RICERCA ITALIANA     

Research program

The peopling of the European continent: the mitochondrial DNA and Y chromosome perspectives

University Co-ordinator

Università degli Studi di PAVIA - GENETICA E MICROBIOLOGIA 'A.BUZZATI-TRAVERSO' - ()

Research Unit Leader

Antonio Torroni

Description

As it is mentioned above, we believe that the absence of “Neolithic branches” in the European portion of the mtDNA tree is only due to inadequacy in the current phylogeny, and that in order to identify Neolithic mtDNA inputs in Europe, which occurred only 8-10,000 years ago, the level of resolution of certain European haplogroups needs to be extensively improved. Indeed, we have some very preliminary (and unpublished) data, based on complete mtDNA sequences, supporting such a scenario. For instance, a partial dissection of haplogroup J2 [16] has allowed us to identify a novel clade (J2a1), whose distribution and age (10.9 ± 2.6 ky) are strongly suggestive of a Neolithic origin from the Near East. Furthermore, this clade is not uncommon at all in Europe, reaching a frequency peak of about 6% (Fig. 4).



Taking into account that certain haplogroups (for instance H, V, U5, etc) have already been extensively analyzed by us and others [10-15], we here propose to perform a detailed molecular dissection of those haplogroups – J1, J2, T1, T2, U3 and U7 – which are shared between Europe and the Near East (Fig. 1) but have been rather neglected until now. We expect that some (hopefully many) of their internal branches might represent the mtDNA legacy of the Neolithic transition in Europe.

This project, built on our own track-record in phylogeographic studies of human mtDNA data, will be performed at the maximum level of molecular resolution by sequencing entire mitochondrial genomes belonging to the six haplogroups J1, J2, T1, T2, U3 and U7, accurately selected on the basis of the preliminary molecular data. This step is essential to avoid redundancies and to allow the inclusion of those rare sub-clades that would most likely be missed if a random selection were performed. In this way we are confident to highly increase the resolution of the six haplogroups identifying all internal clades that differentiated in western Eurasia prior ~7,000 years ago. This will include many of the lineages that diffused while the first farmers were moving from the Levant. To reach this objective, four main steps need to be accomplished:

(a) Identification of the DNA samples (using preliminary molecular data) to be completely sequenced. This will be accomplished by using preliminary molecular data that are largely already available in the lab (see below);
(b) Detection of all sub-clades within haplogroups J1, J2, T1, T2, U3 and U7 that are older than 7,000 years and identification of their diagnostic (coding and control-region) markers;
(c) Population survey and evaluation of the frequency distributions of each sub-clade;
(d) Estimation of the coalescence time for each sub-clade and comparative data elaboration.

In particular, after the selection of the samples, their mtDNA will be completely sequenced according to an established protocol [17], and complete sequences will be analysed following the approach and methods that we have already used for other haplogroups [4, 12, 14]. The distinctive mutations of the newly identified clades will then be surveyed in all available samples from a wide range of populations and geographic areas (through RFLP and/or sequence analysis), in order to evaluate the frequency and geographical distribution of the novel clades in European and Near Eastern populations (and others from surrounding regions), and distinguish those lineages that arose and expanded in the Palaeolithic from the Neolithic ones.

The lab work for this project will follow an overall plan that the laboratory of Pavia has been developing over the last few years. In detail, our plan is to sequence about 500 entire mitochondrial genomes – about 100 for each of J1, J2, T1 and T2, and about 50 for the less common U3 and U7 – carefully selected among a collection of more than 35,000 subjects from more than 200 western Eurasian populations. More than a half of these samples are located in our laboratory, the remainders are available thanks to the contribution of the other four Research Units of this project and the numerous fruitful international collaborations consolidated over the course of years. All samples have already been molecularly characterized in terms of control region sequence variation and haplogroup affiliation.

We expect to produce most of the 500 sequences (at least 400) in the first year of the project, while most of phylogenetic and statistical analyses will be performed and completed in the second year when almost all of the sequence data will be already available.

Why do we believe that we can reach the planned objectives by sequencing 500 complete mtDNA genomes? This number might appear both large and small at the same time. Large, because the simple production of 500 complete mtDNA sequences (the human mtDNA is 16,569 bp) requires a significant number of man-hours and their analysis will be challenging; small, because 500 sequences may appear too few to encompass most of the Neolithic mtDNA input to Europe. However, rather than choosing mtDNAs for complete sequencing simply on the basis of the ethnic/geographic origin, it is worth to further stress that we will make an efficient pre-selection of mtDNAs based on the existing molecular data (SNPs and control-region data). Our experience with other haplogroups (H, V, M1, U5, U6) [4, 11-12, 14] indicates that 500 mtDNAs should be sufficient to bring haplogroups J1, J2, T1, T2, U3 and U7 at a resolution boundary of about 7,000 years. In this way, with "only" 500 novel mtDNA sequences we will be able to roughly quantify the relative (female) contributions of Neolithic versus Palaeolithic gene pools in modern Europeans, at a still acceptable cost. Our mtDNA results will be then compared with those obtained from the male counterpart (Y chromosome) that is going to be concomitantly analyzed by the other Research Units in this project.

As we plan to remain in this field of research for many years to come, the project is deliberately broad and could easily be extended and deepened to analyze additional rare haplogroups (for instance HV and R0a; see Achilli et al. [18]) and population samples in the course of the two years: experience has indeed shown that new questions constantly arise during these studies. Moreover, one of the goals of this project is to further develop a long-lasting and very productive collaboration between all the Research Units that contribute to this application. The sharing of DNA samples, data, technical procedures and students is a way to attain this result.