Expression and trans-specific polymorphism of self-incompatibility RNases in Coffea (Rubiaceae)

Self-incompatibility (SI) is widespread in the angiosperms, but identifying the biochemical components of SI mechanisms has proven to be difficult in most lineages. Coffea (coffee; Rubiaceae) is a genus of old-world tropical understory trees in which the vast majority of diploid species utilize a mechanism of gametophytic self-incompatibility (GSI). The S-RNase GSI system was one of the first SI mechanisms to be biochemically characterized, and likely represents the ancestral Eudicot condition as evidenced by its functional characterization in both asterid (Solanaceae, Plantaginaceae) and rosid (Rosaceae) lineages. The S-RNase GSI mechanism employs the activity of class III RNase T2 proteins to terminate the growth of "self" pollen tubes. Here, we investigate the mechanism of Coffea GSI and specifically examine the potential for homology to S-RNase GSI by sequencing class III RNase T2 genes in populations of 14 African and Madagascan Coffea species and the closely related self-compatible species Psilanthus ebracteolatus. Phylogenetic analyses of these sequences aligned to a diverse sample of plant RNase T2 genes show that the Coffea genome contains at least three class III RNase T2 genes. Patterns of tissue-specific gene expression identify one of these RNase T2 genes as the putative Coffea S-RNase gene. We show that populations of SI Coffea are remarkably polymorphic for putative S-RNase alleles, and exhibit a persistent pattern of trans-specific polymorphism characteristic of all S-RNase genes previously isolated from GSI Eudicot lineages. We thus conclude that Coffea GSI is most likely homologous to the classic Eudicot S-RNase system, which was retained since the divergence of the Rubiaceae lineage from an ancient SI Eudicot ancestor, nearly 90 million years ago.United States National Science Foundation [0849186]; Society of Systematic Biologists; American Society of Plant Taxonomists; Duke University Graduate Schoolinfo:eu-repo/semantics/publishedVersio

DNA-based S-genotyping of Japanese plum and pluot cultivars to clarify incompatibility relationships

Diploid japanese plum (Prunus salicina Lindl.) cultivars are commonly self-incompatible. To date, 14 incompatibility alleles (S-alleles) have been identified and labeled with alphabetical (S-a-S-n) and 5 with numeric codes (S-1, S-3-S-6). We applied polymerase chain reaction amplification of the S-RNase alleles with degenerate and allele-specific primers in 10 japanese plum cultivars and two pluots of unknown incompatibility alleles. Besides DNA sequencing, an additional method for the exact length determination of the first intron region was used for the first time for S-genotype japanese plums. The S-3-allele was shown to correspond to S-k in the alphabetic nomenclature, S-4 to S-c, S-5 to S-e, and S-6 to S-f. The S-5-allele-specific primer can be used as a reliable marker for self-compatibility in japanese plum. 'Black Amber', 'October Sun', 'TC Sun', and 'Super Giant' share the SbSc genotype, which was confirmed by test crosses. These cultivars belong to the widest incompatibility group currently known in japanese plum. An additional incompatibility group (ScSh) was established, including 'Green Sun' and 'Queen Rosa', a cultivar formerly known as a universal donor. By incorporating all previous and recent results, a table was assembled including 49 cultivars assigned to I-VII incompatibility groups, to the self-compatible group and to the group O of unique genotypes. These data may considerably contribute to further growing and breeding activities

S-genotyping in Japanese plum by PCR and capillary gel electrophoresis detection

In this work a PCR S-genotyping method using capillary electrophoresis detection was assayed in Japanese plum. Sweet cherry primers designed for S-RNase and SFB intron length polymorphism detection by capillary electrophoresis were evaluated in Japanese plum cultivars. Amplification of both genes was successful and amplified sizes were correlated with Japanese plum S-alleles. The S-RNase genotype of 58 Japanese plum type cultivars previously determined by other methods was confirmed using this technology and the SFB alleles of these cultivars were also determined. Allele sizes of both genes are reported for 13 different S-alleles found in Japanese plum and will allow efficient S-genotype characterization in this species.Peer Reviewedgamenophytic self-incompatibilityS-RNaseSFBS-allelePrunus salicina LindlPublishe

RNase H enables efficient repair of R-loop induced DNA damage.

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I

Allelic diversity of S‑RNase alleles in diploid potato species

S-ribonucleases (S-RNases) control the pistil specificity of the self-incompatibility (SI) response in the genus Solanum and several other members of the Solanaceae. The nucleotide sequences of S-RNases corresponding to a large number of S-alleles or S-haplotypes have been characterised. However, surprisingly few S-RNase sequences are available for potato species. The identification of new S-alleles in diploid potato species is desirable as these stocks are important sources of traits such as biotic and abiotic resistance. S-RNase sequences are reported here from three distinct diploid types of potato: cultivated Solanum tuberosum Group Phureja, S. tuberosum Group Stenotomum, and the wild species Solanum okadae. Partial S-RNase sequences were obtained from pistil RNA by RT-PCR or 3’RACE (Rapid Amplification of cDNA Ends) using a degenerate primer. Full length sequences were obtained for two alleles by 5’RACE. Database searches with these sequences, identified sixteen S-RNases in total, all of which are novel. The sequence analysis revealed all the expected features of functional S-RNases. Phylogenetic analysis with selected published S-RNase and S-like-RNase sequences from the Solanaceae revealed extensive trans-generic evolution of the S-RNases and a clear distinction from S-like-RNases. Pollination tests were used to confirm the self-incompatibility status and cross-compatibility relationships of the S. okadae accessions. All the S. okadae accessions were found to be self-incompatible as expected with crosses amongst them exhibiting both cross-compatibility and semi-compatibility consistent with the S-genotypes determined from the S-RNase sequence data. The progeny analysis of four semi-compatible crosses examined by allele-specific PCR provided further confirmation that these are functional S-RNases

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

The effect of sheltered load on reproduction in Solanum carolinense, a species with variable self-incompatibility

In previous studies, we have investigated the strength of self-incompatibility (SI) in Solanum carolinense, a highly successful weed with a fully functional SI system that inhabits early successional and other disturbed habitats. We have found that the SI response in S. carolinense is a plastic trait—its strength being affected by the age of the flowers, and the presence of developing fruits and that there are genetic differences among families in their self-fertility. However, in species with a fully functional SI response, selfing would not be that common. As a result, deleterious recessives scattered though the genome of horsenettle are only occasionally exposed to selection. It has been suggested that deleterious recessives accumulate near S-alleles in strong SI species because the S-locus is located in a non-recombining region of the genome and because strong S-alleles are never in the homozygous state, thus sheltering some of the genetic load near the S-locus from selection. We performed a series of laboratory and greenhouse experiments to determine the extent to which sheltered load adds to the overall magnitude of inbreeding depression in horsenettle. Specifically, we amplified and sequenced the S-alleles from 16 genets collected from a large population in Pennsylvania and performed a series of controlled self-pollinations. We then grew the selfed progeny in the greenhouse; recorded various measures of growth and reproductive output; and amplified and sequenced their S-allele(s). We found that the heterozygous progeny of self-pollinations produce more flowers and have a greater ability to set both self and cross seed than S-homozygous progeny. We also found evidence of variation in the magnitude of load among S-alleles. These results suggest that sheltered load might slow the fixation of weak (partially compatible) S-alleles in this population, thus adding to the maintenance of a mixed mating system rather than leading to the fixation of the selfing allele

Molecular and genetic characterization of novel S-RNases from a natural population of Nicotiana alata

Self-incompatibility in the Solanaceae is mediated by S-RNase alleles expressed in the style, which confer specificity for pollen recognition. Nicotiana alata has been successfully used as an experimental model to elucidate cellular and molecular aspects of S-RNase-based self-incompatibility in Solanaceae. However, S-RNase alleles of this species have not been surveyed from natural populations and consequently the S-haplotype diversity is poorly known. Here the molecular and functional characterization of seven S-RNase candidate sequences, identified from a natural population of N. alata, are reported. Six of these candidates, S 5 , S 27 , S 70 , S 75 , S 107 , and S 210 , showed plant-specific amplification in the natural population and style-specific expression, which increased gradually during bud maturation, consistent with the reported S-RNase expression. In contrast, the S 63 ribonuclease was present in all plants examined and was ubiquitously expressed in different organs and bud developmental stages. Genetic segregation analysis demonstrated that S 27 , S 70 , S 75 , S 107 , and S 210 alleles were fully functional novel S-RNases, while S 5 and S 63 resulted to be non-S-RNases, although with a clearly distinct pattern of expression. These results reveal the importance of performing functional analysis in studies of S-RNase allelic diversity. Comparative phylogenetic analysis of six species of Solanaceae showed that N. alata S-RNases were included in eight transgeneric S-lineages. Phylogenetic pattern obtained from the inclusion of the novel S-RNase alleles confirms that N. alata represents a broad sample of the allelic variation at the S-locus of the Solanaceae.Fil: Roldán, Juan Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Quiroga, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Goldraij, Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

SPT5 affects the rate of mRNA degradation and physically interacts with CCR4 but does not control mRNA deadenylation

The CCR4-NOT complex has been shown to have multiple roles in mRNA metabolism, including that of transcriptional elongation, mRNA transport, and nuclear exosome function, but the primary function of CCR4 and CAF1 is in the deadenylation and degradation of cytoplasmic mRNA. As previous genetic analysis supported an interaction between SPT5, known to be involved in transcriptional elongation, and that of CCR4, the physical association of SPT5 with CCR4 was examined. A two-hybrid screen utilizing the deadenylase domain of CCR4 as a bait identified SPT5 as a potential interacting protein. SPT5 at its physiological concentration was shown to immunoprecipitate CCR4 and CAF1, and in vitro purified SPT5 specifically could bind to CAF1 and the deadenylase domain of CCR4. We additionally demonstrated that mutations in SPT5 or an spt4 deletion slowed the rate of mRNA degradation, a phenotype associated with defects in the CCR4 mRNA deadenylase complex. Yet, unlike ccr4 and caf1 deletions, spt5 and spt4 defects displayed little effect on the rate of deadenylation. They also did not affect decapping or 5\u27 - 3\u27 degradation of mRNA. These results suggest that the interactions between SPT5/SPT4 and the CCR4-NOT complex are probably the consequences of effects involving nuclear events and do not involve the primary role of CCR4 in mRNA deadenylation and turnover