Bio-fortification potential of global wild annual lentil core collection

Lentil, generally known as poor man’s’ meat due to its high protein value is also a good source of dietary fiber, antioxidants and vitamins along with fast cooking characteristics. It could be used globally as a staple food crop to eradicate hidden hunger, if this nutritionally rich crop is further enriched with essential minerals. This requires identification of essential mineral rich germplasm. So, in the present study, a core set of 96 wild accessions extracted from 405 global wild annual collections comprising different species was analyzed to determine its bio-fortification potential. Impressive variation (mg/100 g) was observed for different minerals including Na (30–318), K (138.29–1578), P (37.50–593.75), Ca (4.74–188.75), Mg (15–159), Fe (2.82–14.12), Zn (1.29–12.62), Cu (0.5–7.12), Mn (1.22–9.99), Mo (1.02–11.89), Ni (0.16–3.49), Pb (0.01–0.58), Cd (0–0.03), Co (0–0.63) and As (0–0.02). Hierarchical clustering revealed high intra- and inter-specific variability. Further, correlation study showed positive significant association among minerals and between minerals including agro-morphological traits. Accessions representation from Turkey and Syria had maximum variability for different minerals. Diversity analysis exhibited wide geographical variations across gene-pool in core set. Potential use of the identified trait-specific genetic resources could be initial genetic material, for genetic base broadening and biofortification of cultivated lentil.


Introduction
On earth, at least half the human population do not get sufficient daily amounts of essential minerals, a condition leading to 'hidden hunger' either due to their low concentrations in commonly eaten staple food crops or their reduced bioavailability [1]. Among minerals, deficiencies specifically of iron exists within nearly 3.7 billion people worldwide comprising about 60% of the whole population [2], whereas zinc affecting about one-third of the world population [3], are the most common. Adequate amounts of copper, manganese, molybdenum, cobalt, nickel, sodium and potassium are also required in body as these act as components of a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Mineral analysis
Sodium (Na) and potassium (K) were determined using flame photometer 128 (Systronics), phosphorus (P) by using spectrophotometer DR5000 (HACH). Calcium (Ca) and magnesium (Mg) were determined by using Atomic Absorption spectrophotometer (AnalytiKjena ZEEnit 700, Germany). Other mineral elements namely, iron, copper, zinc, manganese, molybdenum, nickel, lead, cadmium, cobalt and arsenic were determined using ICP-Mass Spectrophotometer (NexIon 1 300X Series, Perkin-Elmer). All the standards of different mineral elements used in the study were of analytical grade. All the observations were recorded in two replications.

Statistical and diversity analysis
Descriptive statistics analysis was done using Windows 2007 Microsoft Excel Data Analysis Pack [15]. Phenotypic coefficient of variation (PCV) for different elements were calculated as PCV = p V P / mean x 100as per Burton [16]. Genetic diversity among accessions based on elemental composition data was determined from the Euclidean distances. Hierarchical clustering and correlation analysis along with Principal Component Analysis (PCA) was carried out using SAS software [17].
The maximum variability for Ca content was observed in L. nigricans with 10.47 mg/100g (EC718266) to 188.75 mg/100 g (ILWL9) whereas, minimum variability was observed in L. culinaris ssp. culinaris from 4.74mg/100 g (ILL8006) to 5.63 mg/100 g (ILL10829) followed by L. culinaris ssp. odemensis with 8.48 mg/100 g (ILWL357) to 37.34 mg/100 g (ILWL35). Accessions ILWL9 of L. nigricans from Syria and ILWL278 and ILWL343 of L. culinaris ssp. orientalis from Turkey and Syria, had Ca content >100 mg/100 g with 188.75, 136.63, and 113.37 mg/100 g, respectively. Species L. ervoides and L. culinaris ssp. orientalis showed substantial variability with 36.25 to 158.33 and 46 to 158 mg/100 g, respectively, for the Mg  Table 3. In case of Zn, maximum variability was observed in L. culinaris ssp. orientalis followed by L. culinaris ssp. odemensis as evident from the Table 2. Accessions from L. lamottei and L. nigricans showed almost identical range for Zn with 4.12 to 12.60 and  Table 3.

Diversity analysis
Diversity analysis was done using data of all the 15 minerals analysed in the study. Euclidean distances ranged from 0.845 to 14.291 as given in S2 Table. The maximum distance of 14.291 was observed between accessions ILWL7 from Turkey and ILWL227 from Syria of L. culinaris ssp. orientalis, which was followed by L. culinaris ssp. orientalis accessions ILWL227 from Syria and ILWL278 from Turkey with a value of 11.248, ILWL7and ILWL101, both from Turkey with a distance value of 10.518, and ILWL278 of L. culinaris ssp. orientalis from Turkey and ILWL16 of L. nigricans from Alpes-Cote d'Azur with 8.268. The minimum distance or maximum similarity was observed between ILWL95 of L. culinaris ssp. orientalis and ILWL35 of L. culinaris ssp. odemensis, both from Turkey with a Euclidean distance of 0.845 followed by L. nigricans accessions ILWL19 from Spain and EC718270 from Croatia with Euclidean distance of 0.90, and ILWL75 of L. culinaris ssp. orientalis from Israel and ILWL198 of L. culinaris ssp. tomentosus from Syria with a distance of 1.238. The hierarchical clustering of accessions resulted into9different clusters A-J as shown in different colors in Fig 1. All the clusters occupied by the accessions of different species, but in some clusters majority of the accessions were observed species specific. Cluster C was mainly occupied by L. nigricans, E by L. culinaris ssp. odemensis, F by L. ervoides and L. culinaris ssp. culinaris, G by L. ervoides, H by L. culinaris ssp. orientalis. ILWL384 forms a separate cluster I. The clustering pattern indicates that grouping of accessions was not according to their geographical origins as shown in S2 Table.

Principal Component Analysis
Principal Component Analysis was used to assess the patterns of variation by considering all variables simultaneously. First four components have eigen value >1.0 which together explained 65.09 per cent of total variability Table 4. PC 1 accounted for 23.2% of the total variation, and, Mn, Cd, Cu and Mo had the highest positive coefficients means that they were the  Table 4. Furthermore, the two dimensional scattering of core set accessions according to PCA and various traits contributing to the variation are shown in Fig 2a and 2b, respectively.

Association between mineral elements and with agro-morphological traits
The correlation coefficients among the different mineral elements and with agro-morphological traits are shown in S3 Table. Correlation analysis indicated numerous significant positive and negative correlations. As test power increased with large number of observations, giving

Elemental composition of core set accessions
The wild annual lentil core accessions used in the present study developed from the global wild lentil germplasm accessions introduced from twenty seven counties [12]. An idea was to study the core set accessions for detailed evaluation, which seems to be much useful from utilization point of view. Further, it makes easy to handle reduced number of accessions for detailed studies including biochemical, physiological and molecular analysis. Earlier also, core and mini core collections have been evaluated for nutritional traits [18,19]. The accessions showed high variability for different minerals because core set includes accessions from five different wild species belonging to the diverse originswith different genetic backgrounds. Further, core set retains the genetic diversity of the whole population from which it is developed [20]. The partitioning of mineral elements is affected by grain morphology including grain size, embryo size, and thickness of the tissue layers. In wild and cultivated wheat, variability in grain for Fe and Zn contents have been found related to allelic variation at a chromosomal locus involved in remobilization of protein, Fe, Zn and Mn from senescing leaves to seeds [21]. Recently, Gupta et al. [22] developed markers for some iron metabolism related genes (Ferritin-1, BHLH-1, IRT-1) in lentil. Thus, the intra-as well as interspecific variability observed for seed elemental composition in the present study also highlights the hidden role of tight genetic control besides influence of plant morphology. Thus, the data generated provides a valuable resource for mapping studies to identify genes or points of regulation for elemental variation.
In general, species L. nigricans and L. culinaris ssp. orientalis showed wide variability for Ca content, whereas L. ervoides and L. culinaris ssp. orientalis, for Mg content. Species L. culinaris ssp. odemensis, L. culinaris ssp. orientalis and L. ervoides showed remarkable variability for different minerals viz. Fe, Zn, Cu, Mn and Mo. This may be occurred due to diverse genetic makeup of these species or may be due to the sound expression of respective genes of particular species under given environmental conditions. However, L. culinaris ssp. culinaris had the minimum variability for some of the minerals as it is a cultivated species. Singh et al. [23] revealed significant effects for genotype, environment and genotype by environment interaction for both grain Fe and Zn concentration in 50 lentil accessions.
In the present study, Ni, Pb, Cd, Co and As were found almost within recommended range except two-three accessions ( Table 2). The data suggests that wild Lens accessions are not good accumulators of these minerals. Leshe and Tessema [26] reported mineral composition of Ethiopian lentil samples in the range Ni (0.120 to 0.244 mg/100 g), Pb (0.142 to 0.176 mg/ 100 g), Cd (0.009 to 0.013 mg/100 g) and Co (0.285 to 0.360 mg/100 g).
The Comparison of mineral data of lentil wild species obtained in the present study with that of the literature values of cultivated material in S1 Table highlighted that wild species are rich source of various minerals. The variation reported from earlier studies could be due to diverse genetic constitution, species-specificity, origin and environmental differences. These untapped gene sources identified in the present study can be hybridized with cultivated lentil varieties for genetic improvement of nutritional traits [28]. Earlier also, value rich accessions have been identified for various minerals and other nutritional constituents viz. 10 accessions each for high iron and zinc in sorghum seed, 1 accession for iron and 2 accessions for zinc in pearl millet seed, 10 accessions each for zinc, iron, protein, calcium, and beta-carotene contents in finger millet, 5 accessions for high seed protein in chickpea, 14 accessions for zinc in pigeon pea seed [18]. Upadhyaya et al. [19] also carried out evaluation studies of core collection of foxtail millet and obtained protein content in the range of 10.7 to 18.5%, calcium from 90.3 to 288.7 mg/kg, iron from 24.1 to 68.0 mg/kg and zinc from 33.6 to 74.2 mg/kg.

Diversity among core set accessions
A high level of variation was obtained for various mineral nutrients as revealed by the dendrogram. Results revealed that maximum variability among accessions was inter-specific and not limited to a particular country. The maximum diversity was observed among the accessions with origin from Syria and Turkey. The Euclidian distances calculated based on data from 0.845 to 14.291 showed large genetic distances among the accessions. Vieira et al. [29] reported genetic distances up to 196.61 in 19 wheat accessions analyzed for 17 phenotypic characters. A large genetic distance between heterotic germplasm can be useful for developing lines with good combining ability in hybrid breeding [30]. Earlier also, based on morphological and molecular studies, it has been established that Turkish lentil landraces had substantial genetic diversity at the genotypic and phenotypic levels [31]. Singh et al. [12] also reported maximum genetic diversity among wild Lens accessions with Turkey origin. Singh et al. [23] analyzed 50 lentil accessions for Fe and Zn content along with diversity analysis using 20 genomic and 54 EST-SSR markers which revealed role of geographic origin in diversity. The knowledge of the genetic diversity among accessions provides clues about the wider genetic base of accessions/ species and that can be exploited through wide hybridization in order to develop polymorphic populations for various traits of interest.

Principal Component Analysis
In present study of mineral composition of wild Lens accessions, PCA has been used to assess the pattern of variations among wild lentil accessions by considering all variables simultaneously where first four principal components accounted for 65.09% of the total variability. Similar findings were observed by Karakoy et al. [32] where first four PCs accounted for 79.45% of the variability. They reported PC1 accounted for 36.90% of the total variation, and P, Zn, Mg, and K had the highest positive coefficients, whereas, PC2 explained 20.38% of the total variation, and seed size, 100-seed weight, Mn, and Cu had the highest positive coefficients.

Association studies between mineral elements and agro-morphological traits
The concentrations of several minerals, particularly Fe, Cu, Mn and Mo, were positively correlated with other minerals suggesting that similar pathways or transporters control the uptake and transport of these minerals. Karakoy et al. [32] reported similar observation for Zn in lentil samples. Cabrera et al. [33] also found a direct statistical correlation between Cu-Cr, Zn-Al and Cr-Ni and Al-Pb. The positive correlations among various minerals suggest the possibility of breeding for increased concentrations of these elements simultaneously. The variations in results from earlier studies can be explained on the basis that mineral composition is influenced by a number of factors, genetic background of the materials, and environmental adaptability [28].

Conclusions and future perspectives
High variability was recorded for various essential minerals in wild lentil core accessions developed from global collection. Species L. culinaris ssp. odemensis, L. culinaris ssp. orientalis and L. ervoides showed remarkable variability for different minerals viz. Fe, Zn, Cu, Mn and Mo. In general, species, L. nigricans and L. culinaris ssp. orientalis showed wide variability for Ca content, whereas L. ervoides and L. culinaris ssp. orientalis, for Mg content. Accessions belonging to Turkey and Syria region showed maximum diversity for various minerals. The results highlight the underlying tight genetic control of physiological and molecular mechanisms involved in the mineral portioning. The genetic diversity analysis revealed intra-as well as inter-specific variability and relationships among the wild annual Lens core set accessions. Recently, some initiatives have been made for improvement of domesticated lentil through bio-fortification of minerals using cross breeding approach. But efficient pre-breeding efforts for utilizing wild Lens species for mineral bio-fortification of domesticated lentil improvement have not been undertaken at the global level. The present study clearly indicates the potential of wild lentil species through identification of essential mineral rich accessions including ILWL15, ILWL480 and ILWL401 etc. which can be used for bio-fortification of cultivated lentil using appropriate cross breeding techniques.
Supporting information S1