-glum^s allele via pollen was observed, whereas a low frequency of transmission of S22B-glum^s was observed. Cytological analysis showed that the sterile pollen grains caused by S22A could reach the bicellular or tricellular stage, and the nearly-sterile pollen grains caused by S22B could reach the tricellular stage. Our genetic analysis showed repulsion linkage effect is possible to induce strong reproductive barrier by high pollen sterility based on recombination value and transmission ratio of hybrid sterility gene to the progeny was influenced by frequency of competitors on fertilization.     

硬粒小麦品种Lpx-B1位点等位变异的分子鉴定及其脂肪氧化酶活性

硬粒小麦籽粒中脂肪氧化酶(LOX)与硬粒小麦面制品的加工品质关系密切相关, 而Lpx-B1位点不同变异类型对LOX活性有重要影响。对来自不同国家和地区的167份硬粒小麦品种的LOX活性进行测定, 并对其Lpx-B1位点不同变异类型进行分子鉴定。不同品种间LOX活性差异明显, 变幅为0.20～7.98 AU min^-1 g^-1。在Lpx-B1.1位点鉴定出3种等位变异, 分别为Lpx-B1.1a、Lpx-B1.1b和Lpx-B1.1c, 以Lpx-B1.1a所占比例最高(55.1%), 其次为Lpx-B1.1c (37.1%), 而Lpx-B1.1b仅占7.8%; Lpx-B1.2和Lpx-B1.3二者总是互补出现在不同的品种中, 146份品种为Lpx-B1.2型, 其余21份品种均为Lpx-B1.3型, 表明二者可能互为一对等位因子。在Lpx-B1.1位点的3种等位变异类型中, Lpx-B1.1b类型品种的LOX活性显著高于Lpx-B1.1a和Lpx-B1.1c类型品种, 而Lpx-B1.1c类型品种的LOX活性最低。Lpx-B1.3类型品种的LOX活性显著高于Lpx-B1.2类型的品种。参试品种共有6种Lpx-B1基因型组合, 其中Lpx-B1.1b/Lpx-B1.3基因型的LOX活性显著高于其他基因型, 而Lpx-B1.1c/Lpx-B1.2和Lpx-B1.1c/Lpx-B1.3基因型的LOX活性最低。这些观测结果为硬粒小麦品质育种提供了重要信息。