Italy has long been a crossroads of the Mediterranean sea, where different peoples and cultures have come into contact over time, and in particular during the Metal Age, due to the intensification of the commercial maritime routes. However, the genetic knowledge of the peoples who have contributed to the modern model of Italian genetic variability is rather scarce. In addition, almost all the data currently available are based on the analysis of mitochondrial DNA, which can explain only part of the complex migratory framework. Therefore, together with the matrilinear marker, the genetic investigation of the Y chromosome (Y-chr) variation in ancient population is essential to better contextualize past migratory events. Indeed the Y-chr displays properties such as the haploidy, the uniparental transmission and the non-recombination for most of its length, that have significant influences on the genetic structure, mutational processes and diversity between and within populations. In this work, a total of 79565 SNPs located in the X-degenerate region of the Y-chr have been isolated. The selected positions are considered crucial to provide inferences related to the male genealogy but are also suitable for the identification of individuals who share the same paternal ancestry. Two different experimental design were carried out for the production of RNA probes aiming to enrich a subset of the total positions previously identified but which still exhibit an excellent level of resolution. The efficiency, specificity and the total yield of the probes produced were tested through 4 different enrichment protocols of the selected regions. In particular, experimental analysis were carried out both through the protocols suggested by the probes’ manufacturer, as well as through two protocols specifically developed for degraded material. The analysis were conducted on 4 modern and 4 ancient (previously successfully analyzed) samples, and showed that with both the set of probes, is possible to perform a detailed haplogroups (Hg) assignation. In addition, the performed analysis showed that the use of experimental designs and protocols specifically developed for damaged substrates , significantly increases the specificity of the probes and the deducible information. After the development of the best method to provide maximum performance in the target enrichment phases, 101 samples recovered in 8 Italian necropolis associated with the Iron Age were screened for the genetic sex. The analysis conducted on the Y-chr variation among 42 samples (selected from the total amount of male available) highlighted the population distribution into four main Hg (R1b, I2, G2a and J); these Hg are typical of the modern genetic makeup of the Italian males. Furthermore, although the marked presence of the Hg R1b (which is homogeneously distributed among Europe), it is possible to hypothesize a well-established genetic variability cline that would differentiate the variability of the Italian Y-chr in two latitudinal regions, the north and the south, as proposed in previous works. The Y-chr Hg variation of the analyzed population were compared with those of ancient and modern European populations recovered in the literature. The results obtained showed a genetic closeness of the Iron Age Italian peoples with those of the same period recovered by different Italian contexts, and with the Metal Age populations from the Pontic-Caspian steppes. A particular affinity with Italian individuals of the subsequent Roman period was also found. It will be necessary to both broaden the Y-chr analysis on a larger set of samples of the same periods, as well as radiocarbon dating in order to better clarify the historical context and the genetic situation during the Iron Age.

L’Italia ha rappresentato a lungo un crocevia del Mediterraneo, dove popoli e culture diverse hanno trovato facile approdo nel corso del tempo, giungendo ad un apice durante l’età dei Metalli. Attualmente le conoscenze genetiche dei popoli che hanno concorso al moderno modello di variabilità genetica italiana sono piuttosto scarse. Inoltre, la quasi totalità dei dati disponibili si basa sull’analisi del DNA mitocondriale, in grado di spiegare solo in parte il complesso quadro migratorio. L’indagine genetica delle caratteristiche del cromosoma Y (Y-chr) nelle popolazioni antiche risulta pertanto essenziale per contestualizzare gli eventi migratori passati. Esso presenta proprietà che hanno influenze sulla struttura genetica, sui processi mutazionali e sulla diversità intra- ed inter-popolazionistica. Inoltre è un marker d'elezione in genetica di popolazioni grazie a qualità come l’aploidia, la trasmissione uniparentale e la non ricombinazione per la maggior parte della sua lunghezza. In questo lavoro sono stati isolati un totale di 79565 SNPs localizzati nella regione X-degenerata del Y-chr e considerati essenziali per produrre inferenze relative alle linee di discendenza per via paterna, ed idonei anche per l’identificazione di individui imparentati. Sono stati condotti due differenti disegni sperimentali per la costruzione di sonde a RNA su un subset delle posizioni totali, ma in grado di fornire le informazioni ricercate con un ottimo livello di risoluzione. L’efficienza, specificità e resa delle sonde prodotte sono state confrontate mediante l’applicazione di 4 diversi protocolli per l’arricchimento delle regioni selezionate; in particolare sono stati adottati i protocolli suggeriti dalle aziende produttrici delle sonde, oltre a due modificati per fornire maggior resa in caso di substrati degradati. I test comparativi, eseguiti su un set di 4 campioni moderni e 4 antichi, hanno mostrato che è possibile effettuare analisi di determinazione degli aplogruppi (Hg) estremamente dettagliate. I risultati ottenuti mostrano che entrambi gli assetti valutati presentano una buona resa, ma l’utilizzo di disegni sperimentali e protocolli ad hoc per substrati degradati aumenta significativamente l’efficienza delle sonde e le informazioni deducibili. Con questo approccio ottimizzato è possibile giungere alla lettura di un elevato numero di posizioni del Y-chr, rendendo la metodologia innovativa ed applicabile ad una varietà di contesti. È stato successivamente eseguito uno screening per la determinazione genetica del sesso, su 101 campioni associati al periodo dell’età del Ferro, recuperati da 8 siti del Centro e Sud Italia. Le analisi condotte sul Y-chr di 42 campioni selezionati tra i maschi disponibili, hanno messo in luce la differenziazione genetica della popolazione in 4 principali Hg (R1b, I2, G2a e J) tipici dell’attuale variabilità della popolazione italiana maschile. Inoltre, se si esclude la componente maggioritaria ottenuta con l’Hg R1b, omogeneamente diffuso in tutta Europa, è possibile ipotizzare un cline di variabilità genetica distinto tra Nord e Sud Italia, già proposto in lavori precedenti. La comparazione delle frequenze degli Hg del Y-chr della popolazione in analisi, con quelle di popolazioni antiche e moderne recuperate in letteratura, ha permesso di mostrare una vicinanza genetica della popolazione italiana dell’età del Ferro con quelle dello stesso periodo recuperate da diversi contesti italiani, e con le popolazioni dell’età dei metalli provenienti dalle steppe pontico-caspiche. È stata inoltre riscontrata una particolare affinità con individui italiani del successivo periodo romano. Per poter chiarire in maggior dettaglio la complessa situazione italiana dell’età del Ferro sarà necessario genotipizzare un numero maggiore di individui dello stesso arco temporale e consolidare i dati ad oggi ottenuti mediante datazioni al radiocarbonio.

UTILIZZO DELLA METODOLOGIA NGS ASSOCIATA ALLA CATTURA DEL CROMOSOMA Y PER LA CARATTERIZZAZIONE DELLA POPOLAZIONE ITALIANA NELL’ETÀ DEL FERRO

VERGATA, Chiara
2021

Abstract

Italy has long been a crossroads of the Mediterranean sea, where different peoples and cultures have come into contact over time, and in particular during the Metal Age, due to the intensification of the commercial maritime routes. However, the genetic knowledge of the peoples who have contributed to the modern model of Italian genetic variability is rather scarce. In addition, almost all the data currently available are based on the analysis of mitochondrial DNA, which can explain only part of the complex migratory framework. Therefore, together with the matrilinear marker, the genetic investigation of the Y chromosome (Y-chr) variation in ancient population is essential to better contextualize past migratory events. Indeed the Y-chr displays properties such as the haploidy, the uniparental transmission and the non-recombination for most of its length, that have significant influences on the genetic structure, mutational processes and diversity between and within populations. In this work, a total of 79565 SNPs located in the X-degenerate region of the Y-chr have been isolated. The selected positions are considered crucial to provide inferences related to the male genealogy but are also suitable for the identification of individuals who share the same paternal ancestry. Two different experimental design were carried out for the production of RNA probes aiming to enrich a subset of the total positions previously identified but which still exhibit an excellent level of resolution. The efficiency, specificity and the total yield of the probes produced were tested through 4 different enrichment protocols of the selected regions. In particular, experimental analysis were carried out both through the protocols suggested by the probes’ manufacturer, as well as through two protocols specifically developed for degraded material. The analysis were conducted on 4 modern and 4 ancient (previously successfully analyzed) samples, and showed that with both the set of probes, is possible to perform a detailed haplogroups (Hg) assignation. In addition, the performed analysis showed that the use of experimental designs and protocols specifically developed for damaged substrates , significantly increases the specificity of the probes and the deducible information. After the development of the best method to provide maximum performance in the target enrichment phases, 101 samples recovered in 8 Italian necropolis associated with the Iron Age were screened for the genetic sex. The analysis conducted on the Y-chr variation among 42 samples (selected from the total amount of male available) highlighted the population distribution into four main Hg (R1b, I2, G2a and J); these Hg are typical of the modern genetic makeup of the Italian males. Furthermore, although the marked presence of the Hg R1b (which is homogeneously distributed among Europe), it is possible to hypothesize a well-established genetic variability cline that would differentiate the variability of the Italian Y-chr in two latitudinal regions, the north and the south, as proposed in previous works. The Y-chr Hg variation of the analyzed population were compared with those of ancient and modern European populations recovered in the literature. The results obtained showed a genetic closeness of the Iron Age Italian peoples with those of the same period recovered by different Italian contexts, and with the Metal Age populations from the Pontic-Caspian steppes. A particular affinity with Italian individuals of the subsequent Roman period was also found. It will be necessary to both broaden the Y-chr analysis on a larger set of samples of the same periods, as well as radiocarbon dating in order to better clarify the historical context and the genetic situation during the Iron Age.
CARAMELLI, David
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2487907
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