Image analysis of O viciifolia seeds

Seeds were characterised by image analyses. Software (developed by Mr R. Farrell, NIAB) was used to measure several seed morphometric traits. Areas were measured in cm2, length and width in mm. Indexes were created to assess the shape of seeds. The shape index measurement is based on the idea of boundary length to area, normalised so that a circle has a shape of 1.0 and anything else has a shape of < 1.0. The shape change gives a measure of the irregularity of the seeds (e.g. spines), the higher it is the more indented the seed is. All of these values are given in Appendix 5 for the 75 accessions. These morphometric traits were all found to be significantly different among the 75 accessions (p <0.001).

Cultivation status has a significant effect on seed area and dimensions. Seeds from wild accessions were longer and their area is greater than seeds from cultivated/cultivar accessions (Table 32).

Table 32: Means for area, length and width/length ratio according to cultivation status

Cultivar Cultivated Wild F pr

Area cm2 0.2384 0.2611 0.273 0.012

Width/length 0.7305 0.7113 0.7055 0.014

Length mm 6.66 7.081 7.237 0.005

The geographic origin of each accession was shown to have a significant effect on the width to length ratio (Table 33). Accessions from China, Hungary and the USA appear to have a reduced length to width ratio, thus to be more elongated. Accessions from Bulgaria, Switzerland and the UK have a high length to width ratio, thus to be more rounded.

Table 33: Means of the width to length ratio according to countries Country F pr=0.034 Armenia 0.7166 Bulgaria 0.7855 China 0.6758 Czech Republic 0.713 Former Soviet Union 0.7166

Germany 0.7118 Hungary 0.6909 Iran 0.7024 Italy 0.7216 Lithuania 0.7137 Morocco 0.7085 Norway 0.704 Poland 0.7166 Romania 0.7197 Russia 0.7077 Spain 0.7097 Switzerland 0.7399 Turkey 0.7052 UK 0.7309 Ukraine 0.7242 USA 0.6787

All other morphometric characteristics and accession origin data were not found to be statistically significant.

A linkage tree analysis was performed. Four different clusters were given by this analysis (Figure 36). Clusters were not found to be generally linked to the geographical or climatic origin or the cultivation status. The upper cluster is made of mostly of cultivated accessions from the UK.

Figure 36: Linkage similarity tree based on all morphometric traits measured on the seeds from the 75 accessions.

These different clusters probably countain seeds possessing similar morphometric characteristics (small versus big - see Figure 37 for an example or - spiked versus rounded) that are not related to the accession origin.

Figure 37: Comparison of small seeds (accession 1116) and large seeds (accession 1005) showing that important variations in seed size can greatly affect the result of

image analyses

4.8. Discussion

As observed for the agronomic traits, morphological traits were highly variable among the accession. Morphological characterisation was also a challenging task as reproducibility of measurements was crucial but difficult because of the intra- accession variability. Very significant differences were observed in colour shape and size of the different organs. The most unusual individuals were characterised by white flowers. A few accessions were characterised by red stems or totally prostrate habit, although these traits were sparsely found in some other individuals. A high morphological variability was also observed among a limited germplasm from central Italy (Negri and Cenci, 1988).

Diversity in leaf shape and length can be important from an agronomic perspective, as it can help to decide whether landraces are more adapted to pasture, hay production or silage. Colour diversity might be linked to the polyphenolic composition and/or quantities, and therefore be a good indicator of specific beneficial properties (e.g. anti-bloat, anthelmintic). The normal distributions observed in morphological traits shows that similarly to the agronomical traits, only a few accessions were characterised by extreme values. These traits might be of interest for breeding purposes.

Significant differences in morphological traits were seen between accessions of the germplasm collection. Few correlations with geographic and climatic origin were found probably due to the limited accession number analysed. Still, solid correlations were shown on crucial traits, and it appears that geographic origin was a strong driver of O. viciifolia morphological characteristics. A clear division (more than 50% of differences) was found between Eastern European accessions, which were characterised by more inflorescences, and Western European ones characterised by more leaflets. This might reflect general agricultural uses with pasture dominating in Eastern Europe, and hay production, with preference to foliage production, in Western Europe.

The most striking morphological difference was shown for mountainous accessions. These accessions were characterised by lower organ length but higher number of stems. This reduced organ size was also recorded for mountainous accessions in a germplasm from central Italy (Negri and Cenci, 1988). This is a typical adaptation to mountainous condition which has been observed for a quantity of alpine plants. These morphological features are due to the shorter warm season and important UV and temperature stresses. Mountain-adapted O. viciifolia constitute a very important forage option as they could be optimal forages for pasture in mountainous zones affected by drought and poor soils.

Cultivated and cultivar accessions were characterised by thick stems and a higher number of leaflets. These characteristics were probably selected for hay production and silage, because such characteristics allow production of more biomass.

The cluster analysis, combining all morphological traits and flowering data, strongly support (more than 50% difference between the two clusters) a general

distinction between Western European accessions and accessions from the rest of the world. This distinction might be explained by climatic factors as the accessions from Western Europe, generally characterised by a temperate climate, tends to have an earlier flowering date. There might also be an effect of agricultural history. Accessions from Western Europe were characterised by a greater number of thick stems with more leaflets, characteristics that have probably been selected for hay production or silage. Other accessions were characterised by longer organs and higher numbers of leafs and inflorescences, characteristics probably more adapted for livestock pasture.

As observed during the germplasm collection, seeds were very variable in size and shape. Most morphometric traits were found to differ significantly among these accessions. Seeds derived from certain countries (China, Hungary, USA) were more elongated compared to seeds from other countries (Bulgaria, UK and Switzerland) which were more rounded, but no coherent cluster was found. A significant trend was shown for wild accessions to have bigger seeds. This might be crucial for wild accessions as establishement is made difficult by competition from other plants. Thus larger nutrient stocks are necessary to ensure some germination success. Different groups of seeds were shown to exist, based mainly on varying size and shape. Somewhat surprisingly, this variation does not seem to be linked to the origin and cultivation status of different O. viciifolia accessions.