Evaluation of wheat germplasm from the Madeira and Canary archipelagos using a single molecular marker. A rapid screening method for identiﬁcation of durum wheat accessions

This work aims to present a rapid and precise screening method of wheat germplasm and identification of durum wheat accessions in germplasm collections. Fifty-two wheat accessions maintained in the ISOPlexis germplasm bank at the Madeira University, Portugal, and 72 accessions from the Centre for Conservation of Crop Biodiversity of Tenerife (CCBAT), have been screened for their specific ploidy status using the molecular marker Dgas44. We have demonstrated that the Dgas44 sequence is effective in the screening of Madeiran and Canarian wheat accessions. This screening method permitted the detection of 10 and 11 durum accessions among Madeiran and Canarian wheats, accounting for 19.2 and 15.3% of screened collections, respectively. The obtained results have shown a 100% of cases correspondence with the previously performed morphological identification of the Madeiran wheats. It also permitted rectification and clarification of previous classification of some accessions based only on the morphological traits. The PCR based assay was directly applicable to the screening of seeds and was suitable for detecting seed mixtures in accessions. This rapid method has been proven to be a useful tool in gene bank accessions management including verification of their ploidy status and detection of seed lots adulteration.


Introduction
The current wheat genetic diversity on the Archipelago of Madeira is the result of several germplasm introductions from various geographical locations (Vieira 1983). These introductions, as well as cultivar adaptation to local conditions, tra- 0133-3720/$20.00 © 2007Akadémiai Kiadó, Budapest Cereal Research Communications 35(3), pp. 1397-1404(2007 DOI: 10.155610. /CRC.35.2007 ditional agricultural practices and the artificial selection performed by farmers, have determined the evolution of a crop diversity possessing unique morphological and agronomic features (Pinheiro de Carvalho et al. 2003). Wheat diversity of the Madeira and Canary archipelagos has been monitored, and its phytogenetic resources conserved as germplasm collections in the ISOPlexis germplasm Bank, at the University of Madeira and in the Centre for Conservation of Crop Biodiversity of Tenerife (CCBAT). The ISOPlexis and CCBAT banks make an effort to preserve traditional wheat landraces on both archipelagos, through an extensive program of diversity monitoring and germplasm management. The characterization and identification of germplasm accessions are primarily done based on morphological and physiological traits. These morphological and physiological descriptors are useful but may be affected by environmental factors and their analysis is time consuming. For such reason, the use of molecular markers as a fast technique for fingerprinting and screening of varieties or accessions with agricultural relevance have recently received researcher's attention. Molecular markers can complement morphological characterization because they are plentiful and independent of tissue or environmental effects (Manifesto et al. 2001;Perry 2004). The accessions characterization aims to promote the crop varieties description, seed certification and, probably the most important, their germplasm protection (Dograr et al. 2000). It is also necessary to assess the distinctness, uniformity and stability of new cultivars for certification (Pasqualone et al. 1999). To keep the accessions value unchanged in a germplasm bank, seed contaminations or mixture must be eliminated. At the same time, additional attention needs to be paid to avoid the genetic erosion phenomena of germplasm collections. Usually, to prevent these phenomena samples need to be periodically in situ collected or regenerated, which could lead to the adulteration of the accessions. To avoid the possibility of seed mixture and accidental contaminations occurring during either wheat harvest or storage routine quality control, tests for screening of accessions are essential. PCR based markers that require very small amounts of DNA seem to be a relatively rapid tool for germplasm screening. Bryan et al. (1998) have reported a PCR method based upon a D genome amplified sequence, which can be used to detect the presence of the wheat D genome and distinguish tetraploid from hexaploid accessions. The screening of the wheat species in germplasm collection using molecular markers facilitate further morphological characterization and can act as a substitute for cariotype analysis as it is faster and more reliable.
In this work, we present the results of screening for the presence of D genome in 52 and 72 wheat accessions from the Madeira and Canary Archipelagos, re-spectively. The aim of this work is to establish a rapid and accurate screening method for wheat accessions using a single molecular marker.

Seed material
Fifty-two and seventy-two accessions conserved in the ISOPlexis and CCTAB collections were used as representatives of wheat diversity from the Madeira and Canary Islands (Tables 1 and 2). Avonlea (durum wheat) cultivar was used as an out group marker.

DNA extraction and PCR amplification
Samples were ground in a mortar to a fine powder and DNA was extracted from five dry seeds of each accession by the modified Dellaporta method (Dellaporta et al. 1983). DNA concentration and quality was assessed by gel electrophoresis in 0.8% agarose gels prepared and run with 1X TAE buffer.
Amplification was performed in 30 µl volumes using a PxE (ThermoHybaid) Thermocycler. Each reaction consisted of 1X Taq PCR buffer, 1 unit of Taq DNA polymerase, 0.2 mM of each deoxyribonucleotide, 0.25 µM of each primer and 20 ng of genomic DNA. The amplification protocol consisted of an initial 4 min denaturation step at 94ºC, followed by 30 cycles of denaturation at 94 ºC for 1 min, annealing for 1 min at 65ºC and extension at 72ºC for 2 min. A final extension step was performed at 72ºC for 5 min. The oligonucleotides used as primers were synthesized on the basis of published PCR primers designed to Dgas44 DNA sequence (Bryan et al. 1998), with the following sequences: N, 5' CTTCTGACGGGTCAGGGGCAC 3'; P, 5' CTGAATGCCCCTGCGGCTTA AG 3'. Once amplified the samples were analyzed by electrophoresis in 1.2% agarose gels and visualised by ethidium bromide staining.

Results and Discussion
The analysis of DNA extracted from five seeds from 52 Madeiran and 72 Canarian wheat accessions allowed us to distinguish durum wheat from hexaploid accessions (Tables 1 and 2). An earlier evaluation of the Madeira wheat diversity using morphological and cytological criteria resulted in the recognition of 2 Triticum species (dos Santos and Pinheiro de Carvalho 2006) and the classification of accessions among these species. However, some doubts arose in the dis-tinction of T. aestivum from T. turgidum in several accessions, which results for morphological traits that could be interpreted as durum or bread wheat. These problems persisted even after caryotype analysis. This situation prompted us to find a rapid and reliable PCR-based solution to screen and distinguish between durum and bread wheat accessions. The proposed method is based on the amplification of the sequence Dgas44, which demonstrated to be an effective tool for detection of the D genome in wheat samples acting as a specific marker for bread wheat identification. Bryan et al. (1998) applied this sequence to a large number of wheat cultivars and demonstrated that it has a wide applicability in the identification of D genome in wheat. Dgas44 is a repetitive sequence that exists in a high copy number (200-300) within the D genome of bread wheat (McNeil et al. 1994). This marker has been shown to be effective in the detection of adulteration among bread wheat flour (Bryan et al. 1998, Alary et al. 2002. Tilley (2004) used Dgas44 in the amplification of wheat sequences from DNA extracted during milling and baking. Other authors have also used this marker to analyse the composition of genome of Triticale (Tams et al. 2004).
In the present work, the amplification of Dgas44 target sequence resulted in a band of the expected molecular size of 286 bp (Fig. 1). All DNA samples extracted from the previously identified Madeiran T. turgidum accessions failed to produce the noticeable band of the D genome, including the Avonlea marker, whereas DNA from T. aestivum cultivars produced a distinctive amplification product. The Dgas44 sequence was demonstrated to be effective to characterize the absence or presence of D genome in all studied accessions. Ten accessions have been identified as durum cultivars (accessions 1, 6, 9, 18, 21, 38, 39, 43, 47 and 51), corresponding to 19.2% of screened collection (Table 1 and Fig. 1). This approach allowed us to achieve 100% cases confirmation of the previous morphological and cytological classification. This method also proved to be efficient in the detection of seed mixtures and contamination as we managed to detect contamination in one of the examined accessions (# 9). It presumed to be durum wheat, while molecular analysis revealed in the sample presence of genetic material originated from the hexaploid wheat. This particular accession has been collected in situ again to verify its ploidy status. The newly collected sample has been proven to be durum wheat. This incident instantly validated the suitability of the test for verification of seed contamination in our gene banks. This approach was also applied to screen 72 not previously classified wheat accessions from the Canary Islands (Table 2). The collected samples were subjected to the Dgas 44 marker screening followed by the morphological and cytological characterization. We were able to rapidly identify 11 durum wheat cultivars in the 72 studied accessions, corresponding to 15.3% of the screened collection (data not shown). A large quantity of time consuming tasks needed for full ANDRADE et al.: Durum Wheat Accessions Screening Method characterization of the accessions was avoided, and thus improved the management and quality control of the germplasm collection. This approach can also be used for a first verification of seed contamination in gene bank sample management and can be important for industry and flour analysis.
Although we are convinced that we have successfully identified the durum accessions among the Madeiran and Canarian wheat germplasm based on morphological evidences, the existence of false negatives selected by the use of Dgas 44 marker cannot be entirely excluded. To avoid a remote possibility of misidentification and to assure unconditional reliability of this test the Dgas44 negative accessions need to be further screened with a second molecular marker specific for high molecular weight glutenins (HMWGs) for example the primers allele-specific for identification of Dy10 or Dy12 (Ahmad 2000). Durum accessions exhibit the lack of these subunits and it would be the ultimate proof of the nature of these accessions.
However, one has to remember that full and comprehensive accessions studies, including a complete morphological, biochemical and molecular characterization need to be made in order to obtain passport data and complete variety characterization.
This work has contributed to the knowledge of the agricultural heritage of the Madeira and Canary archipelagos, specifically to the wheat diversity in the framework of a long germplasm monitoring and characterization study of the cultivated wheat varieties of the Archipelago of Madeira and Macaronesian Region.