# Genetic Variation of a Lentil (_Lens culinaris_) Landrace during Three Generations of Breeding
## 4 Discussion  
The objective of this study was to examine genetic variation within and between pedigree sister lines derived from the commercially cultivated lentil landrace \"Evros\", using SSR molecular markers and HRM analysis. The 15 SSR molecular markers applied with HRM analysis based on melting curves and difference plot of the markers efficiently discriminated the 40 lentil lines according their genealogy. Researchers have used molecular markers to study genetic diversity in lentil and other legumes [22; 23; 24; 25; 26], but genetic differentiation among successive generations of breeding populations has been considered granted and has not been thoroughly investigated. Generally, it is considered that genetic diversity decreases through the breeding of landraces and their transformation to inbred varieties after the first cycle of breeding. The transient fluctuations in the variability led to the bottleneck effect, possibly due to industry and consumers' preferences of seed quality characteristics [27]. All plant species went through two major bottlenecks, the first during domestication and the second through breeding [28]. However, some breeding programs reversed the bottleneck effect by increasing the level of genetic variation. According to studies, genetic diversity increased through breeding over the years or at certain breeding cycles [29; 30; 31; 32; 33]. In addition, there are studies where genetic diversity through breeding is preserved, such as in wheat, barley, maize, and flax [34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44] while others indicated that genetic diversity decreases, such as in wheat and maize [45; 46; 47; 48; 49]. Several studies examining lentil polymorphism using agromorphological traits, molecular markers and imaging analysis indicate that genetic variation between lentil species, varieties, and within or between landraces is high [50; 51; 52; 53; 54; 55]. Even if genetic variation during breeding decreases, there is evidence that this reduction in variation is minor while the allelic reduction at individual chromosomal segments is significant [10]. In this study, the level of diversity of the 14 lentil families was high enough as determined by the presence of 48 alleles, indicating adequate variation in the \"Evros\" landrace to encourage the breeding effort. Although the progenies were selected from a limited number of mother plant-families in successive breeding generations, allelic variation was not diminished. Lost alleles in the second cycle were at the level of 18.75%, while at the end of the third cycle 29.1% of allelic richness (14 alleles) of IGSLs was lost. Considering that the final selection (3 GSLs) discarded 11 out of the 14 initially selected plants (IGSLs), allelic richness was not substantially affected. This could be due to similarities of the discarded families with the selected families 2 and 15, as revealed in the UPGMA dendrogram and PCoA analysis. As evident in Figures 3 and 4, the unique genotypic category that is not represented in the 3 GSLs, is the family 5. Diversification of this family is characteristic and could point to the utility of molecular markers in better management of genetic diversity during selection. This family could be included in the successive generations to avoid further loss of genetic diversity in the breeding population. Family 8 and its progeny are grouped in a separate clade, and the rest of 1 GSLs are grouped with families 2 and 15, indicating a common pool of alleles in these materials. Crossing genetically different plants leads to significant genetic diversity, which could be further material for selection. In such a way, breeders can develop uniform new varieties, which replace polymorphic landraces [9]. Selection within a landrace may similarly result in uniform new varieties. Our study found that genetic diversity through breeding a landrace decreased but not diminished.