The Molecular Determination of Hybridity and Homozygosity Estimates in Breeding Populations of Lettuce (Lactuca sativa L.)

The development of new varieties of horticultural crops bene\ufb01ts from the integration of conventional and molecular marker-assisted breeding schemes in order to combine phenotyping and genotyping information. In this study, a selected panel of 16 microsatellite markers were used in di\ufb00erent steps of a breeding programme of lettuce (Lactuca sativa L., 2 n = 18). Molecular markers were \ufb01rst used to genotype 71 putative parental lines and to plan 89 controlled crosses designed to maximiserecombinationpotentials. Theresulting871progenyplantswerethenmolecularlyscreened, and their marker allele pro\ufb01les were compared with the pro\ufb01les expected based on the parental lines. Theaveragecross-pollinationsuccessratewas68\ub133%,so602F1hybridswerecompletelyidenti\ufb01ed. Unexpected genotypes were detected in 5% of cases, consistent with this species\u2019 spontaneous out-pollination rate. Finally, in a later step of the breeding programme, 47 di\ufb00erent F3 progenies, selected by phenotyping for a number of morphological descriptors, were characterised in terms of their observed homozygosity and within-population genetic uniformity and stability. Ten of these populations had a median homozygosity above 90% and a median genetic similarity above 95% and are, therefore, particularly suitable for pre-commercial trials. In conclusion, this study shows the synergistic e\ufb00ects and advantages of conventional and molecular methods of selection applied in di\ufb00erent steps of a breeding programme aimed at developing new varieties of lettuce

TRANSCRIPTOMIC ANALYSIS OF WHOLE PISTILS AND OVULE CELLS TO IDENTIFY GENES RELATED TO APOSPORY IN HYPERICUM PERFORATUM L.

St. John\u2019s wort (Hypericum perforatum L.) is a medicinal plant that produces important metabolites with antidepressant and anticancer activities. Beside the pharmaceutical interest, recently gained information has shown that H. perforatum is also an attractive model system for the study of aposporous apomixis, that is a reproductive strategy, which, unlike sexual reproduction, permits the inheritance of the maternal genome over generations without genetic recombination events. This asexual mode of seed formation is believed to be a trait with enormous economic and social potential in agriculture. Its innovative use in this area relies upon the idea that indefinitely fixing highly complex genotypes, including hybrid cultivars, through apomixis would have tremendous advantages in plant breeding, biomass and seed production. During the last decades, the understanding of the molecular basis of apomixis in this species has been complicated by the lack of biological data, e.g. genomic or even transcriptomic sequences. The aim of our research project was the sequencing, annotation and comparative investigation of the H. perforatum flower transcriptome, as critical steps toward a better understanding of the genetic control of aposporic and sexual reproduction in the facultative apomict H. perforatum. To this end, next generation sequencing technologies have been used to sequence the flower transcriptomes of obligate sexual and unrelated apomictic H. perforatum genotypes. This approach has enabled the assembly and annotation of large cDNA repositories and their exploitation to design a custom array to be used in flower expression studies. Global gene expression analysis of H. perforatum was initially performed on ovaries collected from sexual and aposporic plant accessions for the purpose of identifying genes and processes potentially associated with apomixis in this model species. Overall, across two selected developmental stages, 224 and 973 unigenes were found to be significantly upand down-regulated. Ontological annotation of differentially expressed genes indicated that terms related to cell cycle, single-organism cellular process DNA (cytosine-5-)-methyltransferase activity, among others, were significantly enriched. In a following step, a laser-capture microdissection approach was adopted in combination the RNA-seq technology with the aim of identifying genes differentially expressed in the ovule cell types primarily involved in the differentiation of the megaspore mother cells and aposporous initials. On the whole, our data suggest that phenotypic expression of apospory is concomitant with the modulation of key genes involved in the sexual reproductive pathway and the responsive to hormonal stimuli. Annotation of all identified flower transcripts as well as their qualitative and quantitative expression data will be presented and critically discussed as they prove a far better understanding of molecular bases of pistil development, embyo sac and egg cell formation in sexual and apomictic H. perforatum

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness‐induced CCN1 activates β‐catenin nuclear translocation and signaling and that this contributes to upregulate N‐cadherin levels on the surface of the endothelium, in vitro. This facilitates N‐cadherin‐dependent cancer cell–endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness‐induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium

YesTumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates β-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.Cancer Research UK (CRUK Beatson Institute C596/A17196, CRUK Glasgow Centre C596/A18076 and S.Z. C596/A12935

Genetic Structure of Cultivated Varieties of Radicchio (Cichorium intybus L.): A Comparison between F1 Hybrids and Synthetics

Cichorium intybus L., well known in Italy with the common name “Radicchio”, is an important leafy vegetable that is prevalently reproduced by allogamy due to very efficient barriers of self-incompatibility. Marker-assisted breeding is widely used by seed firms to develop new hybrid varieties that manifest genetic distinctiveness, uniformity and stability. A total of 29 mapped microsatellite markers were used for genotyping 504 samples of the Red of Chioggia biotype: First, two synthetics, four F1 hybrids and two derived F2 populations were compared to assess the distinctiveness of their gene pool and structure; then, the uniformity and stability of 3 years of production of a commercial F1 variety were also investigated. Genetic similarity and diversity statistics as well as the genetic structure of populations were analysed, including allele and genotype frequencies. The mean estimates and ranges of genetic similarity enabled the molecular discrimination of OP synthetics from F1 varieties and their F2 progenies and the determination of individual plant memberships. Moreover, the genetic structure of F1 hybrids produced in 3 years unexpectedly revealed two main clusters that discriminate the first 2 years from the 3rd, mainly because of the presence of uncommon specific alleles and different allele frequencies. Overall, this molecular information will enable breeders to determine the genetic distinctness, uniformity and stability of commercial and experimental varieties, as well as their genetic relationships and relatedness. Hence, this work provides a useful tool for achieving the molecular characterisation and genetic identification of different radicchio populations

Breeding new varieties of horticultural species by means of conventional genetics and biotechnological methods

The growing demand for new vegetable varieties is forcing seed companies to plan faster and more efficient breeding programs. Therefore, conventional breeding methods are increasingly supported by biotechnological methods. In particular, marker-assisted breeding (MAB) reduces the time needed to develop new varieties thanks to the use of molecular assays. The crucial role played by molecular markers in breeding new varieties is discussed in the three works carried out in different horticultural species belonging to Asteraceae family. In a first case study, conventional breeding methods are complemented by molecular methods throughout a typical breeding program in lettuce (Lactuca sativa L.). A total of 16 SSR (Simple Sequence Repeats) markers or microsatellites were efficiently used to genetically characterise 71 putative parental lines and to plan 89 crossings, designed to maximise the yield deriving from each manual pollination. After that, the resulting 871 progeny plants were screened, and their molecular profiles were compared with those expected considering their putative parents. The out-pollination success rate resulted in being, on average, 68 ± 33 % and 602 F1 hybrids were identified. Finally, in an advanced step of this breeding program, 47 different experimental populations (F3 generation) were evaluated in terms of observed homozygosity and genetic similarity within the population. Three of them resulted particularly suitable for pre-commercial trials due to observed median homozygosity above 90 % and an intra-genetic similarity value always higher than 95%. Hence, this study shows the synergetic effect of conventional and biotechnological methods in different steps of a breeding program. In addition to lettuce, different Cichorium intybus var. foliosum L. varieties were compared and molecularly characterised, both for the economic value and the great cultural interest of this species in Veneto region. C. intybus var. foliosum, better known in Italy as radicchio, is an important locally cultivated leafy vegetable that prevalently reproduces by allogamy. Considering that marker-assisted breeding is widely used by seed firms to develop new F1 hybrid varieties that are distinguished by high plant uniformity and crop yields, in this second case study we performed the molecular characterisation and the genetic structure investigation of different commercial populations of Radicchio. A total of 29 mapped microsatellite markers were used for genotyping 504 samples of the Red of Chioggia biotype. Two synthetics, four F1 hybrids and two derived F2 populations were initially characterised and compared in terms of genetic similarity and diversity statistics. Then, the uniformity and the stability of three years of production of an F1 variety were investigated by applying the same panel of molecular markers. As main finding, the mean similarity estimates enabled the clear molecular discrimination of OP synthetics from F1 varieties and their F2 progenies and the determination of individual plant memberships. Moreover, the genetic structure of F1 hybrids produced in 3 years unexpectedly revealed two main clusters that discriminate the first 2 years from the 3rd, mainly because of the presence of uncommon specific alleles and different allele frequencies. Overall, this molecular information will enable breeders to determine the genetic distinctness, uniformity and stability of commercial and experimental populations, as well as their genetic relationships and relatedness. Unlike the previous two more relevant and more studied species (lettuce and chicory), the amount of biological and molecular data available for endive (Cichorium endivia Lam.) is incredibly scarce. This species is a self-pollinated leafy green vegetable, belonging to the Asteraceae family and it is characterized by two cultivar types: curly endive (C. endivia var. crispum Lam.) and escarole or smooth endive (C. endivia var. latifolium Lam.). For seed firms the genetic diversity characterisation of elite breeding material is crucial for the registration and protection of future varieties. In our case study, we verified the distinctiveness of 32 experimental materials belonging to the two cultivar types (escarole and curly endive). In a first SSR-based attempt, only 14 out of 29 microsatellite markers belonging to C. intybus, were successfully transferred to endive and only 8 of them resulted polymorphic. Due to the limited level of discrimination of this molecular assay, an alternative approach based on single nucleotide polymorphisms (SNP) was applied using the restriction site-associated DNA sequencing (RADseq). Overall, a set of 4,621 SNP markers was able to separate curly endive from escarole endive and, in particular 50 of them were able to discriminate the two cultivar types. Moreover, the resulting SNP-supported dendrogram and the PCoA analysis divided the two cultivar types of endive into two distinct clusters, discriminating univocally all the plant materials. Overall, our research was able to evaluate the distinctiveness requirement of the DUS testing. We also evaluated the observed homozygosity to predict the uniformity and stability of progenies, two additional requirements of the DUS testing: individuals with the highest homozygosity are known to produce more uniform and stable populations over generations. In conclusion, this molecular information enabled breeders to determine the genetic distinctness of the 32 elite breeding materials and to reconstruct their genetic relationships.La crescente domanda di nuove varietà sta spingendo le ditte sementiere a pianificare programmi di miglioramento genetico sempre più rapidi ed efficienti. Pertanto, i metodi convenzionali di costituzione di nuove varietà sono sempre più supportati da quelli biotecnologici. In particolare, la costituzione di nuove varietà assistita da marcatori (marker assisted breeding, MAB) riduce il tempo necessario per sviluppare nuove varietà grazie all’utilizzo di saggi molecolari. Nei seguenti tre lavori è discusso il ruolo cruciale svolto dai marcatori molecolari in questi programmi di miglioramento genetico, che sono stati condotti in diverse specie orticole appartenenti alla famiglia delle Asteraceae. Nel primo caso studio, i metodi convenzionali sono integrati dai metodi molecolari attraverso un tipico schema di miglioramento genetico in lattuga (Lactuca sativa L.). Sono stati impiegati marcatori SSR (Simple Sequence Repeats) o microsatelliti, 16 loci in totale, al fine di caratterizzare geneticamente 71 putative linee parentali e di pianificare 89 incroci, i quali sono stati progettati per massimizzare la resa delle impollinazioni manuali e il potenziale di ricombinazione genetica. Successivamente, la progenie risultante, costituita da 871 piante, è stata selezionata e i profili molecolari dei campioni sono stati confrontati con quelli dei loro putativi parentali. Il tasso di successo di questi incroci è risultato in media pari a 68 ± 33 % e il numero di ibridi F1 è risultato pari a 602. Infine, in una fase avanzata di questo programma genetico, sono state valutate 47 popolazioni sperimentali (generazione F3) in termini di omozigosi osservata e di similarità genetica. Tre di queste popolazioni sono risultate particolarmente idonee per accedere ai test pre-commerciali. In particolare questo materiale sperimentale presentava un elevato grado di omozigosi, superiore al 90 %, e gradi di similarità intra-popolazione superiori al 95 %. In conclusione, questo studio evidenzia l’effetto sinergico dei metodi convenzionali e biotecnologici in diverse fasi di un programma di miglioramento genetico. Oltre alla lattuga, sono state confrontate e caratterizzate molecolarmente diverse varietà di Cichorium intybus var. foliosum L., sia per il loro valore economico che per il grande interesse culturale di questa specie in Veneto. C. intybus var. foliosum, meglio conosciuto in Italia come radicchio, è un importante ortaggio a foglia coltivato localmente che si riproduce prevalentemente per allogamia. Le aziende sementiere si avvalgono ampiamente di marcatori molecolari per la costituzione di nuove varietà, soprattutto di ibridi F1 che si distinguono per la loro elevata uniformità e le rese colturali. In questo secondo caso studio abbiamo eseguito una caratterizzazione e un’indagine molecolare sulla struttura genetica di diverse popolazioni commerciali di Radicchio. Sono stati impiegati 29 marcatori microsatellite per la genotipizzazione di 504 campioni del biotipo rosso di Chioggia. In particolare, sono state inizialmente caratterizzate e confrontate in termini di similarità genetica, ricorrendo anche all’uso di statistiche sulla diversità genetica: due sintetiche, quattro ibridi F1 e due popolazioni F2 derivate. Utilizzando il medesimo saggio molecolare sono state valutate anche l’uniformità e la stabilità di tre lotti di tre anni di produzione di una varietà F1. I gradi medi di similarità risultanti hanno consentito la chiara discriminazione molecolare delle sintetiche OP dalle varietà F1 e dalla loro progenie F2, oltre che la determinazione delle singole popolazioni. Inoltre, la struttura genetica degli ibridi F1 prodotti in 3 anni ha rivelato inaspettatamente due gruppi principali che discriminano i primi due anni dal terzo, principalmente a causa della presenza di alleli specifici non comuni e di diverse frequenze alleliche. Nel complesso, queste informazioni molecolari consentiranno ai costitutori di determinare la distinzione genetica, l'uniformità e la stabilità di popolazioni commerciali e sperimentali, nonché le loro relazioni genetiche. A differenza delle due precedenti specie più rilevanti e più studiate (lattuga e cicoria), la quantità di dati biologici e molecolari disponibili per l’indivia (Cichorium endivia Lam.) è incredibilmente minore. Questa specie è un’orticola a foglia verde con un sistema riproduttivo di tipo autogamo. L’indivia appartenente alla famiglia delle Asteraceae ed è caratterizzata da due tipi di cultivar: indivia riccia (C. endivia var. crispum Lam.) e indivia scarola o liscia (C. endivia var. latifolium Lam.). Per le ditte sementiere la caratterizzazione genetica del loro materiale è fondamentale sia al momento della registrazione che in seguito per la protezione delle loro varietà. Nel nostro caso di studio, abbiamo verificato il carattere distintivo di 32 materiali sperimentali appartenenti ai due tipi di cultivar (indivia scarola e riccia). In un primo tentativo basato su marcatori SSR, solo 14 loci marcatori su 29 appartenenti a C. intybus sono stati trasferiti con successo in indivia e solo 8 di essi sono risultati polimorfici. A causa del livello limitato di discriminazione di questo saggio molecolare, è stato applicato un approccio alternativo basato su marcatori SNP (Single Nucleotide Polymorphisms) utilizzando il sequenziamento del DNA associato al sito di restrizione (RADseq). Complessivamente 4.621 marcatori SNP sono stati in grado di separare tutti gli individui di indivia riccia e scarola, in particolare, 50 di loro hanno discriminato i due tipi di cultivar. Inoltre, il dendrogramma e l’analisi PCoA, supportati dagli SNP, hanno diviso i due tipi cultivar di indivia in due sottogruppi distinti, discriminando in modo univoco tutti i materiali vegetali. Nel complesso, la nostra ricerca è stata in grado di valutare la distinguibilità di tutti i campioni analizzati, che rappresenta il primo requisito del test DUS. É stato valutato, inoltre, anche il grado di omozigosi, in modo da prevedere l'uniformità della progenie, ovvero il secondo requisito del DUS test, in quanto gli individui con un elevato grado di omozigosi sono noti per produrre popolazioni maggiormente uniformi. In conclusione, queste informazioni molecolari hanno permesso i costitutori di determinare la distinguibilità genetica dei 32 materiali d’élite e ogni possibile loro relazione genetica

Genotyping by RAD Sequencing Analysis Assessed the Genetic Distinctiveness of Experimental Lines and Narrowed down the Genomic Region Responsible for Leaf Shape in Endive (Cichorium endivia L.)

The characterization of genetic diversity in elite breeding stocks is crucial for the registration and protection of new varieties. Moreover, experimental population structure analysis and information about the genetic distinctiveness of commercial materials are essential for crop breeding programs. The purpose of our research was to assess the genetic relationships of 32 endive (Cichorium endivia L.) breeding lines, 18 from var. latifolium (escarole) and 14 from var. crispum (curly), using heterologous Cichorium intybus-derived simple sequence repeats (SSR) markers and single-nucleotide polymorphisms (SNP) markers. We found that 14 out of 29 SSR markers were successfully amplified, but only 8 of them were related to polymorphic loci. To overcome the limitation of the low number of informative SSR marker loci, an alternative SNP-based approach was employed. The 4621 SNPs produced by a restriction site-associated DNA marker sequencing approach were able to fully discriminate the 32 endive accessions; most importantly, as many as 50 marker loci were found to distinguish the curly group from the escarole group. Interestingly, 24 of the marker loci mapped within a peripheral segment of chromosome 8 of lettuce (Lactuca sativa L.), spanning a chromosomal region of 49.6 Mb. Following Sanger sequencing-based validation, three genes were determined to carry nonsynonymous SNPs, and one of them matched a putative ortholog of AtELP1, subunit 1 of the Elongator complex. Considering that several previously characterized Elongator complex subunit mutants exhibited elongated and/or curly leaf phenotypes, this gene should be taken into consideration for a better understanding of the underlying mechanism controlling leaf shape in endive

Marfan’s syndrome and pregnancy: a good maternal and fetal outcome

This case report highlights the important role of a multidisciplinary team’s task in the care of pregnant women with Marfan’s syndrome (MFS), a systemic disorder of connective tissue that is transmitted as an autosomal dominant trait. Case: a 42 year-old italian pregnant woman with Marfan’s syndrome and degenerative heart disease (aneurysmatic dilatation of the aortic root, mitral regurgitation and prosthetic mitralic valve) was clinically assessed jointly by an obstetrician and a cardiologist, ‘the obstetric team specialised in management of high risk pregnancy’, every 2-3 weeks from the 21th week of gestation. The first ambulatory monitoring echocardiogra- phy revelead aneurysmatic dilatation of the aortic root (41 mm), good function of the previously replaced mitral valve, cardiac ejection fraction 51% and telediastolic volume 116 ml. The echocardio-graphies showed no changes up to 32 weeks gestation. At the 34th week of gestation she had a slight decrease in cardiac ejection fraction and minimal increase of left ventricular diastolic volume. Therefore she underwent elective cesarean section under general anesthesia at 35 weeks’gestation. The post-partum course was uneventfull for the patient and the baby. Conclusion: pregnant women with heart disease benefit from an appropriate antenatal management, which may result in a favourable outcome