Molecular Characterization of Apricot Germplasm from an Old Stone Collection

Citation: Martn C, Herrero M, Hormaza JI ( Molecular Characterization of Apricot Germplasm from an Old Stone Collection Carolina Martn 0 Mara Herrero 0 Jose I. Hormaza 0 Edward Newbigin, University of Melbourne, Australia 0 1 IHSM ''La Mayora'' - CSIC , Algarrobo-Costa, Ma laga, Spain, 2 Pomology Department, EE Aula Dei, CSIC, Zaragoza , Spain Increasing germplasm erosion requires the recovery and conservation of traditional cultivars before they disappear. Here we present a particular case in Spain where a thorough prospection of local fruit tree species was performed in the 1950s with detailed data of the origin of each genotype but, unfortunately, the accessions are no longer conserved in ex situ germplasm collections. However, for most of those cultivars, an old stone collection is still preserved. In order to analyze the diversity present at the time when the prospection was made and to which extent variability has been eroded, we developed a protocol in apricot (Prunus armeniaca L.) to obtain DNA from maternal tissues of the stones of a sufficient quality to be amplified by PCR. The results obtained have been compared with the results from the profiles developed from apricot cultivars currently conserved in ex situ germplasm collections. The results highlight the fact that most of the old accessions are not conserved ex situ but provide a tool to prioritize the recovery of particular cultivars. The approach used in this work can also be applied to other plant species where seeds have been preserved. - Funding: Financial support for this work was provided by the Spanish Ministry of Science and Innovation European Regional Development Fund (Project Grants AGL2010-15140, AGL2009-12621-CO2-01 and RF2008-00028-CO2-02), and Gobierno de Arago n (Grupo de Excelencia A-43). C.M. was supported by a grant from the Mediterranean Agronomic Institute of Zaragoza (IAMZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The development of new technologies, the substitution of local varieties by foreign improved varieties or changes in cultural techniques have resulted in an increasing erosion of germplasm resources that leads to the need of optimizing the conservation of endangered germplasm [1]. In fact, conservation and use of plant genetic resources should be a priority in agricultural research [2-5]. However, this task is often hindered by the abundance of homonymies and synonymies in germplasm collections and the lack of information available on local germplasm erosion. In this work we present a case study in apricot (Prunus armeniaca L.) in Spain. Apricot is an economically important member of the Rosaceae cultivated in Mediterranean climates worldwide. Apricot is a diploid species, with eight pair of chromosomes (2n = 16) and a small genome (5.96108 bp) [6] that is believed to have originated in the Tien-Shan Mountains, in Central Asia, from where it was disseminated both east and westward [7]. The species can be classified into six main ecogeographical groups [8]: Central Asian, East Chinese, North Chinese, Dzhungar-Zailij, Irano-Caucasian and European. However, due the introduction of new cultivars derived from crosses between genotypes of the different groups, the assignment of new cultivars to one of these groups is difficult [9]. In the last ten years a clear effort has been made to characterise apricot germplasm in different parts of the world [10 19] generally showing a regional distribution that probably reflects independent selection in each region and later vegetative propagation of selected genotypes through grafting. Apricot was introduced in the Mediterranean region from Iran or Armenia around the first century BC [20], although more recently new introductions were made from the Middle East, especially into Southern Europe [9]. Spanish apricot cultivars could have been derived from genotypes of both the European and the Irano-Caucasian groups, the latter introduced from Northern Africa by the Arabs [21]. Under that scenario we would expect a high level of variability among the Spanish cultivars; however, this is not the case [10,22] and probably reflects an erosion of the variability present in the Spanish cultivars due to the small geographical area where apricot has been traditionally cultivated in Spain, to the generalized use of grafting in the last two centuries and to the predominance of few cultivars such as Bu lida, Canino or Moniqu that could be ancestors of most of the cultivars currently available in Spain [22]. One of the main limiting factors to analyze genetic erosion is the lack of knowledge on the genetic composition of the cultivars that have been lost, since for most of them we only have written records but it is not possible to ascertain if those cultivars have been preserved in ex situ collections under a different name. In this sense, a thorough inventory of cultivars of different fruit tree species, including apricot, was performed in Spain in the 1950s with detailed data of the collected site of each genotype [23]. Although most of the genotypes are no longer conserved, at least with the same name, in ex situ collections, old stones from some of those genotypes are still preserved. This situation is not particular of apricot but is generalized in a good number of woody perennials, since the stone collections were commonly used at that time for morphological identification purposes. Since the fruit of Prunus species is a drupe where the mature stony endocarp together with the seed forms a propagation unit, there are two tissues of maternal origin that should represent the genetic profile of these old cultivars: the endocarp that derives from the inner layer of the ovary, and the testa that derives from the integuments of the ovule. But we wondered if DNA extracted from this old material could still reveal a genetic profile and thus reflect the genetic variability present at the time of the collection in the field. Thus, as a first step to evaluate the loss of old apricot material in the last decades in Spain we optimized a method to extract DNA from two maternal tissues (the endocarp and the testa) of old stones of apricot to allow the fingerprinting of the old cultivars that originated these fruits. For molecular analyses we used Simple Sequence Repeat (SSR) markers that have been successfully used in apricot germplasm characterization in different works [1019]. In a second step we evaluated the variability of this material in relation to cultivars currently preserved in ex situ collections. Results shed light on how to prioritize recovery of old cultivars. Materials and Methods Plant material Two different local Spanish apricot sample sets were used in this work, one from an old apricot stone collection, and the other from young leaves of apricot cultivars conserv (...truncated)