Introduction

Traditional crops are generally viewed by consumers as more natural and healthier options (Rascio et al., 2015; Rascio et al., 2016). Their reintroduction into cultivation and reinforcing their value chain can play a vital role in bolstering conservation efforts and elevating the value of their cultivation environment. This, in turn, can substantially increase the economic benefits for rural communities (Pallante, Drucker, & Sthapit, 2016). Before the onset of the breeding era in Italy, initiated by Strampelli in the early twentieth century (Scarascia-Mugnozza, 2005), cultivated wheat primarily consisted of autochthonous varieties. Although there is not a worldwide consensus on this definition, these wheats are often referred to as old/ancient wheats, or landraces (Negri, Maxted, & Veteläinen, 2009).

Autochthonous wheats are regarded as valuable sources of alleles for breeding programmes (Terzi et al., 2005). Indeed, over time, environmental conditions and, to a certain extent, purposeful farmer selection have resulted in the cultivation of plants exhibiting high adaptability and superior performance within their respective cultivation regions (Zeven, 1998). For these reasons, the EU (EU, 2009) defines landraces or varieties which are naturally adapted to local and regional conditions as conservation varieties (Spataro & Negri, 2013). They are frequently composed of mixed genotypes (populations) and exhibit a high degree of genetic diversity (Zeven, 1998), in contrast to modern breeding varieties (cultivars or pure lines).

Following the ratification of the International Treaty on Plant Genetic Resources for Food and Agriculture by the UN Food and Agriculture Organization (FAO, 2009) in 2001, most Italian regions implemented laws aimed at protecting and promoting indigenous crops. They also provided funding for projects dedicated to studying the conservation varieties’ distinctive characteristics. Their registration involves a formal process where these varieties are officially recognized, documented, and often included in seed catalogues or databases. Once registered, conservation varieties are often conserved in community seedbanks or similar facilities where they can be accessed by farmers. Enhancing access to these varieties is important to support agricultural biodiversity, improve resilience in the face of environmental challenges, and contribute to the long-term sustainability of food production systems. Some countries, by officially recognizing and documenting these varieties, provide legal protection to conservation varieties to prevent unauthorized use or commercial exploitation (EU, 2009). This protection is designed to encourage the continued conservation and sustainable use of these valuable genetic resources.

In Southern Italy, the cultivation of a wheat type known as ‘Saragollo forte’ which is particularly well-suited for pasta production, has been extensively documented in various commercial agreements dating back to the seventeenth century (Fiore, 2013). As introduced by old botanists (Cillis, 1927; Draghetti, 1927), the use of the plural noun ‘Saragolle,’ highlights the presence of multiple forms of ‘Saragolla’ wheat, all falling under the Saragolla (Sar.) leucurum botanical variety, which was one of the 22 botanical varieties of Triticum turgidum documented at the start of the 1900s (Percival, 1921). In 2004, the ‘Produttori Sementi Bologna’ company registered a variety also named ‘Saragolla’. This variety resulted from crosses between the ‘Iride’ cultivar and the ‘0114’ elite line. Therefore, this ‘Saragolla’ pure line is an enhanced variety and not a local one.

Today, a significant number of farmers in Central and Southern Italy are cultivating the old ‘Saragolla’ wheat, either for personal consumption or to establish short food supply chains of autochthonous wheat. The resurgence of interest in ‘Saragolla’ can be attributed to its adaptability to low-fertility soils and its suitability for cultivation with minimal input methods, making it especially attractive for agriculturally marginal regions. Moreover, this revived interest in ‘Saragolla’ has been magnified by online sources (Eccellenze D’abruzzo, 2024), which suggest that it can be an Italian alternative to Khorasan wheat, marketed under the name ‘Kamut’ (Piergiovanni, 2013). Kamut is a registered variety belonging to the tetraploid species Triticum turanicum Jacubz, initially described byPercival (1921) as T. orientale. This hypothesis is reinforced by the morphological resemblance between the elongated seeds of ‘Saragolla’ cultivated in the Abruzzo Region and the seeds of ‘Kamut’ T. orientale Percival.

Considering the complexity of the situation, in the years 2018–2020, the local authorities of the Italian Region Abruzzo funded the SARAB project, ‘Characterization of local Saragolla durum wheat populations’, to characterize the ‘Saragolla’ wheat currently cultivated. Through intensive cataloguing based on morphology, the project focused on the main species and botanical varieties of ‘Saragolla’ cultivated in 11 different sites within the Abruzzo Region (Rascio, Codianni, Paone, Fiorillo, & Marone, 2021). The results revealed a heterogeneous botanical composition both within and among these sites (Rascio, Fiorillo, Paone, Santis, & Sorrentino, 2022). Nine botanical varieties of durum wheat were observed, the majority belonging to the italicum or apulicum botanical varieties, primarily differing in glume pigmentation intensity. There was also a smaller number of genotypes falling into the leucurum or affine botanical varities (referred to here as Saragolla (Sar.) leucurum), which exhibited variations in seed pigmentation intensity.

The main goals of this study were to conduct genetic and morphological characterizations of representative plants belonging to the most prevalent botanical varieties collected in 11 farms that participated in the SARAB project. Additionally, these varieties were compared with a collection of both pure lines (modern) and traditional Italian (old) wheat varieties. The results here shown indicate that, in contrast to the Sar. leucurum, the italicum genotypes shared a close genetic similarity among themselves. They are widespread in the Abruzzo region and genetically distinct from the groups of modern and old genotypes examined in this study, hence they are eligible for registration as a conservation variety. The study also explores the degree of diversity of the four groups of genotypes.

Material and methods

Results

Morphological characterization

As shown in Figure 1 and in Supplemental Table 3, the Sar. italicum/apulicum-like genotypes have rather compact, hairy glumes, lightly pigmented spikes, long red or brown-red awns, yellow-amber and elongated grains. The Sar. leucurum/affine genotypes have white and glabrous glumes, elongated, compact spikes, and white or red seeds.

The mean values of each morphological and phenological trait for genotypes belonging to the four groups (Sar. leucurum, Sar. italicum, modern cultivars or old wheats) show that the Sar. italicum/apulicum genotypes registered the highest values for glume hairiness and 1,000 seed weight (Table 2). In contrast, the Sar. leucurum/affine genotypes exhibited the highest values for the shape of the lower glume beak. Modern varieties displayed smaller height and earlier heading dates, a result of the extensive breeding efforts they underwent.

Results of stepwise discriminant analysis performed using the four groups of genotypes as classification categories and visualized through the biplot of canonical variables (Figure 2) showed that the model had a high discriminatory power (Lambda Wilks: 0,0075245; approx. F (48,90) = 7,8312; p < 0,0000), with two discriminant functions that accounted for 92.9% of the explained variance (Table 3).

Based on the absolute values of standardized coefficients of the canonical variables (Table 3) the main traits that horizontally contributed to the 4-group separation were the glume hairiness, which was absent in Sar. leucurum and the upper neck glaucosity, which was lacking in Sar. italicum (Table 2). The differences in heading date along with the upper neck green glaucosity degree (Table 2) mainly contributed to the vertical separation of Sar. leucurum and italicum from the pool of new and old genotypes.

Table 3: Values of the standardized coefficients for the canonical variables included in the discriminant functions, obtained using the four groups of genotypes (Sar. leucurum, Sar. italicum, pure lines and old wheats) as classification categories.

	root 1	root 2
Glume hairiness	-0,99	-0,26
Heading date (days from 1/4)	0,05	-1,39
Upper neck glaucosity	0,73	-0,98
Growth habit	-0,48	0,64
Grain shape	-0,06	-0,50
1,000 seed weight	0,03	0,44
Spike glaucosity	-0,43	-0,05
Spike shape	-0,27	-0,08
Glume colour	0,18	-0,57
Awn colour	-0,40	0,38
Plant height	0,48	-0,17
Flag leaf lower side glaucosity	0,19	-0,49
Grain weight/plant	0,09	-0,36
Straw: pith in cross section	-0,23	0,20
Lower glume: length of beak	-0,36	-0,29
Spike density	0,21	0,31
Eigenvalue	12.4	3.4
Explained cumulative variance (%)	72.6	92.9

The Mahalanobis distances between groups were all highly significant (Table 4), except between pure lines and old wheats; Sar. leucurum and Sar. italicum were the two most distant and hence morphologically different groups.

Table 4: Pairwise square Mahalanobis distances (plain text) and probability values (italics) for the contrasts between the four groups of genotypes.

	Sar. leucurum	Sar. italicum	Pure lines	Old wheats
Sar. leucurum		0,0000	0,00036	0,00003
Sar. italicum	69,48		0,00000	0,00000
Pure lines	23,76	78,44		0,01962ns
Old wheats	27,19	68,77	20,95

Diversity

The diversity analysis for all cultivars based on SSR markers (Table 7) yielded low mean values (0.28±0.20) of Shannon’s index. The values (0.176±0.5) of Nei’s gene diversity were lower than the minimum observed in 40 winter wheat genotypes (Petrović, Marić, Čupić, Rebekić, & Rukavina, 2017) coming from European countries (Croatia, Austria, France, Italy, and Russia) and lower than that (0.56) resulting for 124 Ethiopian genotypes (Dagnaw et al., 2023).

The among-groups comparison indicated that old wheats and Sar. leucurum exhibited the highest percentage of polymorphic loci, followed by pure lines and old wheats. The measurement of gene diversity, estimated by both Nei's gene diversity and Shannon's information index, yielded similar values for Sar. leucurum, breeding lines, and old wheats, and the lowest values for Sar. italicum.

Table 7: Genetic diversity indices over 15 SSR loci for all 53 Italian genotypes tested in the study, as well as for the four groups categorized by ‘Saragolla’ botanical variety or control group (Means±SD). PIC, Polymorphic Information Content.

	Shannon's information index	Percentage of polymorphic loci	Nei's gene diversity	PIC
Sar. leucurum	0.19±0.19	71.4	0.10±0.13	0.902±0.08
Sar. italicum	0.08±0.18	22.2	0.05±0.13	0.097±0.08
Old wheats	0.17±0.22	39.7	0.10±0.14	0.929±0.10
Modern pure lines	0.19±0.19	61.1	0.11±0.12	0.764±0.11
All genotypes	0.28±0.20	97.63	0.17±0.15	0.673±0.35

In the case of Sar. leucurum, Sar. italicum, old wheats and pure lines, the highest average number of amplified alleles per locus was observed in 1B (long arm), 6B (short arm) and 6B (long arm), respectively, with average values of 9.0, 6.5, 2.5 and 2.3, respectively. It's worth noting that old wheats and modern pure lines exhibited the highest percentage of polymorphisms detected by SSR markers in the B genome (Table 8), likely originating from a species, or several species closely related to Aegilops speltoides Tausch, a cross-pollinating species; while the Sar. leucurum and Sar. italicum sets had the highest percentage of polymorphisms in the A genome, which can be traced back to diploids like T. urartu Thumanjan ex Gandilyan (Wang, Chen, & Liu, 2007).

Table 8: Percentage of polymorphism detected by SSR in A and B genomes of four groups of genotypes

	‘Saragolla’ leucurum	‘Saragolla’ italicum	Old Italian wheats	Modern pure lines
Genome A	54,1	54,5	46,2	48,5
Genome B	45,9	45,5	53,8	51,5

A similar clustering pattern was observed when analyzing both the morphological traits (Figure 2) and molecular marker profiles (Figure 3) of all 53 genotypes. This analysis employed a hierarchical grouping method, without missing data in the dataset. The resulting dendrogram revealed four major clusters (Figure 3). Cluster 1A comprised all 13 Sar. italicum genotypes, 9 out of 22 Sar. leucurum genotypes of Abruzzo and 1 Sar. leucurum genotype from Puglia. The second major cluster, 1B, could be further subdivided into two subclusters: 1B1 and 1B2. The 1B1 cluster included two subgroups: the first subgroup contained four closely related breeding lines (‘Colosseo’, ‘Simeto’, ‘Ciccio’ and ‘Capeiti’), two Sar. leucurum genotypes, and the old wheat ‘Vallelunga pubescent’; the second subgroup was larger, consisting of modern varieties (‘Duilio’, ‘Realforte’, ‘Svevo’ and the ‘Saragolla’ pure line), some old wheats (‘Russello’, ‘Scorsonera’, ‘Sammartinara’, ‘Cappelli’ and the T. turanicum line), along with eight Sar. leucurum genotypes from Puglia or Abruzzo. Cluster 1B2 included six strongly related Sar. leucurum genotypes: one was from Puglia and five from Abruzzo.

Discussion

The morphological and genetic characterization of autochthonous wheats serves the dual purpose of safeguarding the economic interests of farmers and increasing consumers’ trust in the origin and quality of food products entering the market (Terzi et al., 2005).

A recent morphological analysis conducted by the SARAB project on wheat crops in 11 farms across the Abruzzo Region revealed significant morphological diversity within the cultivated ‘Saragolla’ variety (Rascio et al., 2022). This diversity poses a challenge in accurately defining their distinct traits.

The morphotypes that are both quantitatively and widely spread, across most of the 11 wheat farms included in the SARAB project, belong to the Sar. italicum/apulicum or the Sar. leucurum/affine botanical varieties (Rascio et al., 2022). The genetic characterization described here aimed to validate whether the observed morphological similarity among the genotypes from Abruzzo corresponds to genetic similarity and to develop a tool to differentiate them.

The results presented here confirm the efficacy of SSR markers to characterize wheat genotypes (Dagnaw et al., 2023; Wang et al., 2007). In fact, the 15 SSR primers used in this experiment showed detectable polymorphisms in all the 53 genotypes and their mean polymorphic information content (PIC = 0.77) makes their use very informative. ‘Saragolla’ genotypes, belonging to the italicum/apulicum botanical varieties can be eligible as conservation varieties. These genotypes are widely cultivated across the Abruzzo Region, and they also exhibit a noteworthy genetic similarity, as indicated by the low values of Nei's gene diversity and Shannon's information index. Additionally, Sar. italicum genotypes display both a distinct phenotype and genotype in comparison to Sar. leucurum, older wheats and pure lines.

It is worth noting that Ward's clustering analysis of the 53-genotype pool revealed significant genetic diversity between Sar. italicum and Sar. leucurum genotypes. Out of the 26 Sar. leucurum genotypes examined, only 9 displayed a significant genetic resemblance to Sar. italicum. Six were categorized within the broader groups of pure lines and old wheats, while five formed a distinct group of genotypes very closely related genetically, but distinct from all others. The clustering analysis also revealed a stronger genetic similarity between most Sar. leucurum genotypes and three out of four ‘Saragolla’ genotypes from Puglia and a somewhat lesser degree of affinity with the oldest genotypes. Expanding on the hypothesis (Zeven, 1998), that factors such as geographic distance, environmental conditions and the selection made by farmers can shape the genetic composition of local wheats, it is plausible to infer that the migration of wheat commenced from Sicily. In fact, the leucurum genotypes were documented in Southern Italy as early as the beginning of the 1900s (Cillis, 1927; Draghetti, 1927; Percival, 1921) and were likely among the oldest cultivated in Sicily (Porceddu, Vannella, & Perrino, 1981). From Sicily, it is plausible that these wheats initially spread to the nearby region of Puglia and then reached Abruzzo where the cross with indigenous wheat occurred as well as the selection of alleles improving adaptability, productivity and quality. In terms of affinities with 'Kamut', the results suggest that the Sar. italicum genotypes, despite having elongated and large seeds similar to T. turanicum, formed a distinct cluster and showed a closer genetic relationship to Sar. leucurum and old durum wheats.

Conclusions

The ‘Saragolla’ wheat presently grown in the Abruzzo Region is characterized by its rich diversity, predominantly comprising Sar. italicum/apulicum and Sar. leucurum-like durum wheats, some of which share close morphological and genetic traits. Recently, these genotypes have been officially registered as ‘Saragolla’ conservation varieties from Abruzzo. The genetic distance observed among 39 ‘Saragolla’ genotypes, representative of only two out of the nine previously identified botanical varieties of durum wheat, exceeded that found among the other 13 modern, or old durum wheats used in this study, which differ for age of cultivation and origin. Consequently, at the Maiella National Park seedbank, targeted ex situ conservation measures will be implemented to preserve the currently cultivated populations. A more extensive genetic characterization will enable the assessment of existing variability within the ‘Saragolla’ landrace, for adaptive and agronomically valuable traits, useful for breeding improved varieties.

Supplemental data

Supplemental Table 1. Geographic coordinates of the cultivation sites for the 12 Saragolla wheats.

Supplemental Table 2. Passport details of old and modern wheats used in the present work.

Acknowledgements

The authors wish to thank the farm owners who provided the studied materials, Dr Maurizio Odoardi and Dr Daniela Codoni (Department of Rural Development and Fisheries Policies – Promotion of Knowledge and Innovation in Agriculture – DPD022) of the Abruzzo Regional Authorities, for their valuable contribution to the ‘SARAB project: Characterization of ancient ‘Saragolla’ populations from the Abruzzo Region’. The authors also wish to thank Mr Leonardo Morrone and Mr Vito De Gregorio for their valuable assistance in conducting the experimental trials.

Funding

This work was in part supported by the Abruzzo Region.

Author contributions

AR, study conception and manuscript draft; VDS, molecular analysis; LG, analysis and interpretation of results; SP, data collection; MT, field trials; GS, manuscript revision.

Conflict of interest statement

The authors declare no conflict of interest.