Self-incompatibility Mechanism Research Articles

Do s genes or deleterious recessives control late-acting self-incompatibility in Handroanthus heptaphyllus (Bignoniaceae)? A diallel study with four full-sib progeny arrays.

Genetically controlled self-incompatibility (SI) mechanisms constrain selfing and thus have contributed to the evolutionary diversity of flowering plants. In homomorphic gametophytic SI (GSI) and homomorphic sporophytic SI (SSI), genetic control is usually by the single multi-allelic locus S. Both GSI and SSI prevent self pollen tubes reaching the ovary and so are pre-zygotic in action. In contrast, in taxa with late-acting self-incompatibility (LSI), rejection is often post-zygotic, since self pollen tubes grow to the ovary, where fertilization may occur prior to floral abscission. Alternatively, lack of self fruit set could be due to early-acting inbreeding depression (EID). The aim of our study was to investigate mechanisms underlying the lack of selfed fruit set in Handroanthus heptaphyllus in order to assess the likelihood of LSI versus EID. We employed four full-sib diallels to study the genetic control of LSI in H. heptaphyllus using a precociously flowering variant. We also used fluorescence microscopy to study the incidence of ovule penetration by pollen tubes in pistils that abscised following pollination or initiated fruits. All diallels showed reciprocally cross-incompatible full sibs (RCIs), reciprocally cross-compatible full sibs (RCCs) and non-reciprocally compatible full sibs (NRCs) in almost equal proportions. There was no significant difference between the incidences of ovule penetrations in abscised pistils following self- and cross-incompatible pollinations, but those in successful cross-pollinations were around 2-fold greater. A genetic model postulating a single S locus with four S alleles, one of which, in the maternal parent, is dominant to the other three, will produce RCI, RCC and NRC full sib situations each at 33 %, consistent with our diallel results. We favour this simple genetic control over an EID explanation since none of our pollinations, successful or unsuccessful, resulted in partial embryo development, as would be expected under a whole-genome EID effect.

The identification of the Rosa S-locus and implications on the evolution of the Rosaceae gametophytic self-incompatibility systems

In Rosaceae species, two gametophytic self-incompatibility (GSI) mechanisms are described, the Prunus self-recognition system and the Maleae (Malus/Pyrus/Sorbus) non-self- recognition system. In both systems the pistil component is a S-RNase gene, but from two distinct phylogenetic lineages. The pollen component, always a F-box gene(s), in the case of Prunus is a single gene, and in Maleae there are multiple genes. Previously, the Rosa S-locus was mapped on chromosome 3, and three putative S-RNase genes were identified in the R. chinensis ‘Old Blush’ genome. Here, we show that these genes do not belong to the S-locus region. Using R. chinensis and R. multiflora genomes and a phylogenetic approach, we identified the S-RNase gene, that belongs to the Prunus S-lineage. Expression patterns support this gene as being the S-pistil. This gene is here also identified in R. moschata, R. arvensis, and R. minutifolia low coverage genomes, allowing the identification of positively selected amino acid sites, and thus, further supporting this gene as the S-RNase. Furthermore, genotype–phenotype association experiments also support this gene as the S-RNase. For the S-pollen GSI component we find evidence for multiple F-box genes, that show the expected expression pattern, and evidence for diversifying selection at the F-box genes within an S-haplotype. Thus, Rosa has a non-self-recognition system, like in Maleae species, despite the S-pistil gene belonging to the Prunus S-RNase lineage. These findings are discussed in the context of the Rosaceae GSI evolution. Knowledge on the Rosa S-locus has practical implications since genes controlling floral and other ornamental traits are in linkage disequilibrium with the S-locus.

Breeding Systems and Pollen-Ovule Ratios inEricaSpecies (Ericaceae) of the Cape Floristic Region

Premise of research. Breeding systems dictate the potential for self-fertilization in plants and may therefore have a strong influence on evolutionary and ecological processes. Previous investigations of breeding systems in Ericaceae indicate variation in self-sterility, but studies from the megadiverse genus Erica are almost entirely lacking. Here we quantify the degree of self-sterility and autofertility in Cape Erica species, quantify pollen-ovule ratios (P/Os), and test whether these are related to each other and vary across species with bird- versus insect-pollination syndromes.Methodology. We used controlled hand-pollinations, analyses of pollen tube development, and P/Os to investigate the breeding systems of 15 taxa (comprising 13 species) of Erica in the Cape Floristic Region of South Africa, spanning most main clades across the phylogeny.Pivotal results. In all but one species, seed set from autonomous selfing was almost absent and significantly lower than that from cross-pollination, and only one species had an autofertility index that was higher than 0.2. In 12 taxa, seed set following self-pollination was significantly reduced compared with that following cross-pollination, and the index of self-incompatibility (ISI) was higher than 0.8 for 10 taxa. Following self-pollination, pollen tubes reached the ovary in all species, and for two species with very low seed set in selfed flowers, fluorescence images revealed that self-pollen tubes entered into the ovules. P/Os ranged from 68 to 320 and are therefore low for obligate outcrossing species. These ratios did not correlate with the ISI and did not vary according to whether species conformed to insect- or bird-pollination syndromes.Conclusions. This study indicates that self-sterility is frequent in Cape Erica species and either involves a late-acting mechanism of self-incompatibility or is due to early inbreeding depression. P/Os in these species are consistently low and do not reflect variation in breeding systems and pollination syndromes.

Screening and functional analysis of potential <i>S</i> genes in <i>Chrysanthemum morifolium</i>

<i>S</i> genes are the key genes that cause plant self-incompatibility, to find out the key <i>S</i> genes and understand molecular mechanism of self-incompatibility in chrysanthemum, the stigmas and anthers at different developmental stages of 'Q10-22-2'—a self-incompatible chrysanthemum cultivar, were used for RNA sequencing. After bioinformatics analysis, 13 candidate pistil <i>S</i> genes and five candidate pollen <i>S</i> genes were excavated. A potential pistil <i>S</i> gene was cloned and named as <i>CmSRK1</i>. Meanwhile, a potential pollen <i>S</i> gene was cloned and named as <i>CmPCP1</i>. qRT-PCR revealed that <i>CmSRK1</i> was specifically expressed in mature stigmas, and <i>CmPCP1</i> was specifically expressed in anthers 3 d before maturation. Subcellular localization showed that both CmSRK1 and CmPCP1 were located in the nucleus and the cell membrane. Transcriptional activation activity analysis indicated that both of the two proteins had no transcriptional activation activity. Yeast two hybrid assay showed that there was no interaction between CmSRK1 and CmPCP1. <i>CmSRK1</i> was constructed on the expression vector containing stigma-specific promoter, and <i>CmPCP1</i> was constructed on the expression vector containing pollen-specific promoter, they were then transformed into <i>Arabidopsis thaliana</i>. Artificial hybridization was performed with transgenic lines containing <i>CmSRK1</i> as the female parents, and transgenic lines containing <i>CmPCP1</i> as the male parents. The hybridization results showed that seed sets of two transgenic lines were 19.62% and 11.64%, respectively, while cross-pollinated seed sets of Col-0 was 84.43%. Therefore, it was speculated that <i>CmSRK1</i> and <i>CmPCP1</i> might be pistil and pollen <i>S</i> genes of chrysanthemum, respectively, and SI of chrysanthemum belonged to SSI.

SKP1-like protein, CrSKP1-e, interacts with pollen-specific F-box proteins and assembles into SCF-type E3 complex in 'Wuzishatangju' (Citrus reticulata Blanco) pollen.

S-ribonuclease (S-RNase)-based self-incompatibility (SI) mechanisms have been extensively studied in Solanaceae, Rosaceae and Plantaginaceae. S-RNase-based SI is controlled by two closely related genes, S-RNase and S-locus F-box (SLF), located at a polymorphic S-locus. In the SI system, the SCF-type (SKP1-CUL1-F-box-RBX1) complex functions as an E3 ubiquitin ligase complex for ubiquitination of non-self S-RNase. Pummelo (Citrus grandis) and several mandarin cultivars are suggested to utilize an S-RNase-based SI system. However, the molecular mechanism of the non-S-factors involved in the SI reaction is not straightforward in Citrus. To investigate the SCF-type E3 complex responsible for the SI reaction in mandarin, SLF, SKP1-like and CUL1 candidates potentially involved in the SI reaction of ‘Wuzishatangju’ (Citrus reticulata Blanco) were identified based on the genome-wide identification and expression analyses. Sixteen pollen-specific F-box genes (CrFBX1-CrFBX16), one pollen-specific SKP1-like gene (CrSKP1-e) and two CUL1 genes (CrCUL1A and CrCUL1B) were identified and cloned from ‘Wuzishatangju’. Yeast two-hybrid (Y2H) and in vitro binding assays showed that five CrFBX proteins could bind to CrSKP1-e, which is an ortholog of SSK1 (SLF-interacting-SKP1-like), a non-S-factor responsible for the SI reaction. Luciferase complementation imaging (LCI) and in vitro binding assays also showed that CrSKP1-e interacts with the N-terminal region of both CrCUL1A and CrCUL1B. These results indicate that CrSKP1-e may serve as a functional member of the SCF-type E3 ubiquitin ligase complex in ‘Wuzishatangju’.

Molecular cloning and expression analysis of hybrid hazelnut (Corylus heterophylla×Corylus avellana) ChaSRK1/2 genes and their homologs from other cultivars and species.

The self-incompatibility system of Corylus is a sporophytic type that is phenotypically similar to that of Brassica. While the molecular mechanism of sporophytic self-incompatibility (SSI) has been investigated extensively in Brassica (Brassicaceae), little is known about the corresponding mechanism in Corylus (Betulaceae). Here, we discuss the SSI mechanism with respect to S-locus receptor kinase (SRK) gene homologs. To obtain two SRK candidate unigenes, we compared all of the unigenes in a transcriptional protein database from our previous study with BnSRK-1 (AB270767) using BLASTX with a cutoff e-value of 10-5. We then cloned the full-length cDNA of ChaSRK1 and ChaSRK2 genes from Ping'ou hybrid hazelnut (Corylus heterophylla×Corylus avellana) using RACE techniques. Bioinformatics approaches were used to analyze the cDNA sequences, protein sequences, and domains of the encoded proteins. The full-length ChaSRK1 cDNA was 2883 base pairs (bp) with a coding sequence (CDS) of 2,547bp encoding 849 amino acid residues. The full-length ChaSRK2 cDNA was 2,693bp, with a CDS of 2,433bp encoding 811 amino acids. The ChaSRK1/2 proteins contained an S-domain (extracellular domain), a transmembrane domain, a Ser/Thr protein kinase active site (kinase domain), and DUF3660 and/or DUF3403 domains. The lengths of 18 partial SRK homologs ranged from 1347 to 1451bp, and they contained the same structural domains as ChaSRK1 and ChaSRK2. Phylogenetic analysis revealed that all SRK homologs could be divided into two categories that were similar to the classification of SRKs in Brassica. The expression patterns of ChaSRK1 and ChaSRK2 differed: ChaSRK2 was predominantly expressed in mature stigmatic styles, while ChaSRK1 was expressed in other tissues with the highest in the root tips of Corylus. Using dual-color fluorescence in situ hybridization, ChaSRK1/2 expression was found to be localized in papillar cells. Collectively, these results revealed that SRKs from Corylus had similar characteristics to SRKs from Brassica. We therefore speculated that the SSI mechanism in Corylus might be more similar to the Brassica mechanism than to other SSI types.

Self-(In)compatibility Systems: Target Traits for Crop-Production, Plant Breeding, and Biotechnology.

Self-incompatibility (SI) mechanisms prevent self-fertilization in flowering plants based on specific discrimination between self- and non-self pollen. Since this trait promotes outcrossing and avoids inbreeding it is a widespread mechanism of controlling sexual plant reproduction. Growers and breeders have effectively exploited SI as a tool for manipulating domesticated crops for thousands of years. However, only within the past thirty years have studies begun to elucidate the underlying molecular features of SI. The specific S-determinants and some modifier factors controlling SI have been identified in the sporophytic system exhibited by Brassica species and in the two very distinct gametophytic systems present in Papaveraceae on one side and in Solanaceae, Rosaceae, and Plantaginaceae on the other. Molecular level studies have enabled SI to SC transitions (and vice versa) to be intentionally manipulated using marker assisted breeding and targeted approaches based on transgene integration, silencing, and more recently CRISPR knock-out of SI-related factors. These scientific advances have, in turn, provided a solid basis to implement new crop production and plant breeding practices. Applications of self-(in)compatibility include widely differing objectives such as crop yield and quality improvement, marker-assisted breeding through SI genotyping, and development of hybrids for overcoming intra- and interspecific reproductive barriers. Here, we review scientific progress as well as patented applications of SI, and also highlight future prospects including further elucidation of SI systems, deepening our understanding of SI-environment relationships, and new perspectives on plant self/non-self recognition.

Three multi-allelic gene pairs are responsible for self-sterility in the ascidian Ciona intestinalis

Many hermaphroditic organisms possess a self-incompatibility system to avoid inbreeding. Although the mechanisms of self-incompatibility in flowering plants are well known, little is known about the mechanisms of self-sterility in hermaphroditic marine invertebrates. Ascidians are hermaphroditic sessile marine invertebrates that release sperm and eggs into the surrounding seawater. Several species, including Ciona intestinalis type A (Ciona robusta), exhibit strict self-sterility. In a previous study, we found that the candidate genes responsible for self-sterility in Ciona reside in chromosome 2q (locus A) and chromosome 7q (locus B). Two pairs of multi-allelic genes, named s(sperm)-Themis-A and v(vitelline-coat)-Themis-A in locus A and s-Themis-B and v-Themis-B in locus B, are responsible for self-sterility. In this study, we identified a third multi-allelic gene pair, s-Themis-B2 and v-Themis-B2, within locus B that is also involved in this system. Genetic analysis revealed that the haplotypes of s/v-Themis-A, s/v-Themis-B and s/v-Themis-B2 play essential roles in self-sterility. When three haplotypes were matched between s-Themis and v-Themis, fertilization never occurred even in nonself crossing. Interestingly, gene targeting of either s/v-Themis-B/B2 or s/v-Themis-A by genome editing enabled self-fertilization. These results indicate that s/v-Themis-A, -B and -B2 are S-determinant genes responsible for self-sterility in the ascidian C. intestinalis type A.

Buckwheat heteromorphic self-incompatibility: genetics, genomics and application to breeding.

Common buckwheat (Fagopyrum esculentum Moench 2n = 2x = 16) is an outcrossing crop with heteromorphic self-incompatibility due to its distylous flowers, called pin and thrum. In pin plants, a long style is combined with short stamens and small pollen grains; in thrum plants, a short style is combined with long stamens and large pollen grains. Both the intra-morph self-incompatibility and flower morphology are controlled by a single genetic locus named the S locus; thrum plants are heterozygous (Ss) and pin plants are homozygous recessive (ss) at this locus. Self-incompatibility is an obstacle for establishing pure lines and fixation of agronomically useful genes. Elucidation of the molecular mechanism of heterostylous self-incompatibility of common buckwheat has continued for a quarter of a century. Recent advances in genomic and transcriptomic analyses using next-generation sequencing have made it possible to determine the genomic region harboring the buckwheat S locus and to identify novel genes at this locus. In this review, we summarize the current knowledge on buckwheat heterostyly gained from conventional and molecular genetics and genomics. We also discuss the application of these studies to breeding of common buckwheat.

Assessment of Mating System in Helianthus annuus and H. petiolaris (Asteraceae) Populations

Abstract Helianthus annuus subsp. annuus and H. petiolaris are wild North American species that have been naturalized in central Argentina. They have a sporophytic self-incompatibility genetic system that prevent self-fertilization but the occurrence of self-compatible plants in Argentina was observed in both species and could in part explain their highly invasive ability. Their geographical distribution coincides with the major crop area. The domestic sunflower is self-compatible, can hybridize with both species and presents a considerable amount of gene flow. The aim of this study is to understand the self-incompatibility mechanism in both wild Helianthus species. Reciprocal crossing and seed production were used to identify self-compatible genotypes, the number and distribution of self-incompatibility alleles within populations and the type and extent of allelic interactions in the pollen and pistil. The behaviour of S alleles within each population was explained by five functional S alleles and one non-functional allele in each species, differing in their presence and frequency within accessions. In both species, the allelic interactions were of dominance/recessiveness and codominance in pollen, whereas it was only codominance in the pistil. Inbreeding effects in wild materials appeared in the third generation of self-pollination, with lethal effects in most plants. The number of S alleles is low and they behave in a similar way of other Asteraceae species. The self-compatibility was addressed to non-functional S alleles introgressed in wild Helianthus plants through gene flow from self-compatible sunflower.

Reverse genetics analysis of the 55-kDa B regulatory subunit of 2A serine/threonine protein phosphatase (PP2A) related to self-incompatibility in Chinese cabbage

The type 2A serine/threonine protein phosphatases (PP2As) are key components in regulating signal transduction and controlling cell metabolism. In particular, PP2A has a role in pollen-pistil interaction during pollination in Brassica. In this study, reverse genetics screening was used to obtain a T-DNA-inserted Brassica rapa mutant line with the self-compatible phenotype. The type 2A PP2A 55-kDa B regulatory subunit gene (PR55/B) in this mutant was knocked out by T-DNA insertion, and its expression level was decreased, which led to self-compatible phenotypes, such as seed setting by flower self-pollination. For functional analysis of this self-compatible mutant line, the full-length PR55/B gene related to the self-incompatibility mechanism was isolated and the down-regulation vector (pPPi) was constructed for introduction into Chinese cabbage. Chinese cabbage lines transformed with pPPi showed significantly decreased PR55/B mRNA accumulation, which yielded self-compatible phenotypes. These transgenic lines were found to set pods and seeds by the self-pollination in both buds and flowers. Down-regulation of PP2A, which is related to self-incompatibility signal transduction, led to higher levels of the phosphorylated S-locus receptor kinase (SRK) substrate, which phosphorylated SRK blocks activation of armadillo-repeat-containing 1 (ARC1). Thus, the self-incompatibility response by ARC1 was not activated, and this led to the self-compatible response. Finally, PP2A is proposed to be a significant factor in self-incompatibility of Chinese cabbage.

Identification of Single Nucleotide Polymorphism in Red Clover (Trifolium pratense L.) Using Targeted Genomic Amplicon Sequencing and RNA-seq.

Red clover (Trifolium pratense L.) is a diploid, naturally cross-pollinated, cool-season species. As a perennial forage legume, red clover is mostly cultivated in temperate regions worldwide. Being a non-model crop species, genomic resources for red clover have been underdeveloped. Thus far, genomic analysis used in red clover has mainly relied on simple sequence repeat (SSR) markers. However, SSR markers are sparse in the genome and it is often difficult to unambiguously map them using short reads generated by next generation sequencing technology. Single nucleotide polymorphisms (SNPs) have been successfully applied in genomics assisted breeding in several agriculturally important species. Due to increasing importance of legumes in forage production, there is a clear need to develop SNP based markers for red clover that can be applied in breeding applications. In this study, we first developed an analytical pipeline that can confidently identify SNPs in a set of 72 different red clover genotypes using sequences generated by targeted amplicon sequencing. Then, with the same filtering stringency used in this pipeline, we used sequences from publicly available RNA-seq data to identify confident SNPs in different red clover varieties. Using this strategy, we have identified a total of 69,975 SNPs across red clover varieties. Among these, 28% (19,116) of them are missense mutations. Using Medicago truncatula as the reference, we annotated the regions affected by these missense mutations. We identified 2,909 protein coding regions with missense mutations. Pathway analysis of these coding regions indicated several biological processes impacted by these mutations. Specifically, three domains (homeobox domain, pentatricopeptide repeat containing plant-like, and regulator of Vps4 activity) were identified with five or more missense SNPs. These domain might also be a functional contributor in the molecular mechanisms of self-incompatibility in red clover. Future in-depth sequence diversity analysis of these three genes may yield valuable insights into the molecular mechanism involved in self-incompatibility in red clover.

Ontogenesis of ovary and fruit of Acianthera johannensis (Pleurothallidinae, Orchidaceae) reveals a particular female embryology

We carried out an investigation on the female ontogenesis of Acianthera johannensis, a species with partial self-incompatibility, aiming to report the typical development of ovule and seed. We describe here the development of ovary and fruit after cross-pollination and record the time between pollination and seed formation. In this species, we verify the formation of triads of megaspores and five-nucleate megagametophytes, a very rare association in orchids. The species presents anatropous, tenuinucellate and bitegmic ovules, characters recorded for most of the orchids. Fertilization occurred 40 days after pollination, however some ovules were observed in early stages of development even in fully developed fruits. Our data indicate that unfertilized ovule can be found in mature fruits, reinforcing the hypothesis that these could be the structures often referred in the literature as seeds without embryos. This information is important to the following studies that aim to clarify the mechanism involved in the formation of structures without embryo observed in fruits developed after self-pollination and the nature of the complex mechanism of self-incompatibility in Acianthera.

Gametophytic self-incompatibility in Rosaceae fruit trees

Rosaceae fruit trees are characterized by gametophytic self-incompatibility, with their production typically requiring artificial pollination or pollination tree is required in production. Both of these solutions cause reductions in production efficiency, and self-incompatibility has become a major issue in agricultural biology, and as such, has been extensively studied. In this review, we discuss the relationship between S-RNase content in the style and self-incompatibility, and the role of the SLF gene in stamen-determining factor. Considering mutations in self-compatibility-related genes and self-compatibility in polyploid fruit trees, we discuss the potential mechanisms of self-incompatibility. Based on a preliminary study of the role of pollen tube Ca2+ gradients in self-incompatibility in Pyrus, we propose a new mechanistic model of self-incompatibility taking into account the effect of Ca2+. We also discuss the potential for hormone regulation to be used to control self-incompatibility in Rosaceae fruit trees.

Functional Analysis of M-Locus Protein Kinase Revealed a Novel Regulatory Mechanism of Self-Incompatibility in Brassica napus L.

Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional homolog of BrMLPKs in Brassica rapa, controls SI in B. napus. We identified four paralogue MLPK genes in B. napus, including BnaA3.MLPK, BnaC3.MLPK, BnaA4.MLPK, and BnaC4.MLPK. Two transcripts of BnaA3.MLPK, BnaA3.MLPKf1 and BnaA3.MLPKf2, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that BnaA3.MLPK, especially BnaA3.MLPKf2, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of BnaMLPKs in B. napus SI line S-70. Phenotypic analysis revealed that SI response is partially suppressed in RNA-silencing lines and is completely blocked in quadruple mutants. These results indicate the importance of BnaMLPKs in regulating the SI response of B. napus. We found that the expression of SI positive regulators S-locus receptor kinase (SRK) and Arm-Repeat Containing 1 (ARC1) are suppressed in bnmlpk mutant, whereas the self-compatibility (SC) element Glyoxalase I (GLO1) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of MLPK in the SI of B. napus.

Genetic insights into the modification of the pre-fertilization mechanisms during plant domestication.

Plant domestication is the process of adapting plants to human use by selecting specific traits. The selection process often involves the modification of some components of the plant reproductive mechanisms. Allelic variants of genes associated with flowering time, vernalization, and the circadian clock are responsible for the adaptation of crops, such as rice, maize, barley, wheat, and tomato, to non-native latitudes. Modifications in the plant architecture and branching have been selected for higher yields and easier harvests. These phenotypes are often produced by alterations in the regulation of the transition of shoot apical meristems to inflorescences, and then to floral meristems. Floral homeotic mutants are responsible for popular double-flower phenotypes in Japanese cherries, roses, camellias, and lilies. The rise of peloric flowers in ornamentals such as snapdragon and florists' gloxinia is associated with non-functional alleles that control the relative expansion of lateral and ventral petals. Mechanisms to force outcrossing such as self-incompatibility have been removed in some tree crops cultivars such as almonds and peaches. In this review, we revisit some of these important concepts from the plant domestication perspective, focusing on four topics related to the pre-fertilization mechanisms: flowering time, inflorescence architecture, flower development, and pre-fertilization self-incompatibility mechanisms.

Identification of a gene encoding polygalacturonase expressed specifically in short styles in distylous common buckwheat (Fagopyrum esculentum)

Common buckwheat (Fagopyrum esculentum) is a heteromorphic self-incompatible (SI) species with two types of floral architecture: thrum (short style) and pin (long style). The floral morphology and intra-morph incompatibility are controlled by a single genetic locus, S. However, the molecular mechanisms underlying the heteromorphic self-incompatibility of common buckwheat remain unclear. To identify these mechanisms, we performed proteomic, quantitative reverse-transcription PCR, and linkage analyses. Comparison of protein profiles between the long and short styles revealed a protein unique to the short style. Amino-acid sequencing revealed that it was a truncated form of polygalacturonase (PG); we designated the gene encoding this protein FePG1. Phylogenetic analysis classified FePG1 into the same clade as PGs that function in pollen development and floral morphology. FePG1 expression was significantly higher in short styles than in long styles. It was expressed in flowers of a short-homostyle line but not in flowers of a long-homostyle line. Linkage analysis indicated that FePG1 was not linked to the S locus; it could be a factor downstream of this locus. Our finding of a gene putatively working under the regulation of the S locus provides useful information for elucidation of the mechanism of heteromorphic self-incompatibility.

Progress on deciphering the molecular aspects of cell-to-cell communication in Brassica self-incompatibility response.

The sporophytic system of self-incompatibility is a widespread genetic phenomenon in plant species, promoting out-breeding and maintaining genetic diversity. This phenomenon is of commercial importance in hybrid breeding of Brassicaceae crops and is controlled by single S locus with multiple S haplotypes. The molecular genetic studies of Brassica 'S' locus has revealed the presence of three tightly linked loci viz. S-receptor kinase (SRK), S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11), and S-locus glycoprotein (SLG). On self-pollination, the allele-specific ligand-receptor interaction activates signal transduction in stigma papilla cells and leads to rejection of pollen tube on stigmatic surface. In addition, arm-repeat-containing protein 1 (ARC1), M-locus protein kinase (MLPK), kinase-associated protein phosphatase (KAPP), exocyst complex subunit (Exo70A1) etc. has been identified in Brassica crops and plays a key role in self-incompatibility signaling pathway. Furthermore, the cytoplasmic calcium (Ca2+) influx in papilla cells also mediates self-incompatibility response in Brassicaceae, but how this cytoplasmic Ca2+ influx triggers signal transduction to inhibit pollen hydration is still obscure. There are many other signaling components which are not well characterized yet. Much progress has been made in elucidating the downstream multiple pathways of Brassica self-incompatibility response. Hence, in this review, we have made an effort to describe the recent advances made on understanding the molecular aspects of genetic mechanism of self-incompatibility in Brassicaceae.

Transcriptomics Investigation into the Mechanisms of Self-Incompatibility between Pin and Thrum Morphs of Primula maximowiczii

Heteromorphic self-incompatibility (SI) is an important system for preventing inbreeding in the genus Primula. However, investigations into the molecular mechanisms of Primula SI are lacking. To explore the mechanisms of SI in Primula maximowiczii, the pollen germination and fruiting rates of self- and cross-pollinations between pin and thrum morphs were investigated, and transcriptomics analyses of the pistils after pollination were performed to assess gene expression patterns in pin and thrum SI. The results indicated that P. maximowiczii exhibits strong SI and that the mechanisms of pollen tube inhibition differ between pin and thrum morphs. While self-pollen tubes of the pin morph were able to occasionally, though rarely, enter the style, those of the thrum morph were never observed to enter the style. The transcriptomics analysis of the pistils revealed 1311 and 1048 differentially expressed genes (DEGs) that were identified by comparing pin self-pollination (PS) vs. pin cross-pollination (PT) and thrum self-pollination (TS) vs. thrum cross-pollination (TP). Notably, about 90% of these DEGs exhibited different expression patterns in the two comparisons. Moreover, pin and thrum DEGs were associated with different Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways following enrichment analyses. Based on our results, the molecular mechanisms underlying the pin and thrum SI in P. maximowiczii appear to be distinct. Furthermore, the genes involved in the SI processes are commonly associated with carbohydrate metabolism and environmental adaptation. These results provide new insight into the molecular mechanisms of Primula SI.

A molecular dating service: finding the perfect match for every apple

Despite being perfect hermaphrodites, most angiosperms have developed self-incompatibility as an important genetic mechanism to avoid self-fertilization and inbreeding, and to promote allogamy and outcrossing. The molecular mechanisms of gametophytic self-incompatibility (GSI) in the Rosaceae have been studied intensively, since fertilization represents an important agronomic parameter for stable fruit yield and breeding activities. In apple, GSI is controlled by an S locus with a number of S alleles. In the case of identical S alleles between male pollen and female stigma during pollination, stigmatic S-RNases are able to arrest growth of the pollen tube, preventing it from reaching and fertilizing the ovule. Knowledge of compatible cultivars is a basic requirement for the selection of appropriate pollen donors in production orchards as well as in breeding programs. Thus, to identify the S alleles of a cultivar, a series of different protocols have been developed using either allele-specific primer amplifications or allele-specific restriction enzymes. Both require individual assays for the detection of each single allele out of over 30, and thus lack efficiency in terms of time and money. Recently, a high-throughput method for genotyping S alleles via capillary electrophoresis has been developed that allows up to 49 different S alleles to be distinguished through the use of general amplification primers combined with two enzymatic restrictions. In the present study, the method has been optimized and applied to a set of 180 apple cultivars ranging from local cultivars to new commercial cultivars and breeding lines. With this protocol, all known S alleles of Malus × domestica could be differentiated in a single multiplexed run of capillary electrophoresis. Comparing the results to phenotypic fertilization tests allows the compatibility of cultivars to be predicted in future designs of apple orchards, as well as in future breeding activities.