Article

Molecular diversity and population structure of the Ethiopian lentil (Lens Culinaris Medikus) genotype assessment using SSR markers

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Abstract

Knowledge of genetic diversity in germplasm is essential for formulating effective germplasm collection, conservation, utilization strategies in and crop improvement programs. It also provides an opportunity to take corrective steps infusing new genes to avoid risks associated with a narrow genetic bases. Genetic diversity analysis of 119 lentil genotypes of including 83 germplasm and 36 exotic genotypes from International Center for Agricultural Research in the Dry Areas was studied using 27 primers of simple sequence repeat (SSR) marker. Molecular analysis of variance showed variations of 82% within and 18% of the among population variance was explained. Degree of polymorphism observed among the populations was 100%. A total 122 alleles were detected, with 2 to 7 alleles per locus, with a mean of 4.52 alleles per locus. The estimated gene diversity value for 27 loci was 0.64. The average Shannon’s information index value of 1.19 was obtained showed the existence of high genetic variation within the genotypes. The genetic similarity indices ranged from 0.21 to 1.00. The SSR markers showed an average polymorphic information content (PIC) value of 0.58. Cluster analysis grouped the genotypes into five major clusters as distinct genetic populations. Diversity analyses revealed the existence of a high level of genetic variation among genotypes. This molecular diversity information provides a basis for future germplasm collection, utilization, and conservation strategies in gene banks and introducing exotic germplasm to widen the genetic base of the current lentil breeding population.

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... SSRs are 2-5 bp sequences that are repeated nose-to-tail; the number of copies at each locus is what varies [26]. Due to their advantageous characteristics of universal distribution, high density and codominance, SSRs have been used as effective tools in genetic diversity analysis [27][28][29][30][31][32][33], gene mapping [34], and genome-wide association studies (GWASs) of major quantitative-trait loci (QTLs) [35]. Traditional gel electrophoresis is unable to accurately distinguish the number of repeats present in SSR amplicons. ...
... Traditional SSR markers, which are highly polymorphic and codominant, have been widely used in genetic diversity evaluations [27,[29][30][31][32][33], QTL mapping [35], background selection and marker-assisted selection (MAS)-based breeding [48]. The advantages of SSR-seq technology in relation to the use of conventional SSRs and other markers have been demonstrated [36,40]. ...
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Microsatellites have currently become the markers of choice for molecular mapping and marker-assisted selection for key traits such as disease resistance in many crop species. We report here on the mapping of microsatellites which had been identified from a genomic library of lentil (Lens culinaris Medik.). The majority of microsatellite-bearing clones contained imperfect di-nucleotide repeats. A total of 41 microsatellite and 45 amplified fragment length polymorphism (AFLP) markers were mapped on 86 recombinant inbred lines derived from the cross ILL 5588 x L 692-16-1(s), which had been previously used for the construction of a random amplified polymorphic DNA and AFLP linkage map. Since ILL 5588 was resistant to fusarium vascular wilt caused by the fungus Fusarium oxysporum Shlecht. Emend. Snyder & Hansen f.sp. lentis Vasud. & Srini., the recombinant inbreds were segregating for this character. The resulting map contained 283 markers covering about 751 cM, with an average marker distance of 2.6 cM. The fusarium vascular wilt resistance was localized on linkage group 6, and this resistance gene was flanked by microsatellite marker SSR59-2B and AFLP marker p17m30710 at distances of 8.0 cM and 3.5 cM, respectively. These markers are the most closely linked ones known to date for this agronomically important Fw gene. Using the information obtained in this investigation, the development and mapping of microsatellite markers in the existing map of lentil could be substantially increased, thereby providing the possibility for the future localization of various loci of agronomic interest.
Chapter
The genetic resources of lentils comprise the primitive varieties or landraces of the cultivated species (Lens culinaris Med.) and its wild relatives within the genus Lens Miller.
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Molecular genetic markers represent one of the most powerful tools for the analysis of genomes and enable the association of heritable traits with underlying genomic variation. Molecular marker technology has developed rapidly over the last decade and two forms of sequence based marker, Simple Sequence Repeats (SSRs), also known as microsatellites, and Single Nucleotide Polymorphisms (SNPs) now predominate applications in modern genetic analysis. The reducing cost of DNA sequencing has led to the availability of large sequence data sets derived from whole genome sequencing and large scale Expressed Sequence Tag (EST) discovery that enable the mining of SSRs and SNPs, which may then be applied to diversity analysis, genetic trait mapping, association studies, and marker assisted selection. These markers are inexpensive, require minimal labour to produce and can frequently be associated with annotated genes. Here we review automated methods for the discovery of SSRs and SNPs and provide an overview of the diverse applications of these markers.
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A method is presented by which the gene diversity (heterozygosity) of a subdivided population can be analyzed into its components, i.e., the gene diversities within and between subpopulations. This method is applicable to any population without regard to the number of alleles per locus, the pattern of evolutionary forces such as mutation, selection, and migration, and the reproductive method of the organism used. Measures of the absolute and relative magnitudes of gene differentiation among subpopulations are also proposed.
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RAPD markers were used to distinguish between six different Lens taxa, representing cultivated lentil and its wild relatives. Twenty-four arbitrary sequence 10-mer primers were identified which revealed robust and easily interpretable amplification-product profiles. These generated a total of 88 polymorphic bands in 54 accessions and were used to partition variation within and among Lens taxa. The data showed that, of the taxa examined, ssp. orientalis is most similar to cultivated lentil. L. ervoides was the most divergent wild taxon followed by L. nigricans. The genetic similarity between the latter two species was of the same magnitude as between ssp. orientalis and cultivated lentil. In addition, species-diagnostic amplification products specific to L. odemensis, L. ervoides and L. nigricans were identified. These results correspond well with previous isozyme and RFLP studies. RAPDs, however, appear to provide a greater degree of resolution at a sub-species level. The level of variation detected within cultivated lentils suggests that RAPD markers may be an appropriate technology for the construction of genetic linkage maps between closely related Lens accessions.
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AFLP and RAPD marker techniques have been used to evaluate and study the diversity and phylogeny of 54 lentil accessions representing six populations of cultivated lentil and its wild relatives. Four AFLP primer combinations revealed 23, 25, 52 and 48 AFLPs respectively, which were used to partition variation within and among Lens taxa. The results of AFLP analysis is compared to previous RAPD analysis of the same material. The two methods provide similar conclusions as far as the phylogeny of Lens is concerned. The AFLP technique detected a much higher level of polymorphyism than the RAPD analysis. The use of 148 AFLPs arising from four primer combinations was able to discriminate between genotypes which could not be distinguished using 88 RAPDs. The level of variation detected within the cultivated lentil with AFLP analysis indicates that it may be a more efficient marker technology than RAPD analysis for the construction of genetic linkage maps between carefully chosen cultivated lentil accessions.
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Lentil rust caused by Uromyces vicia-fabae (Pers.) Schroet is one of the most important diseases of lentil in South Asia, North Africa and East Africa. This disease is usually observed during late flowering and early podding stages. Early infection accompanied by favorable environmental conditions can result in complete crop failure and huge economic losses. Therefore, breeding for resistance against this pathogen is one of the major challenges for the breeders in those regions. It is important to identify resistance sources and to determine the location of the genes for resistance in the lentil genome. Since field screening is often difficult due to the unpredictable nature of the disease, selectable molecular markers can be useful tools to assist lentil breeding and complement field screening and selection for resistance. To map the genes for resistance, a recombinant inbred line (RILs) population composed of 220 RILs was developed from a cross between a rust resistant line, ILL-4605, and a susceptible line from Bangladesh, ILL-5888. Phenotyping of the RIL population was carried out during 2006–2007 and 2008–2009 cropping seasons at the Pulse Research Center, Ishurdi, Bangladesh. There was a lack of uniformity of disease pressure in the 2006–2007 cropping year causing inconsistencies between replicates. Nevertheless, we were able to choose clearly resistant and clearly susceptible RILs for selective genotyping using markers previously placed on our lentil genetic map. One of the 62 markers used for selective genotyping proved to be linked to the gene for resistance. The identified sequence related amplified polymorphism (SRAP) marker, F7XEM4a, was estimated to be 7.9 cM from the gene for resistance. The F7XEM4a marker could be used for marker assisted selection for resistance; however, additional markers closer to the resistance gene are needed.
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This paper reports the development of new microsatellite markers for lentil (Lens culinaris subsp. culinaris) and their use for genetic diversity analysis of a lentil core collection developed at ICARDA (Aleppo-Syria). Fourteen new markers were developed from microsatellite flanking sequences of a genomic library from a cultivated lentil accession ILL5588. The core collection used comprises 109 accessions from 15 countries representing 57 cultigens (including 18 breeding lines) from 8 countries and 52 wild types of germplasm (L. culinaris subsp. orientalis, L. culinaris subsp. tomentosus and L. culinaris subsp. odemensis) from 11 countries. Total number of alleles detected across all microsatellite loci was 182, with a mean of 13 alleles per locus. The wild accessions were rich in alleles (151 alleles) compared to cultigens (114 alleles). The genetic diversity index for the microsatellite loci in the wild accessions ranged from 0.16 (for locus SSR28 in L. culinaris subsp. odemensis) to 0.93 (for locus SSR66 in L. culinaris subsp. orientalis) with a mean of 0.66, while in the cultigens genetic diversity varied between 0.03 (locus SSR28) and 0.87 (locus SSR207) with a mean of 0.65. The cluster analysis indicated two major clusters, mainly one with the cultigens and the other with the wild accessions.
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In recent years, considerable emphasis has been placed on the development of molecular markers to be used for a variety of objectives. This review attempts to give an account of different molecular markers—restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), sequence-tagged sites (STS), DNA amplification fingerprinting (DAF), amplified fragment length polymorphisms (AFLPs) and microsatellites (STMS)—currently available for genome mapping and for tagging different traits in wheat. Other markers, including microsatellite-primed polymerase chain reaction (MP-PCR), expressed sequence tags (ESTs) and single nucleotide polymorphisms (SNPs) are also discussed. Recent information on synteny in cereal genomes, marker-assisted selection, marker validation and their relevance to cereal breeding in general and wheat breeding in particular are also examined.
Article
With 1 figure and 1 tableAbstractTransferability of microsatellite loci across species offers an opportunity, especially in Lens which suffers from lack of polymorphism and poor genome resources. Transferability of EST and genomic SSR markers from related taxa like Medicago truncatula (Mt), Pisum sativum (Ps) and Trifolium pratense (Tp), which are relatively well characterized genetically, was analyzed. The transferability of microsatellites as determined through successful amplification on six lentil accessions encompassing two subspecies was 36.0%, 62.0% and 25.0% for Mt, Tp and Ps-derived markers, respectively. A total of 22 Mt EST-SSRs produced 21.96% polymorphism, whereas, 98 BAC-SSRs produced marginally higher (24.14%) polymorphism. Twenty-two newly identified cross-species SSR markers and three lentil SSRs previously mapped on lentil genetic map, were used to infer the phylogenetic relationships among 40 accessions belonging to five Lens species (Lens culinaris ssp. culinaris, L. culinaris ssp. orientalis, L. culinaris ssp. odemensis, L. nigricans and L. ervoides). Total number of alleles detected across all microsatellite loci was 80, with a mean of 3.63 alleles per locus. UPGMA dendrogram divided them into six groups with wild subspecies L. nigricans and L. ervoides exhibiting maximum genetic distance, whereas high levels of intraspecific variation was observed among the accessions of L. orientalis, L. odemensis and L. nigricans. A close proximity (57.0%) between cultivated lentil and its supposedly wild progenitor L. culinaris ssp. orientalis was revealed. Our study has implications in large-scale development and identification of SSR markers from related genera which can be used for studying genetic diversity and construction of lentil genetic map and for tagging of various traits of agronomic importance for marker-assisted breeding.
Article
Using random amplified polymorphic DNA (RAPD) analysis we assessed the genetic relationships between 16 accessions and cultivars of lentil (Lens culinaris ssp. culinaris) in the Australian lentil breeding program. All lines exhibited polymorphism with a maximum dissimilarity value of 0.36. This indicated a limited degree of genetic variation. Polymerase chain reaction (PCR) with primers based on the flanking regions of the 5S rRNA gene from Pisum sativum amplified the non-translated spacer (NTS) region from within the 5S rRNA gene of Lens. Three distinct amplification banding patterns differentiated between restricted genomic DNA of Lens spp. L. culinaris ssp. culinaris and L. culinaris ssp. orientalis shared similar markers of two distinctly different NTS sizes. L. nigricans and L. odemensis shared the same amplification pattern of a single sized NTS region. However, L. ervoides contained two separate sizes of NTS, distinct from other Lens species. In an effort to widen the genetic base of cultivated lentil, these species-specific molecular markers may be used to follow potential introgression between species.
Article
The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.
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The diversity and genetic relationship among and within 16 populations of Pogostemon cablin from China were analyzed using ISSR and SRAP markers. High percentage of polymorphism observed by these two markers indicated a high level of genetic diversity existed among P. cablin populations. The percentage of polymorphic bands revealed by these two markers showed genetic variations were much higher between populations of P. cablin than within. The UPGMA clustering results using ISSR, SRAP or ISSR + SRAP combination showed most accessions from the same or adjacent regions were classified together. However, cluster results based on the absolute contents of patchouli alcohol and pogostone did not keep in good accordance with their geographic distributions, which revealed that the forming of patchouli alcohol and pogostone were relevant to the cultivation conditions except for genetic factors and environmental conditions. Finally, an appropriate strategy for conserving the patchouli germplasm was proposed.
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A mathematical model for the evolutionary change of restriction sites in mitochondrial DNA is developed. Formulas based on this model are presented for estimating the number of nucleotide substitutions between two populations or species. To express the degree of polymorphism in a population at the nucleotide level, a measure called "nucleotide diversity" is proposed.
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A method for rapid preparation of plant genomic DNA for PCR analysis was established. A small amount (4~6 mg) of leaf tissue of rice seedling, 200 滋L of TE buffer, and one tungsten bead were put into a 2 mL microcentrifuge tube. After vigorously shaking in a miller for 5 min, 1 滋L of the solution was directly applied to PCR amplification. This method is simple, rapid, high efficient, low cost, and reliable for PCR analysis, thus is es- pecially suitable for genotyping of large number of samples.