MIK2 is a candidate gene of the S-locus for sporophytic self-incompatibility (SSI) in chicory (Cichorium intybus, Asteraceae)

The Cichorium genus offers a unique opportunity to study the sporophytic self incompatibility (SSI) system, being composed of species characterized by highly efficient SI (C. intybus) and complete self compatibility (C. endivia). The chicory genome was used to map 7 previously identified SSI locus-associated markers. The region containing the S locus was restricted to an 4 M bp window on chromosome 5. Among the genes predicted in this region, MDIS1 INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) was promising as a candidate for SSI. Its ortholog in Arabidopsis is involved in pollen stigma recognition reactions, and its protein structure is similar to that of S-receptor kinase (SRK), a key component of the SSI in the Brassica genus. The sequencing of MIK2 in chicory and endive accessions revealed two contrasting scenarios. In C. endivia, MIK2 was fully conserved even comparing different botanical varieties (smooth and curly). In C. intybus, 387 SNPs and 3 INDELs were identified when comparing accessions of different biotypes from the same botanical variety (radicchio). The SNP distribution throughout the gene was uneven, with hypervariable domains preferentially localized in the LRR-rich extracellular region, putatively identified as the receptor domain. The gene was hypothesized to be under positive selection, as the nonsynonymous mutations were more than double the synonymous ones (dN / dS = 2.17). An analogous situation was observed analyzing the first 500 bp of the MIK2 promoter: no SNPs were observed among the endive samples, whereas 44 SNPs and 6 INDELs were detected among the chicory samples. Further analyses are needed to confirm the role of MIK2 in SSI and to demonstrate whether the 23 species-specific nonsynonymous SNPs in the CDS and/or the species-specific 10 bp INDEL found in a CCAAT box region of the promoter are responsible for the contrasting sexual behaviors of the two species

Heterosis in horticultural crop breeding: combining old theoretical bases with modern genomic views

Heterosis in plants has been among the challenging topics for plant scientists worldwide. The production of F1 hybrid varieties of seed-propagated horticultural species is one of the most successful applications of plant breeding techniques. The exploitation of the heterosis phenomenon promotes homogeneity and maximizes crop yields and is a way for breeders to legally control and protect their commercial products. In the past heterosis has been largely studied and explored in cereal crop systems, considering maize as a model for understanding the genetic bases of this phenomenon. To date, crossbreeding in horticultural vegetables has also rapidly progressed. F1 hybrid varieties are available for many horticultural crops, including both allogamous and autogamous species. Several genetic and nongenetic mechanisms have been applied to facilitate the large-scale production of F1 hybrid seeds in vegetable crops to prevent undesirable selfing. Although the development and commercialization of F1 hybrids is currently common in agriculture, this phenomenon is still being investigated at different levels. With the rapid accumulation of knowledge on plant genome structures and gene activities and the advancement of new genomics platforms and methodologies, significant progress has been achieved in recent years in the study of the genetic and molecular bases of heterosis. This paper provides a brief overview of current theoretical advances and practical predictions of the molecular mechanisms underlying heterosis in plants. The aim is to carefully summarize the fundamental mechanisms of heterosis in plants, focusing on horticultural plant breeding, to improve the existing knowledge in this research area. We describe the quantitative genetic model of phenotypic variation and combine evolutionary, phenotypic and molecular genetic views to explain the origin and manifestation of heterosis and its significance for breeding F1 hybrid varieties in horticultural crops. The principles of genomic prediction and its applications in genomic selection are then covered

MIK2 is a candidate gene of the S-locus for sporophytic self-incompatibility in chicory (Cichorium intybus, Asteraceae)

The Cichorium genus offers a unique opportunity to study the sporophytic self-incompatibility (SSI) system, being composed of species characterized by highly efficient self-incompatibility (e.g., C. intybus) and complete self-compatibility (e.g., C. endivia). To this end, the chicory genome was used to map seven previously identified SSI locus-associated markers. The region containing the S-locus was therefore restricted to an ~4 M bp window on chromosome 5. Among the genes predicted in this region, MDIS1 INTERACTING RECEPTOR LIKE KINASE 2 (ciMIK2) was particularly promising as a candidate for SSI. Its ortholog in Arabidopsis (atMIK2) is involved in pollen−stigma recognition reactions, and its protein structure is similar to that of S-receptor kinase (SRK), a key component of the SSI system in the Brassica genus. The amplification and sequencing of MIK2 in chicory and endive accessions revealed two contrasting scenarios. In C. endivia, MIK2 was fully conserved even when comparing different botanical varieties (i.e., smooth and curly endive). In C. intybus, 387 polymorphic positions and 3 INDELs were identified when comparing accessions of different biotypes all belonging to the same botanical variety (i.e., radicchio). The polymorphism distribution throughout the gene was uneven, with hypervariable domains preferentially localized in the LRR-rich extracellular region, putatively identified as the receptor domain. The gene was hypothesized to be under positive selection, as the nonsynonymous mutations were more than double the synonymous ones (dN/dS = 2.17). An analogous situation was observed when analyzing the first 500 bp of the MIK2 promoter: no SNPs were observed among the endive samples, whereas 44 SNPs and 6 INDELs were detected among the chicory samples. Further analyses are needed to confirm the role of MIK2 in SSI and to demonstrate whether the 23 species-specific nonsynonymous SNPs in the CDS and/or the species-specific 10 bp-INDEL found in a CCAAT box region of the promoter are responsible for the contrasting sexual behaviors of chicory and endive

MULTIPLE TECNOLOGIE GENOMICHE APPLICATE ALLA CARATTERIZZAZIONE DEL GERMOPLASMA ED AL MIGLIORAMENTO GENETICO E PROTEZIONE DI VARIETA' VEGETALI

Al giorno d'oggi, la genomica gioca un ruolo importante miglioramento delle colture, nella tracciabilità delle cultivar e nella protezione delle varietà vegetali. La disponibilità di strumenti genomici per caratterizzare molecolarmente le specie coltivate è in continuo aumento, e dimostra la sua utilità per molti scopi, come la selezione delle migliori linee parentali per la costituzione di nuovi ibridi F1, la caratterizzazione di varietà locali di una specie per studiarne il germoplasma locale, la valutazione del pool genico di origine di piante coltivate, la tracciabilità delle cultivar per proteggere i diritti dei coltivatori, o la determinazione della distinguibilità genetica e delle relazioni genetiche di diversi biotipi coltivati. I principali strumenti genomici utilizzati in questo campo sono i marcatori molecolari, che consistono in brevi regioni genomiche polimorfiche utili per determinare le differenze o le somiglianze genetiche tra individui, o per stimare la loro omozigosi e altre importanti statistiche genetiche. Queste informazioni possono poi essere utilizzate per diversi fini, come è già stato menzionato sopra, che possono migliorare l'agricoltura e la tracciabilità dei suoi prodotti, aspetti importanti che vengono considerati non solo dai coltivatori, ma anche dai consumatori. Questo progetto di ricerca si propone di illustrare le potenzialità degli strumenti e delle piattaforme genomiche per fini applicativi nel miglioramento genetici di piante coltivate e nella tracciabilità delle colture. Gli argomenti presentati riguarderanno il miglioramento genetico assistito da marcatori (MAB), che consiste nel migliorare le varietà di colture selezionando le migliori linee parentali da utilizzare nello sviluppo di ibridi F1 destinati al commercio, la caratterizzazione del germoplasma finalizzata a indagare la genetica delle varietà o dei biotipi locali, e la protezione delle varietà vegetali (PVP) incentrata sulla tracciabilità delle cultivar per proteggere i diritti dei coltivatori dalle frodi e dalle appropriazioni indebite da parte dei loro concorrenti.Nowadays, genomics plays an important role in crops’ breeding, cultivars’ traceability, and plants variety protection. The availability of genomic tools to molecularly characterize crop species is continually increasing, and demonstrates its usefulness for many purposes, like selecting the best breeding parental lines for the constitution of new F1 hybrids, characterizing multiple landraces to investigate a species local germplasm, assessing the gene pool of origin of cultivated plants, tracing cultivars in order to protect the plant breeders’ rights, or determining the genetic distinctiveness and genetic relationships of different cultivated biotypes. The main genomic tools used in this field are DNA molecular markers, which consist in short polymorphic genomic regions useful in determining the genetic differences or similarities among individuals or in estimating their homozygosity and other important genetic statistics. These information can then be used for different purposes, as it has already been mentioned above, which can improve agriculture and its products’ traceability, which are important aspects considered not only by cultivators, but also by costumers. This research project aims at illustrating the potentials of genomic tools and platforms for applicative purposes in plant breeding and crops traceability. The topics presented will concern markers-assisted breeding (MAB), which consists in improve crops varieties by selecting the best parental lines to be used in the development of commercially destined F1 hybrids, germplasm characterization aimed at investigating the genetics of landraces or local biotypes, and plant variety protection (PVP) focused on the traceability of cultivars to protect breeders’ rights from frauds and embezzlements from their competitors

Molecular Characterization and Genetic Structure Evaluation of Breeding Populations of Fennel (<i>Foeniculum vulgare</i> Mill.)

Fennel, or Foeniculum vulgare Mill., is an important horticultural crop belonging to the Apiaceae family that is cultivated worldwide and used in the agri-food sector and for pharmaceutical preparations. Breeding strategies in this species usually involve three parental lines, including two maternal lines (one cytoplasmic male-sterile line and an ideotype representative maintainer line) that are crossed to obtain an ideotype representative of the cytoplasmic male-sterile line and one paternal line, used as a pollinator in crosses with the progeny of the derived maternal lines. From this cross, F1 hybrid progenies are obtained, which are characterized by high levels of heterozygosity and hybrid vigor. In this study, over 450 plants, representing 8 breeding populations and their respective 3 parental and 1 progeny line, were genotyped by means of codominant molecular markers. The 12 highly polymorphic microsatellites enabled the analyses of the genetic variability, distinctiveness and stability of each breeding line. Moreover, the genetic structure of the core collection was investigated, which, together with the homozygosity, gene flow and genetic similarity results, allowed the identification of unsuitable lines to be used in breeding plans due to their low homozygosity (10.4% in the pollinator line of population 7). Moreover, the Bayesian reconstruction of the core collection’s genetic structure, based on the codominant markers used, allowed us to confirm the distinctiveness results obtained from the genetic similarity investigation and the computed gene flow estimates. Among these, a trend in hybrid heterozygosity was also observed, that increased when the genetic similarity between the respective parental lines decreased. Thus, this research proposes a suitable method for genotyping fennel populations in pre- and post-breeding approaches, such as marker-assisted breeding or breeding line distinctiveness and stability verifications

Molecular Characterization and Genetic Structure Evaluation of Breeding Populations of Fennel (Foeniculum vulgare Mill.)

Fennel, or Foeniculum vulgare Mill., is an important horticultural crop belonging to the Apiaceae family that is cultivated worldwide and used in the agri-food sector and for pharmaceutical preparations. Breeding strategies in this species usually involve three parental lines, including two maternal lines (one cytoplasmic male-sterile line and an ideotype representative maintainer line) that are crossed to obtain an ideotype representative of the cytoplasmic male-sterile line and one paternal line, used as a pollinator in crosses with the progeny of the derived maternal lines. From this cross, F1 hybrid progenies are obtained, which are characterized by high levels of heterozygosity and hybrid vigor. In this study, over 450 plants, representing 8 breeding populations and their respective 3 parental and 1 progeny line, were genotyped by means of codominant molecular markers. The 12 highly polymorphic microsatellites enabled the analyses of the genetic variability, distinctiveness and stability of each breeding line. Moreover, the genetic structure of the core collection was investigated, which, together with the homozygosity, gene flow and genetic similarity results, allowed the identification of unsuitable lines to be used in breeding plans due to their low homozygosity (10.4% in the pollinator line of population 7). Moreover, the Bayesian reconstruction of the core collection&rsquo;s genetic structure, based on the codominant markers used, allowed us to confirm the distinctiveness results obtained from the genetic similarity investigation and the computed gene flow estimates. Among these, a trend in hybrid heterozygosity was also observed, that increased when the genetic similarity between the respective parental lines decreased. Thus, this research proposes a suitable method for genotyping fennel populations in pre- and post-breeding approaches, such as marker-assisted breeding or breeding line distinctiveness and stability verifications

Pipeline to Design Inbred Lines and F1 Hybrids of Leaf Chicory (Radicchio) Using Male Sterility and Genotyping-by-Sequencing

Chicory, a horticultural crop cultivated worldwide, presents many botanical varieties and local biotypes. Among these, cultivars of the Italian radicchio group of the pure species Cichorium intybus L. and its interspecific hybrids with Cichorium endivia L.—as the “Red of Chioggia” biotype—includes several phenotypes. This study uses a pipeline to address the marker-assisted breeding of F1 hybrids: it presents the genotyping-by-sequencing results of four elite inbred lines using a RADseq approach and an original molecular assay based on CAPS markers for screening mutants with nuclear male sterility in the radicchio of Chioggia. A total of 2953 SNP-carrying RADtags were identified and used to compute the actual estimates of homozygosity and overall genetic similarity and uniformity of the populations, as well as to determine their genetic distinctiveness and differentiation. Molecular data were further used to investigate the genomic distribution of the RADtags among the two Cichorium species, allowing their mapping in 1131 and 1071 coding sequences in chicory and endive, respectively. Paralleling this, an assay to screen the genotype at the male sterility locus Cims-1 was developed to discriminate wild-type and mutant alleles of the causative gene myb80-like. Moreover, a RADtag mapped close to this genomic region proved the potential application of this method for future marker-assisted selection tools. Finally, after combining the genotype information of the core collection, the best 10 individuals from each inbred line were selected to compute the observed genetic similarity as a measure of uniformity as well as the expected homozygosity and heterozygosity estimates scorable by the putative progenies derived from selfing (pollen parent) and full-sibling (seed parent) or pair-wise crossing (F1 hybrids). This predictive approach was conducted as a pilot study to understand the potential application of RADseq in the fine tuning of molecular marker-assisted breeding strategies aimed at the development of inbred lines and F1 hybrids in leaf chicory

Expression of Factors of DNA Methylation in A. thaliana reproductive structures

RNA-directed DNA methylation (RdDM) is a small RNA-mediated epigenetic pathway involved in transcriptional repression of both transposons and genes. The expression of genes involved in this pathway is required for proper differentiation of gametophyte precursors in A. thaliana ovules. Members of the gene family Factors of DNA Methylation (FDM), together with their closely related IDN2, are dsRNA-binding proteins that regulate RdDM at chromatin targets. Recently we found that several FDM-like transcripts are differentially expressed in ovules collected from aposporous apomictic H. perforatum accessions. To gain further insight into the activity of FDM-like genes in gamete formation, we studied the expression of IDN2 and five FDM genes in A. thaliana reproductive structures. Microarray data indicate that expression of FDM genes is higher in flowers at stage 9 and decreases throughout flower development. Among the different flower parts, their expression is enriched in carpels at stage 12 and 15 and, within the pistil, it appears to be higher in the ovary. A protocol allowing reverse-transcription quantitative PCR (RT-qPCR) directly from micro-dissected ovules, without RNA extraction, was developed to monitor the expression levels of FDM genes in ovules at different developmental time points. In doing so, we could confirm the expression of IDN2 together with FDM1, FDM2 and FDM5 in ovules at different time points corresponding to sporogenesis and gametogenesis. Knockout and reporter lines were investigated to address the role of FDM genes in gametes formation and seed production. In doing so, we found that the A. thaliana IDN2 knockout line displays a significant reduction in the number of vital seeds. Similar phenotypes were observed when other effectors of the RdDM pathway were knocked out. Results on FDM expression contribute in paving the way toward a better comprehension of genetic and molecular basis of gametes formation and seed production

Molecular Relationships and Genetic Diversity Analysis of Venetian Radicchio (Leaf Chicory, Cichorium intybus subsp. intybus var. sylvestre, 2n = 2x = 18) Biotypes

Chicory (Cichorium intybus L., 2n = 2x = 18) is naturalized and grows wild in many parts of Europe, South and Central Asia and N. Africa; moreover, this plant is an important leafy vegetable cultivated worldwide. In Italy, this horticultural crop is known as radicchio, and different biotypes of this crop are cultivated, especially in the north-eastern part of the Italian Peninsula. Known to be introduced in and cultivated since the 17th century in the Venice area, the original biotype, still cultivated and named &ldquo;Late Red of Treviso&rdquo;, differentiated over the centuries, and it was also hybridized with endive (C. endivia), giving origin to many other biotypes. Several studies, based on morphological characterizations and historical reports, describe the relationships between the most popular cultivated local varieties of this species, but this work, focused on the use of molecular marker information obtained through DNA fingerprinting, presents validations and new insights into the genetic relatedness and diversity of these biotypes. By means of random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) molecular markers, this study provides insights into the genetic relationship that intercourses among the five most important local biotypes historically cultivated in the Veneto region, which is also the geographic centre of differentiation of this cultivated leafy vegetable. Through the construction of a maximum-likelihood dendrogram and the reconstruction of the genetic structure of a core collection, consisting of 652 samples belonging to five biotypes of radicchio divided into 22 old farmer populations, original data on their genetic origin, distinctiveness, relatedness and differentiation are reported and discussed