Morphological characterization of the Tunisian durum wheat accessions
Phenotypic diversity and morphological characterization
The Shannon-Weaver index (H’) revealed a high morphological diversity among the durum wheat accessions with an overall H’ of 0.80 (Table 1). The most polymorphic characters were the spike length (H’=0.98), the grain size (H’=0.94), grain forms (H’=0.87), the grain color (H’=0.86) and the spike shape (H’=0.86), while the spike color showed the least polymorphic level (H’=0.53).
The 304 durum wheat accessions were grouped into eleven landraces namely Azizi, Jneh Khotifa, Taganrog, Mekki, Richi, Souri, Roussia, Badri, Biskri, Biada and Mahmoudi, according to the catalog of old durum wheat landraces and are part of the 40 durum wheat landraces recorded in Tunisia [10]. These landraces were characterized by the 12 specific morphological traits based on IPGRI [20] and UPOV [21] (Table S5, Table S6). A multitude of spike characteristics has been observed between the durum wheat landraces, whereas these characteristics were homogeneous between accessions of the same landrace. In fact, the Shannon-Weaver index (H’) calculated for each landrace were relatively low, ranging between H’=0.00 for Badri and Jneh Khotifa landraces and H’=0.23 for Richi landrace with an overall mean H’ of 0.11 (Table S7). For instance, the variety Mahmoudi accessions had particularly large spikes with sub-pyramidal shape, very long awns and big grain size; whereas rectangular and very flat spikes characterized Azizi accessions. Biskri accessions had fusiform and big size spikes. The Spike color, length and shape were variable among the studied accessions and varied from dark to light, and from short to long spikes For example, Badri spike was very short and thick with a greyish color, while Biada was characterized by very light (white) spikes and awns color. Souri and Roussia were both characterized by tight and red colored spikes with distinct spike shape characterized as rectangular for Souri and cylindrical for Roussia. The former varieties were also characterized by a distinct orange grain color. Interestingly, Richi accessions had a unique feathery spike, while Mekki was characterized by short and dense spikes with parallel edges. Finally, Taganrog accessions are characterized by white colored spikes washed with black, while Jneh Khotifa accessions had a very dark (black to purple) long and dense spike and awn colors.
Principle Coordinates Analysis (PCoA)
The PCoA performed on the 12 spike morphological traits of the 304 durum wheat accessions showed that axes 1, 2 and 3 accounted for 22.28%, 17.47% and 15.87% of the total genetic variation, respectively (Figure 1). Color-coding of the accessions in 2-dimensional PCoA plots (axis 1 vs. 2 and axis 1 vs. 3) showed a good correlation between the morphological grouping and the landraces denomination. For axes 1 and 2, four subgroups were identified. The first subgroup comprised Mekki, Souri and Roussia; the second subgroup was composed of Azizi, Biskri, Taganrog, Jneh Khotifa and Richi. The first subgroup shared similar color traits of the glumes (red) and awns (red or brownish), while the second subgroup shared similar spike characteristics such as spike length (medium to long) and similar awns color (black or white). Landraces Badri and Mahmoudi were separated in two distinct subgroups and were characterized by unique spike shapes, notably pyramid shaped and very short round shaped spikes, respectively. All landraces were morphologically distinguished using the first three axes based on the 12 spike characteristics, except for landraces Roussia and Souri and for landraces Biskri, Richi, Jneh Khotifa and Taganrog that were not distinct from each other in respect to their spike size and color. Thus, additional morphological traits were considered to classify the latter landraces into distinct subgroups such as glumes form (Table S6).
Genetic diversity and population structure of Tunisian durum wheat accessions
SSR polymorphism
Ten SSR markers were used in this study and were mapped on 8 different chromosomes and considered therefore largely independent (Table 2, Table S2). The percentage of missing data was low and always remained below 10% for each locus. The 10 SSR markers amplified a total of 99 alleles. The number of different alleles per locus (Na) varied from 4 for Xgpw2103 to 16 for Xgwm413, with a mean of 9.9 across all loci. Overall, the PIC value was 0.690. The highest PIC value was obtained for Xgwm413 (0.851), whereas the lowest PIC value was obtained for Xgpw2103 (0.448). The Shannon’s information index (I) also showed the highest value for Xgwm413 (2.182), whereas the lowest I value was obtained for Xgpw2103 (0.781). The fixation index (Fis) was close to 1 for each locus except for Xgwm495 (Fis= -0.373), where a high PIC level was observed (0.659). Pairwise genetic differentiation (Fst value) ranged from 0.201 for Xgwm495 to 0.688 for Xgpw7148.
Population structure analysis and relationship between genetic and morphological characterizations
A population structure analysis was investigated using the 302 Tunisian durum wheat accessions (188 MLG). The maximum likelihood (LnP(K)) and delta K (ΔK) methods indicated that the most likely number of genetic groups (K) was 11 (Figure 2, a and b). The estimated membership coefficients of each accession to the different genetic groups (at K=11) is shown in the population structure plot (Figure 2, c).
Overall, each genetic group corresponds to a landrace. The genetic groups G2, G3, G4, G5, G7, G9, G10 and G11 corresponded to Jneh Khotifa, Taganrog, Mekki, Richi, Badri, Beskri, Biada and Mahmoudi, respectively. Moreover, a significant correlation between the genetic distance matrix and morphological distance matrix was observed (P=0.01; Rxy=0.435). However, a discrepancy between the genetic distance matrix and the morphological distance matrix was observed for the landraces Azizi, Souri and Roussia. In fact, Azizi was clustered by STRUCTURE into two different genetic groups G1 and G8, and the two landraces Souri and Roussia were clustered in one genetic group G6 despite their distinct morphological characters.
Forty-one admixed individuals were observed in the collection. The majority of the admix is composed by G6 (Roussia and Souri) and G10 (Biada) representing 14.6 % of the admix accessions, followed by G1 (Azizi) and G9 (Beskri) representing 9.7 % of the admix.
Mahmoudi G11, Beskri G9 and the admixed accessions were the most frequent groups composing the overall landrace collection with 23.8 %, 12.2% and 14% of the accessions, respectively. Azizi G1, Taganrog G3, Mekki G4, Badri G7 and Biada G10 each accounted for about 8% of the entire collection. However, Jneh Khotifa G2, Richi G5, Roussia and Souri G6 and Azizi G8 were the least represented in the collection and each accounted for solely 3% of the collection.
Analysis of diversity indices and molecular variance
The eleven clusters defined by the STRUCTURE analysis presented different levels of genetic diversity (Table 3). Group G6 showed the highest level of genetic diversity, while G7 represented the lowest level. The number of effective alleles per locus ranged from 1.152 for G7 to 2.379 for G6. Genetic groups with the highest number of MLGs were G6 (100% of different MLGs), G8 (90%) and G3 (85.7%), while G7 and G11 had the lowest number of MLGs, with 27.2% and 34.7%, respectively. The percentage of polymorphism ranged from 40% for G7 to 100% for G6 and G8. Shannon's index varied from 0.166 for G7 to 0.937 for G6 with an average of 0.620 across all accessions. In addition, G6 and G8 had the highest number of private alleles, with 7 and 4 private alleles respectively; while G2 and G7 had no private alleles (Table S8). G10 and G4 had both 2 diagnostic alleles, while G3, G5 and G7 had 1 diagnostic allele with a frequency > 70%. The fixation index (Fis) ranged from 0.698 for G4 to 1 for G7 where Ho was 0.100 and null, respectively. Furthermore, the analysis of variance showed that 59% of the total genetic diversity was observed between the distinct genetic groups, while 41% of the genetic diversity was explained by differences within each group (Table 4).
Network analysis
The genetic relatedness between genotypes was tested using the mininum spanning network (MSN) based on Bruvo’s distance. MSN separated all the accessions into two main clusters (Figure 3). The first cluster named C1 grouped accessions belonging to Azizi G1 and G8, Jneh Khotifa G2, Richi G5, Souri and Rousia G6, Badri G7 and Biskri G9, while the second cluster named C2 grouped accessions belonging to Taganrog G3, Mekki G4, Biada G10 and Mahmoudi G11. In addition, the pairwise Nei’s genetic distances calculated between the 11 genetic groups were also in agreement with the accession clustering by the MSN (Table S9). The highest Nei’s genetic distance value (2.416) was recorded between G10 and G5, followed by the genetic distance value (2.319) recorded between G10 and G7. The lowest genetic distance was registered between G1 and G8 (0.421), between G11 and G3 (0.630), and between G3 and G4 (0.630); indicating that G1 and G8, as well as G11, G3 and G4 were the most genetically related groups respectively. In addition, a morphological comparison between the network groupings revealed a significant difference (p-values< 0.05) between C1 and C2 for spike shape, spike length, awn length, grain color, grain form, the number of spikelet/spike and for awns and glumes colors (Table 5). The cluster C1 had a higher gene diversity (He=0.740) and phenotypic diversity (H’=0.77) than cluster C2 with He=0.425 and H’=0.61 (Table S10 and S11). The C1 cluster presented higher spike shape, and spike length values than C2; while C2 had significantly higher awns length and grain size traits (Table 5).
Diversity analysis by regions and climatic stages
Morphological diversity analysis by regions and climatic stages
Shannon-Weaver index (H’) was assessed based on 12 spike’s morphological traits at the five regions (Sousse, Mahdia, Kairouan, Gabes and Medenine) and the three climatic stages (low semi-arid, high-arid and mid-arid climates) of the designated sites (Table 1). Kairouan had the highest diversity index (H’=0.74) followed by Medenine (H’=0.66), while Sousse had a null diversity index indicating no phenotypic variability between accessions in that region where Richi was the only landrace identified. The most polymorphic characters by regions were spike length (H’=0.69), grain form (H’=0.65), grain color (H’=0.62) and number of spikelets/spike (H’=0.61). Furthermore, high-arid climate had the highest diversity index (H’=0.74), as this climate stage is represented by Kairouan; while low semi-arid climate represented by Mahdia and Sousse had the lowest diversity index (H’=0.59). The most polymorphic characters by climatic stages were awn length (H’=0.90), grain form (H’=0.82), grain color (H’=0.79), and number of spikelets/spike (H’=0.73).
Polymorphism level of some characters differed distinctly among regions excluding Sousse where an overall homogeneity of the morphological traits was recorded. Awns color was variable among regions and ranged between 0.12 and 0.73, for Kairouan and Mahdia, respectively. Simirlarly, the highest spike length was registered in Mahdia (0.99), while the least spike length was recorded in Gabes (0.49). Notwithstanding, the Mahdia region was characterized by the lowest spike color and glumes color indices with 0.00 and 0.48, respectively. However, the highest values of spike color and glumes color were recorded in Mednine (0.53) and Kairoun (0.97), respectively. Morphological traits were also variable from one climate stage to another. The low semi-arid climate is characterized by the lowest records for spike length (0.0) and for glumes color (0.41), in contrast to the high-arid climate where spike length and glumes color were the highest (0.48 and 0.96, respectively). Contrary, awns color registered the least value in the high-arid climate (0.12) and the highest record in the mid-arid climate (0.71). However, no variations were observed between regions for grain color and between climate stages for number of grains/spikelet.
In addition, a dominant phenotypic class of some characters was observed among regions (within more than 70% of accessions), except for Sousse which didn’t show any differences in morphological traits. In Gabes, long (>9 cm) (84%) and lightly colored (92%) spikes with cylindrical shape (79%), awns shorter than the spike (84%), moderatly long grains form (82%) with a small grain size (<0.3 cm) (82%) and an intermediate number of grains/spikelets (2 to 3) (79%) were noted while spikes with medium length (6 to 9 cm) (73%) were dominant in Medenine. For Mahdia spikes with equal awns and spike length (72%) and with small grain size (78%) were largely observed. However, Kairouan was dominated by spikes with awns longer than the spike (72%). Concerning climatic stages, small grain size (<0.3 cm) (72%) were dominant in mid-arid climate zone, whilst, dark colored (72%) spikes with black awns (96%) were dominant in high-arid climate zone. No particular phenotypic classes were observed within the low semi-arid climate (Table S4).
Genetic diversity analysis by regions and climatic stages
The analysis of variance showed that 19% and 10% of the total genetic diversity were observed among regions and among climatic stages, respectively, while 81% and 90% of the genetic variabilities were explained by differences within regions and within climatic stages, respectively (Table 4).
Genetic diversity by region showed a number of effective alleles ranging from 1.366 for Sousse to 3.031 for Gabes (Table 3). Overall and among all investigated regions, Sousse region has shown the lowest genetic diversity indexes, in contrast to the outstanding indexes registered at Gabes. In fact, Gabes region had the highest number of MLG (31) and the highest Shannon's diversity index with 1.296, while Sousse and Medenine had the lowest number of MLG (7), and the lowest Shannon's diversity index registered at Sousse (0.305). Moreover, the percentage of polymorphism was 100% for all regions except for Sousse which was 50%. Gabes had also the highest number of private alleles (17), while Sousse and Medenine had the lowest number of private alleles (1). The fixation index was above 0.800 in each region except for Sousse which was 0.691. Interestingly, solely the diagnostic allele and heterozygosity index were high in Sousse compared to the other regions. In fact, three diagnostic alleles with a frequency that exceeded 70% and a Ho of 0.100 were registered at Sousse, compared to only one at Gabes.
The SSR data analysis by climatic stages revealed that the mid-arid climate was outstanding among the studied climatic stages and had the highest number of effective alleles (3.174), the highest Shannon's diversity index (1.318) and the highest number of private alleles (19). Contrary to the mid-arid climate, the high-arid climatic stage showed the lowest number of effective alleles (2.707), the lowest Shannon's diversity index (1.050) and the lowest number of private alleles (2). However, the fixation index was similar among all studied climatic stages and recorded an index above 0.800 for all climatic regions (Table 3).
Correlations between genetic distance and geographic distance
The Mantel test showed a significant correlation at (P=0.010; Rxy=0.286) between genetic and geographic distances among durum wheat accessions, suggesting that geographicaly close individuals were genetically related. With the exception of the Sousse region, Azizi and Mahmoudi were the most widespread landraces between central and southern Tunisia across all regions and climatic stages. However, Azizi was more frequent in Gabes (25 accessions out of 38), while Mahmoudi was mostly found in Medenine (13 accessions out of 22) and Mahdia (11 accessions out of 27) (Figure 4). In addition, Sousse grouped all G5 genotypes, corresponding to Richi landrace. Kairouan grouped all G7 and G2 genotypes, corresponding to landraces Badri and Jneh Khotifa , respectively. The landrace Taganrog, representative of the genetic group G3, was exclusively found in Mahdia.
Furthermore, and exclusively for the widespread landraces Azizi and Mahmoudi, a comparison of morphological traits between Azizi and Mahmoudi accessions collected from central and southern Tunisia was carried out and has revealed a non-significant difference (p-values > 0.05) for all traits, except for spike density within Mahmoudi (p=0.00). In fact, Mahmoudi accessions from central Tunisia had compact (SD=7) spikes compared to southern Mahmoudi accessions characterized by lax spikes (SD=5) (Table 5).