Pollen-stigma Incompatibility Research Articles

Pollen-stigma incompatibility within and between species: Tread lightly, sedate the dogs, and don't wake the guards!

In a recent issue of Nature, Huang etal. identify and show how to overcome the barriers to successful pollen germination after interspecific crosses.1 Their findings answer a long-standing question about reproductive barriers in flowering plants and open the door to harnessing genetic diversity of distant relatives for crop improvement.

Characterization of self-incompatible Brassica napus lines lacking SP11 expression.

Recognition of self-incompatibility (SI) is regulated by the SRK and SP11 genes in Brassicaceae. Brassica rapa and B. oleracea are self-incompatible, while most cultivated species of B. napus, which arose from hybridization between B. rapa and B. oleracea, are self-compatible. Various studies of the SRK and SP11 genes in self-compatible B. napus have been reported, but details of the mechanism in different B. napus lines are not fully understood. In this study, we confirmed the S haplotypes, SI phenotypes and SP11 expression in 10 representative lines of B. napus, and identified two SI lines (N110 and N343) lacking SP11 expression. In N343 (with BnS1 and BnS6 haplotypes), we confirmed that there is a 3.6-kb insertion in the promoter region of BnSP11-1, and that BnSP11-1 and BnSP11-6 are not expressed, as reported previously (expression of BnSP11-6 is suppressed by the BnS1 haplotype), although this line is self-incompatible. Similarly, in N110, with two novel S haplotypes (BnS8 and BnS9) in addition to BnS6, a 4.3-kb insertion was identified in the promoter region of BnSP11-9, and expression levels of BnSP11-6, BnSP11-8 and BnSP11-9 were all suppressed (BnSP11-6 and BnSP11-8 may be suppressed by BnS8 and BnS9, respectively), although the phenotype was self-incompatible. This observation of an SI phenotype without SP11 expression suggests the existence of unknown factor(s) that induce pollen-stigma incompatibility in B. napus.

INCOMPATIBILITY ALLELES OF HAZELNUT CULTIVARS

Pollen-stigma incompatibility in hazelnut is sporophytic and under the control of a single locus with multiple alleles. To date, 33 alleles have been identified at the S-locus, and a designated pollen tester for each preserved in our collection. Hazelnut is diploid, and nearly all cultivars are heterozygous at the S-locus. Over the past 25 years, we have enlarged our collection by importing cultivars from several countries as scions. As the trees began to flower, we identified their S-alleles using florescence microscopy. In December, we bagged emasculated branches on trees growing in the field to protect the female inflorescences from wind-borne pollen. In January, we collected pollen from our tester trees. In February, females were collected from bagged branches and brought to the lab in petri dishes. The females were pollinated in the afternoon, one or two per tester pollen. The following day, the styles were squashed in aniline blue and observed under UV light. In compatible pollinations, pollen germination was excellent and tubes could be seen growing parallel down the style. In incompatible pollinations, germination was often reduced, pollen tubes were short and did not penetrate the stigmatic surface, and the tubes often ended in bulbs. Each pollination was scored as compatible or incompatible, and the testing of a tree required 1-3 years for completion. In this paper, we report the alleles of 170 cultivars for the first time, and summarize the alleles of 112 cultivars previously typed. Cultivars with different names often have identical microsatellite marker fingerprints and S-alleles. To reduce confusion, a list of cultivars with identical fingerprints is also included.

Mapping the Incompatibility and Style Color Loci in Two Hazelnut Progenies

Pollen–stigma incompatibility in european hazelnut (Corylus avellana L.) is of the sporophytic type and under the control of a single locus with multiple alleles (haplotypes). The S-locus was previously assigned to linkage group 5 (LG5) and linked DNA markers were identified. The loci that control leaf color and style color are linked to the S-locus. We investigated segregation for leaf and style color and S-alleles in two progenies, mapped the loci, and compared the two new maps with the LG5 reference map using simple sequence repeat (SSR) markers. Segregation for color, S-alleles and SSR markers fit expectations. The color loci and the S-locus mapped to LG5 between SSR markers B028 and B774. The three maps aligned and the SSR markers were collinear. The SSR markers closest to the S-locus are KG819, KG847, and BR259. In progeny 05050, which segregated for style and leaf color, no recombination was observed between the two traits. Recombination between the S-locus and the style color locus was 5.4 cM in progeny 05050 and 10.1 cM in progeny 00064. The style color locus was placed very close to SSR marker B028 in both progenies. On the reference map, random amplified polymorphic DNA (RAPD) markers 564-500M, 345-1050dF, and 204-950dF and intersequence simple sequence repeat (ISSR) marker 815-540dF are very close to the S-locus. The identification of closely linked markers will facilitate the map-based cloning of the S-locus and color loci in hazelnut.

Involvement of MLPK Pathway in Intraspecies Unilateral Incompatibility Regulated by a Single Locus With Stigma and Pollen Factors

Plants have evolved many systems to prevent undesirable fertilization. Among these, incompatibility is a well-organized system in which pollen germination or pollen tube growth is inhibited in pistils. We previously found that a novel one-way pollen–stigma incompatibility response [unilateral incompatibility (UI)] occurred between two self-incompatible Brassica rapa plants, a Turkish line, and a Japanese cultivated hybrid variety, “Osome.” Pollen from the Turkish line is rejected on the stigma of the Osome line, but the reverse cross is compatible; such a UI phenotype closely resembles self-incompatibility (SI). The pollen factor of this UI has been genetically explained by a single locus which is different from the S-locus. In this study, we performed further genetic analyses of this intraspecies UI and showed that the stigma factor was also controlled by a single locus, and we named the loci corresponding to the stigma and pollen factors of the intraspecies UI, stigmatic unilateral incompatibility (SUI), and pollen unilateral incompatibility (PUI) loci, respectively. Interestingly, segregation analyses of SUI and PUI indicated that they are closely linked to each other and behave as a single unit. To investigate the effect of an SI-related gene, MLPK in this UI, we produced segregation lines for SUI and mlpk. A distorted segregation ratio of SUI phenotype in an mlpk background indicated involvement of MLPK in SUI, suggesting the existence of an MLPK-dependent novel pollen–stigma recognition mechanism.

COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN ORCHIS MASCULA AND ORCHIS PAUCIFLORA

Studies of the strength and nature of reproductive isolation (RI) between species can greatly contribute to our understanding of speciation. Although the role of RI in speciation is well recognized, there is a dearth of information on the contributions of different barriers between related plant species. Here, we estimated multiple components of RI between two Mediterranean orchid sister species (Orchis mascula and Orchis pauciflora), disentangling the strength and absolute contributions of seven different isolating mechanisms. Our survey includes one prepollination, two postpollination prezygotic (pollen-stigma incompatibility, conspecific pollen precedence), two intrinsic postzygotic (embryo mortality and hybrid sterility) and two extrinsic postzygotic (hybrid habitat differentiation and hybrid pollination) isolating mechanisms. We found strong RI between the investigated species, although none of the barriers were able to completely impede gene flow. Five isolating mechanisms contributed positively to the maintenance of species boundaries. Contrary to most surveys of isolating mechanisms, our data speak against a clear predominance of prepollination or of prezygotic barriers but confirm the emerging pattern of multiple barriers contributing to the maintenance of species integrity. These findings suggest an allopatric condition during early phases of species divergence. We discuss our data in the wider context of previous studies carried out in this orchid group by using a comparative approach.

Incompatibility Alleles in Portuguese Hazelnut Landraces

In many higher plants, selffertilization and genetically related individuals are prevented by pollen-stigma incompatibility. In the genusCorylus, incompatibility is of the sporophytic type and controlled by a single locus with multiple alleles. The objective of this study is to identify theS-alleles present in a collection of Portuguese landraces in order to select the most appropriate landraces for establishment of future orchards and for breeding programmes. Ten major Portuguese hazelnut landraces were submitted to controlled pollinations in the field, with 18 genotypes whoseS-alleles are known. The pollen tubes were observed at 100X under a florescence microscope to evaluate their development. Three landraces were revealed to haveS2allele, two haveS5, and four have one of theS3,S5,S10, andS18alleles. One landrace was compatible with the 18S-alleles tested and for two landraces, it was possible to identify both alleles. The information of the self-incompatibility relationship between these old cultivars is obviously useful for selecting the most suitable pollinators for planning new orchards and for new cultivars development.

A bacterial artificial chromosome library for ‘Jefferson’ hazelnut and identification of clones associated with eastern filbert blight resistance and pollen–stigma incompatibility

European hazelnut (Corylus avellana L.) is the only economically important nut crop in the family Betulaceae. Because of its small genome size (~385 Mb / 1C), relatively short life cycle, availability of a dense linkage map, and amenability to transformation by Agrobacterium, the European hazelnut could serve as a model plant for the Betulaceae. Here we report the construction of a bacterial artificial chromosome (BAC) library for 'Jefferson' hazelnut using the cloning enzyme MboI and the vector pECBAC1 (BamHI site). The library consists of 39,936 clones arrayed in 104,384-well microtitre plates with a mean insert size of 117 kb. The genomic coverage of the library is estimated to be about 12 genome equivalents. This library provides a valuable resource for the map-based cloning of two important genes, the resistance gene from 'Gasaway' that confers resistance to eastern filbert blight caused by the fungus Anisogramma anomala (Peck) E. Müller and the S locus that controls pollen-stigma incompatibility. Fine-resolution mapping near the two loci was carried out using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. Fine mapping at the disease resistance locus showed that markers W07-375 and X01-825 flanked the resistance locus at distances of 0.06 and 0.05 cM, respectively. The S locus is flanked by markers 204-950 and KG819-200 at distances of 0.14 and 0.24 cM, respectively. Assuming that 1 cM corresponds to a physical distance of 430 kb, it will take approximately two to three chromosome walks to assemble BAC contigs that span both loci.

A genetic linkage map for hazelnut (Corylus avellanaL.) based on RAPD and SSR markers

A linkage map for European hazelnut (Corylus avellana L.) was constructed using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers and the 2-way pseudotestcross approach. A full-sib population of 144 seedlings from the cross OSU 252.146 x OSU 414.062 was used. RAPD markers in testcross configuration, segregating 1:1, were used to construct separate maps for each parent. Fifty additional RAPD loci were assigned to linkage groups as accessory markers whose exact location could not be determined. Markers in intercross configuration, segregating 3:1, were used to pair groups in one parent with their homologues in the other. Eleven groups were identified for each parent, corresponding to the haploid chromosome number of hazelnut (n = x = 11). Thirty of the 31 SSR loci were able to be assigned to a linkage group. The maternal map included 249 RAPD and 20 SSR markers and spanned a distance of 661 cM. The paternal map included 271 RAPD and 28 SSR markers and spanned a distance of 812 cM. The maps are quite dense, with an average of 2.6 cM between adjacent markers. The S-locus, which controls pollen-stigma incompatibility, was placed on chromosome 5S where 6 markers linked within a distance of 10 cM were identified. A locus for resistance to eastern filbert blight, caused by Anisogramma anomala, was placed on chromosome 6R for which two additional markers tightly linked to the dominant allele were identified and sequenced. These maps will serve as a starting point for future studies of the hazelnut genome, including map-based cloning of important genes. The inclusion of SSR loci on the map will make it useful in other populations.

Inheritance of Style Color in Hazelnut

The style color of standard hazelnut (Corylus avellana L.) cultivars ranges from pink to dark purple. Styles with an unusual yellow color were first noted in seedlings of the progeny `Goodpasture' × `Compton', and the ratio was ≈3 red: 1 yellow. Controlled crosses were made to investigate the genetic control of style color. The same 3:1 ratio was observed in four additional crosses in which both parents had red styles. Two crosses of a red and a yellow parent gave ≈50% yellow styles, while a cross of two selections with yellow styles gave only seedlings with yellow styles. These segregation ratios indicate control by a single locus, with yellow style color recessive to red. Seedlings with yellow styles have green buds and catkins and a more upright growth habit than their siblings with red styles. Inspection of the pedigrees of these progenies shows that `Daviana', `Willamette', `Butler', `Compton', `Goodpasture', and `Lansing #1' are heterozygous. `Daviana' appears to be the original source of the allele for yellow styles, as it is a known or suspected parent or ancestor of the others. Ratios in a progeny segregating simultaneously for growth habit (normal vs. contorted) and style color indicated independence of the traits. However, in a progeny segregating simultaneously for leaf color (red vs. green) and style color, no redleaf seedlings had yellow styles. The S-alleles of eight genotypes with yellow styles were determined, and indicate a possible linkage between the yellow style locus and the S locus that controls pollen-stigma incompatibility. One explanation is that the yellow style trait is conferred by an allele (ays) at the anthocyanin (A) locus that controls leaf color. A second explanation is that there is a yellow style locus closely linked to the A locus. The A locus is known to be loosely linked to the S locus.

062 Genetic Relationships among Corylus Species

Interspecific hybridization, pollen-stigma incompatibility, and DNA sequence analysis were used to study the relationships among hazelnut (Corylus) species. Interspecific crosses resulted in a wide range of cluster set from 0% to 65%. Reciprocal differences were common. In general, crosses involving C. avellana and C. heterophylla were more successful when used as pollen parents, but crosses involving C. americana were more successful when it was the female parent. C. cornuta, C. californica and C. sieboldiana intercrossed freely in both directions, as did C.colurna and C.chinensis. The Asian species, C. sieboldiana, C.heteropyhlla, and C. chinensis, were not cross-compatible with each other. Fluorescence microscopy showed that pollen-stigma incompatibility exists within and among wild hazelnut species, in addition to the cultivated European hazelnut C. avellana. Pollen-stigma incompatibility and embryo abortion (blank nuts) appear to be major blocks to interspecific gene flow. In addition, the chloroplast matK gene and the Internal Transcribed Spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) were amplified and sequenced. The matK sequence was highly conserved and thus was not informative. However, the ITS sequence was highly informative and parsimony analysis agreed with morphological similarities. Corylus species were placed into four groups: 1) C. avellana, C. maxima, C. americana and C. heterophylla 2) C. colurna, C.chinensis, and C. jacquemontii 3) C. cornuta, C. californica and C. sieboldiana 4)C. ferox.

Biosynthesis of glycoproteins involved in the pollen-stigma interaction of incompatibility in developing flowers of Brassica oleracea L.

De-novo synthesis of the S-allele-specific glycoproteins of Brassica oleracea is demonstrated in stigmas at different developmental stages. Excised stigmas incorporate (14)C-labeled amino acids into their S-glycoproteins early in development and before the self-incompatibility response is acquired, but the rate of synthesis accelerates prior to anthesis, resulting in the accumulation of high levels of the S-glycoproteins in the stigma and coinciding with the acquisition of the pollen-stigma incompatibility response. Since the self-compatible and self-incompatible zones of developing inflorescences are very sharply delineated, a threshold quantity of S-glycoproteins appears to be critical for the onset of self-incompatibility. Incorporation experiments in which [(35S)methionine was applied to intact stigma surfaces indicate that the papillae are the main sites of synthesis of the S-specific glycoproteins.