# Genetic Variation of a Lentil (_Lens culinaris_) Landrace during Three Generations of Breeding
## 4 Discussion
The selection of plants with desirable traits narrows the genetic diversity in marker loci and contributes to selecting co-inherited beneficial characteristics. Moreover, the objective of plant breeding is to combine as many favorable traits as possible in only one genotype or even increase the presence of such traits in a population [9]. Interestingly, examining allele frequencies by generation we observed that unique alleles appeared for markers SSR19, 33, 59, and 154 in 2 GSLs and then lost in 3 GSLs. This finding merits an explanation and could be due to the way heterozygous loci are detected and recorded with HRM-PCR. This analysis assumes each different melting curve as a single different allele. However, heterozygous loci are also recorded as a single allele,and allelic separation in the next generation will give rise to three different allelic HRM patterns: one for each homozygous and one for the heterozygous genotype. In such a way and due to Mendelian segregation, successive generations detect heterozygous loci from the declining genetic variability. As an example, this is evident in the analysis of HRM curves for SSR107. In 1 GSLs, electrophoresis of the PCR products indicates the presence of two alleles in homozygous and heterozygous patterns (Supplement Figure S2). Samples with the one allele (1-4, 6-7, 11-14) display a slow band, while samples 5 and 8 with the other allele display a fast band. Luckily enough, one sample (10) is heterozygous for SSR107 and displays both bands. However, HRM analysis indicates the presence of three curve patterns, which is interpreted as the presence of three alleles. In the consecutive breeding generations, no samples are deriving from family 10. Thus, a bialallelic pattern is observed in HRM and the analysis records a loss of one allele (the heterozygous curve). However, as heterozygosity in lentil is extremely low, this should not significantly affect estimation of genetic variation. Furthermore, the high selection pressure in the breeding population resulted in a small number of lines in succeeding generations with similar genetic background. Selection and fixation of the favorable alleles would decrease the genetic variation. Accordingly, a low genetic distance between GSLs is expected by the way each GSL was formed. The above hypothesis is in accordance with AMOVA, which showed that this low diversity is partitioned rather within (88%), than between (12%) GSLs, which is expected for self-pollinated species. PCoA and analysis with the STRUCTURE software revealed the structure within GSLs. Estimation of hypothetical subpopulations using K-values indicated the presence of three subpopulations that in 1 GSLs prevailed two subpopulations (blue and green colors) while at the end of the breeding (3 GSLs) prevailed the third sub-population (red color) (Figure 4). These results indicate a shift in allelic composition among the populations and the effect of breeding through GSLs. Furthermore, the highest genetic distance between 1 GSLs and 3 GSLs (0.267) could provide support for the efficiency of the breeding methodology. The observed breeding populations' composition throughout breeding cycles indicates the prevalence of selected genotypes with the desirable characteristics. In conclusion, this study provided supportive evidence for the genetic diversity of the breeding population, as was indicated by allele richness of the 1 GSLs. Selection at ultra-low density in consecutive cycles decreased genetic diversity and maintained the genetic relationships of selected plants, revealing proper management of the breeding material. The allelic composition of the improved selected plants compared to the original population was shifted, indicating the prevalence of improved genotypes in the final population. Molecular markers could provide an effective tool for proper management of allelic composition in landrace breeding to avoid extreme loss of diversity and potentially useful alleles for future variety development.