Collection and characterisation of a population of Triticum boeoticum Boiss . , a wild wheat species not previously found in the Mediterranean western region

A population of wild wheat was collected in July 2010 in an abandoned area near Madrid, Spain. This zone possesses a high botanical biodiversity together to a particular type of soil termed “green clays”. A sample of wheat was collected, multiplied and characterised for several agro-morphological traits and glutenin subunits. The 2n chromosome number revealed that it was a diploid wheat species, and characterisation data indicated that the accession was Triticum boeoticum Boiss. This species probably arrived as a weed of the einkorn crop grown in the zone until at least the first half of s. xix. The specific edaphic and climatic characteristics of the habitat and the fact that there were no references so far about this species in the Mediterranean western region make this acquisition very valuable for wheat improvement. The new accession is conserved at the National Plant Genetic Resources Centre and a herbarium sheet has been deposited in the Royal Botanic Garden in Madrid. Additional key words: cereal; germplasm; glutenins; Spain.

Short communication.Collecting a population of Triticum boeoticum Boiss. in Spain from a weather station 2.5 km far from the collecting zone were 765.90 mm for ETP, 404.40 mm for annual rainfall and 14ºC for annual temperature (38-year mean taken since 1961).The soil (Suppl.Fig. 1e [pdf]) termed as "Green Clays" is uncommon in Spain (Martínez Labarga, 2010).It is composed almost exclusively by phyllosilicates and small quantities of quartz and feldspar.The greatest part of the phyllosilicates is magnesium smectite (saponite) of high purity (García Romero et al., 1990;García Romero, 2004).These soils strongly retain the nutrients and water, so they are fertile but the available water for plants is low.The wheat was in a sloping depression where the humidity was higher.The pH of the soil is alkaline which agrees with wild einkorns have affinity for basic soil types like marls, clays and limestones (Zohary & Hopf, 2000).The collecting site possesses a high biodiversity and more than 150 species have been catalogued Diploid wheats comprise three species: two wild, Triticum urartu Tum. and Triticum boeoticum Boiss., and the cultivated Triticum monococcum L. The three species are closely related to the A genome of polyploidy wheats (Kihara, 1924).The wild species are useful genetic resources as donors of genes for valuable traits for wheat improvement, such as disease resistance, stress tolerance and gluten quality (Gale & Miller, 1987;Appels & Lagudah, 1990;Rogers et al., 1997).Most of the wild einkorn was first spread through the northern part of the Fertile Crescent (Mac Key, 2005) but their distribution area was narrower than that of the cultivated einkorn.So, some populations of diploid wild wheats have been found in the countries of the eastern Mediterranean as Greece, Bulgaria, Lebanon, Syria and Turkey (Kimber & Feldman, 1987) but there is none reference for the western countries as Italy, France, Spain and Portugal.In contrast, T. monococcum was cultivated in all Mediterranean countries in the past.On the other hand, the diploid wild wheats presented a wide range of natural habitats, from the sea level in Macedonia to 2000 m in Iran and Iraq, in soils hardly suitable for cultivation, and on virgin and fallow land (Damania, 1998;Mac Key, 2005).High intra-specific variation with different agromorphological types has been also described (Dorofeev, 1968;Filatenko & Hammer, 1997).The objective of this work was to collect, identify and characterise a wild wheat recently found in Spain and not previously described.
In July 2010, a population of wheat with brittle rachis has been located in an abandoned area in the locality of Coslada (Madrid), Spain.The geographic parameters were 40º 26'14'' N of latitude, 3º 33' 55'' W of longitude and 609 m of altitude.The collecting area occupies 10 ha of difficult access, isolated by factory premises and the train rails.Suppl.Figs. 1 b and c (pdf) shows two photographs of the zone at the present time.The topographic map of the region of 1875 reported that the zone was cultivated, and the aerial image of the zone in 1946 shows the crop plots (Suppl.Fig. 1a [pdf]).In the 70's the crops were abandoned and only some grazing continued till 2005.The wheat population was located in an area of about 1,400 m 2 (Suppl.Fig. 1b [pdf]).The plants had brittle rachis, so most of the spikelets were collected from the soil although some spikelets remained in the plant (Suppl.Fig. 1d,e,f [pdf]).The zone has a Mediterranean Continental climate typical of the centre of Spain, with cold in winter and hot and drought in summer.Climatic parameters  by Martínez Labarga (2010).Other wild species of the Triticeae tribe, as Aegilops lorentii Hochst, Aegilops geniculata Roth and Aegilops triuncialis L., were collected and maintained in the CRF-INIA.Dorofeev (1968) reported that wild einkorns were often observed growing together with plants belonging to Ae. biuncialis Vis.(synonym: Ae. lorentii Hochst.) and Ae.triuncialis L. It was also found one of the greatest populations in the world of Cynara tournefortii Boiss.& Reut., and the best Iberian populations of Malvella sherardiana (L.) Jaub.& Spach and Linaria caesia (Pers.)F. Dietr.(Martínez Labarga, 2010).Cynara tournefortii and Malvella sherardiana are catalogued as CR (critically endangered) and VU (vulnerable), respectively in the IUCN Red List of threatened plants (Moreno, 2008).Other dicotyledonous species not located before or infrequent in the Madrid province were discovered, like Teucrium spinosum L. and Convolvulus humilis Jacq., Klasea flavescens (L.) Holub subsp.flavescens, Scolymus maculatus L. or Ziziphora hispanica L. Traditional agricultural and livestock uses and, in particular, the proximity of the enclave to several cattle tracks has surely contributed positively in the richness in species (Martínez Labarga, 2010).
The chromosome number of the collected seeds of wheat showed that it was a diploid species (2n = 14).The ears collected had brittle and hairy rachis, and two awns and two grains (one of them smaller and darker) per spikelet (Suppl.Fig. 1f [pdf] and Fig. 1a).Since cultivated einkorn usually has one awn and one grain per spikelet (Fig. 1b), and weaker rachis hairiness and brittleness (Filatenko et al., 2002) these data point to the wild wheats T. urartu or T. boeoticum.In some cases both wild species are very similar morphologically, but they can be distinguished with some molecular markers as glutenins and short-sequence DNA repeats (SSRs) (Waines & Payne, 1987;Hammer et al., 2000).So, High Molecular Weight (HMW) glutenin subunits were analysed to distinguish between T. boeoticum and T. urartu.Fig. 1c shows the glutenin pattern of the collected accession, together with the patterns of other accessions of T. urartu and T. boeoticum conserved at the CRF-INIA.Three Spanish accessions of T. monococcum were also included.Comparisons with T. urartu indicated that the collected accession had less prominent "1Ay" subunits, typical of T. boeoticum, and different from the major one common in T. urartu (Waines & Payne, 1987;Hu et al., 2012).Also, subunit "y" in our sample was clearly slower than subunit 12 of 'Chinese Spring', whereas the mobility was similar in T. urartu.Subunit "1Ax" of the collected accession was slightly faster than subunit 2 of 'Chinese Spring'.According to Waines & Payne (1987) we can conclude that the protein genotype of this new accession did not corresponded to T. urartu but to T. boeoticum.The range of mobilities of the subunits of the Spanish cultivated einkorns and the other T. boeoticum accessions were also in agreement with the ranges obtained by Waines & Payne (1987) and Hu et al. (2012).It can be observed that all the T. boeoticum analysed, ours included, had a prominent faster band (►) not present in the cultivated einkorn.
A sample of the collected seeds was sown for multiplication and agro-morphological characterisation during 2011 in the experimental fields of the CRF-INIA in Alcalá de Henares, 20 km far from the collecting site.Table 1 shows the agro-morphological data of the accession.The variables were measured according to IBPGR (1985) and Ruiz et al. (2007).Some agromorphological traits such as very high hairy leaf, and long and less dense spike were clearly different from the Spanish accessions of cultivated einkorn (Fig. 1b), which had hairless leaf, and shorter and very dense spikes (Ruiz et al., 2007).Agro-morphological data also indicated that the sample was different from other T. boeoticum accessions maintained at the CRF-INIA.Two main eco-geographic races of T. boeoticum have been recognized.One is a relatively small one-seeded type typical of the cooler Balkans and western Anatolia usually referred to as T. boeoticum subsp.aegilopoides.The other race is a larger two-seeded race  2005).In Anatolia all intergradations and intermediates between these two races occur often in mixed stands (Zohary, 1969).Our data indicate that the accession collected was T. boeoticum subsp.tahoudar as proposed Dorofeev et al. (1979) or T. monococcum subsp.aegilopoides var.thaoudar according to Mac Key (2005).A herbarium sheet of the specimen has been deposited in the Royal Botanic Garden in Madrid (No. MA-840865).
Wild wheats carry interesting traits for adaptation to climatic change in the Mediterranean region (Mac Key, 2005).The specific edaphic and climatic characteristics of the habitat make the new accession of T. boeoticum found in Spain a valuable germplasm for wheat improvement.Moreover, this accession is an important genetic resource since there were no references so far about this species in the Mediterranean western region.The current distribution area of wild wheats may be larger than 10,000 years ago because these species were spread as admixture with seeds of cultivated species.Some of them like Hordeum spontaneum, T. boeoticum, T. urartu and Ae.tauschii developed weedy races well adapted to growing in cereal field.We have found a reference about that einkorn was grown in the locality in the past (Madoz, 1845).So, it is possible that T. monococcum was cultivated in the collecting zone and the wild einkorn found in the present work was a weed of the crop similar to that found in other countries.

Figure 1 .
Figure 1.Spikes, spikelet and grains of (a) the collected accession, (b) a Spanish cultivated einkorn, (c) Glutenin electrophoresis of accessions of T. urartu, 'Chinese Spring' bread wheat control, the collected wheat accession, T. boeoticum and T. monococcum.The regions of "1Ax" and "1Ay" subunits in diploid wheats are indicated.Arrowheads show faster subunits present in some T. boeoticum accessions.The subunits of the bread wheat have been numbered according to Payne & Lawrence (1983).

Table 1 .
Agro-morphological data of the collected accession Short communication.Collecting a population of Triticum boeoticum Boiss. in Spain present in the warmer dry summer areas of southern Turkey, Iraq and Iran commonly referred as T. boeoticum subsp.thaoudar (Reut.ex Hausskn.)Grossh.The first grain of this race is smaller and darker (Mac Key,