Identification of Lens Cultivars in Market by Molecular Tools: DNA Barcoding and SSRs

Substitution of plant cultivars of high commercial value with a cheaper, lower-quality one is a common fraud committed against consumers and producers. Since it is one of the most widely grown legumes, lentil (Lens culinaris Medik.) are a suitable for such frauds. This study aimed to identify lentil cultivars which are registered and authorized in the market in Turkey by using current molecular methods. For this purpose, 26 lentil cultivars were analyzed for 15 SSR markers and 2 DNA barcode loci (trnH-psbA and matK). A high allele diversity was observed by 12 scorable SSR markers, and the average number of alleles was determined to be 16. One of the important findings was the presence of “cultivar-specific alleles” that can be used to identify each cultivar in the lentil market in Turkey. At least one “cultivar-specific allele” was obtained for each cultivar. The lentil cultivars were analyzed in terms of 2 DNA barcode regions as trnH-psbA and matK. Sequences that could identify 14 of the 26 cultivars were obtained. While the rate of the intra-species variation for the trnH-psbA region was observed to be low, a higher rate was found for matK. Nevertheless, it was observed that intra-species discrimination can be made more effective when both loci are used together. We expect that the results of this study, especially the cultivar-specific SSR alleles and DNA barcoding sequence data may be used routinely to identify on production and packaged products that are commercially available in markets.

Effect of Kaempferol and Its Glycoside Derivatives on Antioxidant Status of HL-60 Cells Treated with Etoposide

Kaempferol is a well-known antioxidant found in many plants and plant-based foods. In plants, kaempferol is present mainly in the form of glycoside derivatives. In this work, we focused on determining the effect of kaempferol and its glycoside derivatives on the expression level of genes related to the reduction of oxidative stress—NFE2L2, NQO1, SOD1, SOD2, and HO-1; the enzymatic activity of superoxide dismutases; and the level of glutathione. We used HL-60 acute promyelocytic leukemia cells, which were incubated with the anticancer drug etoposide and kaempferol or one of its three glycoside derivatives isolated from the aerial parts of Lens culinaris Medik.—kaempferol 3-O-[(6-O-E-caffeoyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P2), kaempferol 3-O-[(6-O-E-p-coumaroyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P5), and kaempferol 3-O-[(6-O-E-feruloyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P7). We showed that none of the tested compounds affected NFE2L2 gene expression. Co-incubation with etoposide (1 µM) and kaempferol (10 and 50 µg/mL) leads to an increase in the expression of the HO-1 (9.49 and 9.33-fold at 10 µg/mL and 50 µg/mL, respectively), SOD1 (1.68-fold at 10 µg/mL), SOD2 (1.72-fold at 10–50 µg/mL), and NQO1 (1.84-fold at 50 µg/mL) genes in comparison to cells treated only with etoposide. The effect of kaempferol derivatives on gene expression differs depending on the derivative. All tested polyphenols increased the SOD activity in cells co-incubated with etoposide. We observed that the co-incubation of HL-60 cells with etoposide and kaempferol or derivative P7 increases the level of total glutathione in these cells. Taken together, our observations suggest that the antioxidant activity of kaempferol is related to the activation of antioxidant genes and proteins. Moreover, we observed that glycoside derivatives can have a different effect on the antioxidant cellular systems than kaempferol.

Genetic Variation of a Lentil (Lens culinaris) Landrace during Three Generations of Breeding

Genetic differentiation between 40 lentil genotypes was tested using molecular markers. The genotypes were produced from a Greek landrace of commercial interest via the honeycomb breeding methodology, i.e., single-plant selection in the absence of competition, across three successive pedigree generations. The selected genotypes from each generation were examined for genetic relationships using 15 SSR molecular markers with HRM analysis. As expected, low variation among consecutive generations at the level of 2.5–7.7% was detected. Analysis of molecular variance (AMOVA) revealed that partitioning of this variation was at higher percentage within each generation’s population than between them. Population structure analysis indicated that ongoing selection could effectively shift the allelic composition in each generation. The applied honeycomb breeding methodology that effectively improved progeny yield and seed quality increased the percentage of favorable alleles altering allelic composition but not eliminating genetic variation of the breeding population.

​Impact of Cluster Front Line Demonstrations on Productivity and Economics of Lentil (Lens culinaris L.), Chickpea (Cicer aeritinum L.) and Field Pea (Pisum sativum L.) in Old Alluvial Zone of Malda, West Bengal, India

Background: There are significant gaps in yields of pulses among potential, demonstration and farmers’ plots in West Bengal. This is mostly due to adoption of traditional local varieties and poor cultivation practices. Methods: The yield, technology and extension gaps were studied on improved varieties of lentil, chickpea and field pea during rabi seasons of 2017/18-2020/21 in 80 hectare area of Malda, West Bengal with 205 farmers. Result: The highest and lowest magnitude of technology index (TI) was observed in field pea var. IPFD-10-12 (44.5%) during 2018/19 and in lentil var. KL-320 (30.1%) during 2020/21. Among lentil varieties, the highest benefit cost ratio was obtained from KL-320 (2.87) whereas in case of chickpea, it was obtained from NBEG-49 (2.90). For field pea var. IPFD-10-12 the benefit cost ratio was 2.70. Maximum and minimum extension yield gaps were observed in field pea var. IPFD-10-12 (451 kg ha-1) during 2018/19 and in lentil var. IPL-406 (145 kg ha-1) during 2018/19. Overall assessment reflects lentil var. KL-320, chickpea variety NBEG-49 and field pea variety IPFD-10-12 have good potentiality and most suitable for this region. So, it is hypothesized that improved farm technology has enormous potential to scale up pulse productivity through cluster front line demonstration (CFLD).

Insights into the Host-Pathogen Interaction Pathways through RNA-Seq Analysis of Lens culinaris Medik. in Response to Rhizoctonia bataticola Infection

Dry root rot (Rhizoctonia bataticola) is an important disease of lentils (Lens culinaris Medik.).To gain an insight into the molecular aspects of host-pathogen interactions, the RNA-seq approach was used in lentils following inoculation with R.bataticola. The RNA-Seq has generated >450 million high-quality reads (HQRs) and nearly 96.97% were properly aligned to the reference genome. Very high similarity in FPKM (fragments per kilobase of exon per million mapped fragments) values (R > 0.9) among biological replicates showed the consistency of the RNA-Seq results. The study revealed various DEGs (differentially expressed genes) that were associated with changes in phenolic compounds, transcription factors (TFs), antioxidants, receptor kinases, hormone signals which corresponded to the cell wall modification enzymes, defense-related metabolites, and jasmonic acid (JA)/ethylene (ET) pathways. Gene ontology (GO) categorization also showed similar kinds of significantly enriched similar GO terms. Interestingly, of the total unigenes (42,606), 12,648 got assembled and showed significant hit with Rhizoctonia species. String analysis also revealed the role of various disease responsive proteins viz., LRR family proteins, LRR-RLKs, protein kinases, etc. in the host-pathogen interaction. Insilico validation analysis was performed using Genevestigator® and DEGs belonging to six major defense-response groups viz., defense-related enzymes, disease responsive genes, hormones, kinases, PR (pathogenesis related) proteins, and TFs were validated. For the first time some key miRNA targets viz. miR156, miR159, miR167, miR169, and miR482 were identified from the studied transcriptome, which may have some vital role in Rhizoctonia-based responses in lentils. The study has revealed the molecular mechanisms of the lentil/R.bataticola interactions and also provided a theoretical approach for the development of lentil genotypes resistant to R.bataticola.

Enrichment of Zinc and Iron Micronutrients in Lentil (Lens culinaris Medik.) through Biofortification

Biofortification of pulse crops with Zn and Fe is a viable approach to combat their widespread deficiencies in humans. Lentil (Lens culinaris Medik.) is a widely consumed edible crop possessing a high level of Zn and Fe micronutrients. Thus, the present study was conducted to examine the influence of foliar application of Zn and Fe on productivity, concentration, uptake and the economics of lentil cultivation (LL 931). For this, different treatment combinations of ZnSO4·7H2O (0.5%) and FeSO4·7H2O (0.5%), along with the recommended dose of fertilizer (RDF), were applied to the lentil. The results of study reported that the combined foliar application of ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at pre-flowering (S1) and pod formation (S2) stages was most effective in enhancing grain and straw yield, Zn and Fe concentration, and uptake. However, the outcome of this treatment was statistically on par with the results obtained under the treatment ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at S1 stage. A single spray of ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at S1 stage enhanced the grain and straw yield up to 39.6% and 51.8%, respectively. Similarly, Zn and Fe concentrations showed enhancement in grain (10.9% and 20.4%, respectively) and straw (27.5% and 27.6% respectively) of the lentil. The increase in Zn and Fe uptake by grain was 54.8% and 68.0%, respectively, whereas uptake by straw was 93.6% and 93.7%, respectively. Also the benefit:cost was the highest (1.96) with application of ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at S1 stage. Conclusively, the combined use of ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at S1 stage can contribute significantly towards yield, Zn and Fe concentration, as well as uptake and the economic returns of lentil to remediate the Zn and Fe deficiency.

Midyat Yerel Kırmızı Mercimeği (Lens culinaris Medik.) Konusunda Ön Çalışma

Bu çalışma geçmişi yüzyıllara dayanan, tat, aroma bakımından üstün özellikler taşıması dolayısıyla Mardin ili ve çevresinde yaşayan tüketicilerce en çok tercih edilen Midyat Kırmızı Mercimek Yerel çeşidinin bazı tarımsal karakterler ve kalite özellikleri bakımından karakterize edilmesi amacıyla yapılmıştır. Deneme 2020-21 yetiştirme sezonunda Kızıltepe ilçesi çiftçi tarlasında 2 tekerrürlü olarak yürütülmüştür. Varyans analiz sonuçlarına göre incelenen tüm özellikler için genotipler arasındaki fark istatistiki olarak önemli bulunmuştur. Tane verimi bakımından Fırat-87 çeşidi 250 kg/da ilk sırada yer alırken ve Midyat-1 populasyonu ise 220 kg/da ile diğer standart çeşitlere yakın değere sahip olmuştur. Yerel popülasyonlar standart çeşitlere göre geç çiçeklenme ve olgunlaşma özelliklerine sahip olurken, ilk bakla yüksekliği, bitki boyu bakımından standart çeşitlerden daha düşük değerlere sahip oldukları tespit edilmiştir. Midyat- 1 populasyonu kuru ağırlık (5.05 g), Midyat-2 populasyonu yaş ağırlık (9.55 g) , Midyat-3 populasyonu kuru hacim (58.0 ml) ve yaş hacim (112.5 ml) özellikleri bakımından tüm standart çeşitlerden daha yüksek değerlere sahip olmuşlardır. Elde edilen sonuçlar, Midyat yerel kırmızı mercimek çeşidinin kaliteli kırmızı mercimek çeşitlerinin geliştirilmesi için büyük bir potansiyele sahip olduğunu ve ıslah çalışmalarında direk olarak veya genitör olarak kullanılabilecek değerli bir genetik kaynak olduğunu göstermiştir.