Genetic polymorphism assessment in a new lentil (Lens culinaris Medik., 1787) collection using ISSR markers

Аннотация

ISSR markers were used in the study of the genetic diversity of 46 imported lentil varieties. The lentil collection had a strikingly high level of polymorphism (76%). The UBC 810 and UBC 809 primers have the highest polymorphism rates among the primers, exceeding 85.7%. The use of nine ISSR markers resulted in 69 pieces, with 76% displaying polymorphism. The computed average genetic diversity index ranged from 0.56 to 0.81, indicating a wide range of genetic variation among lentil genotypes. Accessions were classified into six unique groups as a consequence of cluster analysis. The most divergent genotypes within their respective clusters were identified as Flip 2010-96 and Flip 2011-41, Flip 2011-32 and Flip 2011-97, 10932 and Flip 2011-20, and Flip 2010-81 and Flip 2011-19. These findings bear noteworthy implications for the future of lentil breeding, cultivation, and protection. The observed genetic diversity imparts valuable insights that can be harnessed to fortify lentil crops, fostering resilience and adaptability. The identified distant genotypes present promising avenues for targeted breeding initiatives, facilitating the development of lentil varieties harboring diverse and desirable traits. In summation, this study contributes pivotal information to the scientific community, establishing a framework for subsequent research and progress in the improvement of lentil crops.

Acta Biologica Sibirica 9: 1181–1188 (2023) doi: 10.5281/zenodo.10421062

Corresponding author: Saltanat A. Aghayeva (saltanat.genetic@wcu.edu.az)

Academic editor: R. Yakovlev | Received 1 December 2023 | Accepted 18 December 2023 | Published 24 December 2023

http://zoobank.org/E1C7240C-F0D0-49C0-8122-E5FE8217FABD

Citation: Mammadova SE, Aghayeva SA (2023) Genetic polymorphism assessment in a new lentil (Lens culinaris Medik., 1787) collection using ISSR markers. Acta Biologica Sibirica 9: 1181–1188. https://doi.org/10.5281/zenodo.10421062

Keywords

ISSR marker, lentil, genotype, Lens culinaris Medik., polymorphism

Introduction

Lentil (Lens culinaris Medic.) is one of the main legume crops cultivated in the semi-arid areas of the world and in Azerbaijan. With high levels of protein, minerals and vitamins, this plant meets the nutritional needs of millions of people around the world. Currently, lentils grown in more than seventy and consumed in more than 120 countries (Erskine et al. 2011). ISSR markers have been used in genetic research of many plants, including legumes (Bornet and Branchard 2001; Rajesh et al. 2003; Tahir et al. 2011; Bhareti et al. 2012 and Wang et al. 2012). Annual diploid lentil is extremely important in human nutrition due to its high protein content (25%), high fiber content (87%) and low fat (Bosmali et al. 2012). Recently, people pay attention to their health and are likely to consume more plant protein than animal proteins. According to FOASTAT 2014, global lentil production increased almost fivefold in the last 50 years (Kumar et al. 2015).

As elsewhere in the world, lentils in Azerbaijan have a special place in providing food security. The main challenges in this area are the creation of new lentil collections with high-quality, resistant to abiotic and biotic stresses and their use in farms, especially in the arid farming systems of the republic.

In the Caucasus region, the first gene pool was established at the Genetic Resources Institute in Azerbaijan, where 256 landraces and introduced breeding species of lentil are mainly conserved (Babayeva et al. 2018). In order to increase the effectiveness of these collections in breeding programs, molecular-genetic characterization is also needed. Molecular markers are an important tool for the detection of polymorphism at the DNA level, for study of nucleotide sequences characteristic for genome analysis, localized in close to genes, managing the phenotype of any trait and not affected by environmental factors.

Study on polymorphism is not only important for determining genetic diversity, but also for finding alleles useful for possible progress in future breeding programs. To date, polymorphism in lentil plants has been investigated by various molecular markers (Fikiru 2007; Gupta et al. 2012; Bosmali 2012; Babayeva 2018). The ISSR (simple sequenced) markers are technically simple and can detect changes in both coding and non-coding sections of the genome (Singh et al. 2002). The study was conducted to evaluate the genetic diversity of landraces and introduced lentil genotypes and to study the accessions important for breeding (Mamedova et al. 2022).

Materials and methods

A total of 46 lentil genotypes, introduced from ICARDA gene bank, were used in this study (Table 1). The rest accessions represent local landraces collected from different regions of Azerbaijan and improved local varieties, which were obtained by selection from ICARDA introductions in different years.

Genomic DNA was extracted from fresh leaves using CTAB protocol by Doyle and Doyle (1987). PCR reactions for ISSR primers were performed in a 20 μl, con- taining 2 μl 10x PCR buffer; 2 μl mixture dNTP (5 mM); 1.5 μl MgCl2 (50mM); 2 μl of each primer (15 pmol/ μl); 0.1 μl of Taq-polymerase enzyme (1 U/ μl) and 2 μl of extracted DNA (50 ng/ μl). The Thermal Cycler (Applied Biosystems, USA) for ISSR markers was programmed as: pre-denaturation at 94°C for 5 minutes; 35 cycles of – denaturation at 94°C for 1 min, annealing for 45 seconds (temperature depended on the primer used), elongation for 5 minutes at 72°C; the final elongation at 72°C for 10 minutes. PCR products were analyzed by agarose gel electrophoresis, following ethidium bromide staining and visualized under UV light using gel documentation system BioRad. The band size was determined by using Photo-Capt version 12.4 with reference to standard 100 bp ladder. ISSR bands were presented in a matrix form of binary data, in which presence or absence of PCR fragments was considered as 1 and 0 respectively. All analysis was performed using the SPSS 16.0 statistical package (SPSS/PC-16, SPSS Inc., Chicago, IL, USA; http://www.spss.com). The genetic diversity index (GDI) (Weir 1990), polymorphism information content (PIC) (Roldan-Ruiz et al. 2000), effective multiplex ratio (EMR), marker index (MI) (Powell et al. 1994), resolution power (RP) and mean resolution power (MRP) (Prevost et al. 1999; El-Nahas et al. 2011) were calculated for the analyses.

Results and Discussion

This study used ISSR markers to evaluate the diversity and relationship between lentil genotypes. 3 of the used 15 ISSR markers, were monomorphic. The research was continued with 9 ISSR markers, providing polymorphic and clear bands. A total of 69 bands were created in 46 lentil genotypes using 9 ISSR primers, and 52 of them have been polymorphic (Table 1). The molecular size of the amplicons ranged between 100 and 1000 bp. The number of bands for each primer was between 6 and 10, on the average was 8, the highest number of bands have been observed in UBC 827 and the lowest number of bands have been in UBC 823 and UBC 812 (Table 1). In other studies, ISSR markers have been used successfully to evaluate genetic diversity in lentil accessions (Fikiru et al. 2007).

In all collection, the polymorphism was 76%. The highest polymorphism was recorded in UBC 810 and UBC 809 primers (> 85.7%) (Table 2). For comparison, in studied lentil collections, El-Nahas et al. (2011) reported 46% polymorphism, Babayeva et al. (2018) 84%, Duran et al. (2004) 98.8% polymorphism. The highest value of the Genetic Diversity Index among the 10 markers was recorded in the UBC 809 (0.77) and the lowest GDI was recorded in the UBC 812 (0.56) primer. The average genetic diversity in the collection was 0.67. the PIC values, depending on the number of detected alleles and their frequency distribution ranged between 0.15 and 0.56, the average value was 0.25.

Genetic Distance and Cluster Analysis: Cluster analysis has created a dendrogram that divides genotypes into 6 major groups (Fig. 1). Genetic distance values range between 0.07 (for 44% of the average collection) and 0.05, which supports the narrow genetic database of the collection again. The genotypes showing 100% genetic similarity among themselves, were not recorded. The high genetic similarity was noted between Arzu and Flip 2011-36 and between 10932 and 10946. Thus, the study has revealed a high genetic similarity between the introduced lentil accessions.

Figure 1.ISSR profile of 46 lentil genotypes generated by UBC 818.

The first cluster consists of 6, the second cluster 18, the third cluster 11, the fourth cluster 7, each of the fifth and sixth clusters consist of two genotypes. Six genotypes have been located in group I, and the genetic distance between 10932 and 10946 genotypes is very close (0.25). The cluster II containing the most accessions has been divided into four subgroups, and the 10934 and 10942 genotypes were located in a separate subgroups. The GD value between these genotypes and other accessions was 0.86 and 0.78, respectively. The III cluster consisted of 11 accessions has been also divided into three subclusters. Flip 2011-29, Flip 2011-37, 10929, 10930 have created the sub-cluster A, and Flip 2011-36, Jasmine, F.2011-26, F.2011- 19, F.2010-81, F.2010-91, F.201094 was located in this subcluster (B). Improved variety Jasmine, located with Flip 2011-36 in the third cluster has shown the highest genetic similarity (GD = 0.21). In cluster III, the genetic distance values between Jasmine and the other accessions were 0.68-0.74. The lowest genetic distance value (GD = 0.49) among genotypes in cluster IV consisted of 7 accessions was found between F.2010-95 and F.2010-10 (Fig. 2).

Figure 2.UPGMA dendrogram based on Jaccard dissimilarity coeficient in 46 lentil genotypes.

The genetic distance value between genotypes of the fifth and sixth clusters was 0.69, and indicated their difference.

In our study, cluster analysis could distinguish distant genotypes, as well as closely related genotypes. Flip 2010-96 and Flip 2011-41, Flip 2011-32, and Flip 2011-97, 10932, and Flip 2011-20, Flip 2010-81, and Flip 2011-19 have been evaluated as the most distant genotypes. The genotypes of Australian origin were grouped into the same group, which was also reported in our previous study (Babayeva et al. 2009). The released variety Jasmine of Azerbaijan and the genotype introduced by ICARDA showed high genetic similarity, which may be explained by the fact that Arzu variety is ICARDA origin.

The study approved the high genetic diversity at the molecular level in the lentil collection. Acquired knowledge about the diversity of genotypes will increase the efficiency of their utilization in the breeding process and accelerate breeding work to create new varieties adapted to the country's environmental and geographical conditions.

References

Babayeva S, Akparov Z, Abbasov M, Mammadov A, Zaifizadeh M, Street K (2009) Diversity analysis of Central Asia and Caucasian lentil (Lens culinaris Medikus) germplasm using SSR fingerprinting. Genetic Resources and Crop Evolution 56(3): 293–298. http://dx.doi.org/10.1007/s10722-009-9414-6

Babayeva S, Akparov Z, Amirov L, Shikhaliyeva K, Hasanova S, Rustamov K, Mirzayev R, Izzatullayeva V, Mirzaliyeva I, Mammadov A, Abbasov M (2018) Genetic relationship among introduced lentil germplasm using agronomic traits and ISSR markers. Genetika 50(2): 575–590. http://dx.doi.org/10.2298/GENSR1802575B

Bhareti P, Singh DP, Khulbe RK (2012) Genetic diversity in urdbean [Vigna mungo (L.) Hepper] revealed by ISSR markers. Journal Food Legumes 25(2): 89–93.

Bornet B, Branchard M (2001) Nonanchored Inter Simple Sequence Repeat (ISSR) markers: reproducible and specific tools for genome fingerprinting. Plant Molecular Biology Reporter 19: 209–215. https://doi.org/10.1007/BF02772892

Bosmali I, Ganopoulos I, Madesis P, Tsaftaris A (2012) Microsatellite and DNA-barcode regions typing combined with high resolution melting (HRM) analysis for food forensic uses: a case study on lentils (Lensc ulinaris). Food Research International 46: 141–147. https://doi.org/10.1016/j.foodres.2011.12.013

El-Nahas AI, El-Shazly HH, Ahmed SM, Omran AAA (2011) Molecular and biochemical markers in some lentil (Lens culinaris Medik.) genotypes. Annals of Agricultural Science 56(2): 105−112. https://doi.org/10.1016/j.aoas.2011.11.001

Erskine W, Sarker A (2004) Lentil. Encyclopedia of Grain Sciences. Elsevier publication, London, UK, 142–150 pp.

Fikiru E, Tesfaye K, Bekele E (2007) Genetic diversity and population structure of Ethiopian lentil (Lens culinaris Medikus) landraces as revealed by ISSR marker. African Journal of Biotechnology 6: 1460–1468.

Gupta M, Verma B, Kumar N, Chahota RK, Rathour R, Sharma SK, Bhatia S, Sharma TR (2012) Construction of intersubspecific molecular genetic map of lentil based on ISSR, RAPD and SSR markers. Journal Genetics 91(3): 279–287. https://doi.org/10.1007/s12041-012-0180-4

Kumar, S.S. Singh, Praveen (2015) Stability by Additive Main Multiplicative Interaction (AMMI) model & genetic diversity studiesin micro macro-sperma lentil (Lens culinaris L) in mid hills of Jammu Kashmir, India. Legume Research - An International Journal 40(4): 635–638. https://doi.org/10.18805/lr.v0i0.7595

Joshi M, Verma SK, Singh JP, Barh A (2013) Genetic Diversity Assessment In Lentil (Lens Culinaris Medikus) Genotypes Through ISSR Marker. Supplement on Genetics & Plant Breeding w8(4): 1529–1532.

Prevost A, Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theoretical and applied Genetics 98: 107–112. https://doi.org/10.1007/s001220051046

Rajesh PN, Sant VJ, Gupta VS, Muehlbauer FJ, Ranjekar PK (2003) Genetic relationships among annual and perennial wild species of Cicer using inter simple sequence repeat (ISSR) polymorphism. Euphytica 129: 15–23. https://doi.org/10.1023/A:1021567821141

Mamedova S, Gasanova S, Agaeva S (2022) Sravnitel'noe issledovanie pokazatelej produktivnosti novoj kollekcii chechevicy na osnove strukturnogo analiza. Nauka i innovacii 1(7): 68–73. https://doi.org/10.29235/1818-9857-2022-7-68-73

Tahir NA, Hamakareem HFH (2011) Determination of Genetic Relationship among Some Varieties of Chickpea (Cicer arietinum L) in Sulaimani by RAPD and ISSR Markers. Jordan Journal Biological Sciences 4(2): 77–86.

Wang H, Zong X, Guan J, Yang T, Sun X., Ma Y, Redden R (2012) Genetic diversity and relationship of global faba bean (Vicia faba L.) germplasm revealed by ISSR markers. Theoretical and applied genetics 124(5): 789–797. https://doi.org/10.1007/s00122-011-1750-1

Weir BS (1990) Genetic data analysis: Methods for discrete population genetic data. Sinauer Associates, Inc., Sunderland, 377 pp.