Relationships among <Emphasis Type="Italic">Leymus</Emphasis> species assessed by RAPD markers

The DNA genetic diversity of 40 accessions of genus Leymus was analyzed by random amplified polymorphic DNA (RAPD) markers. A total of 352 products were amplified by 34 10-mer arbitrary primers, among which 337 products (95.74 %) were found to be polymorphic. 5–14 polymorphic bands were amplified by each polymorphic primer, with an average of 9.91 bands. The data of 352 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Great genetic diversity in genus Leymus was observed, the genetic diversity among the different species more abundant than that of the different accessions, and the different accessions in a species or the species from the same areas were clustered together.     

Identification of Molecular Marker and Aggressiveness for Different Groups of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates Causing Spot Blotch Disease in Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

One hundred fifty-five isolates of Bipolaris sorokiniana of wheat were studied for their morphopathological characterization. These isolates were grouped in five categories--black, brown/dull black, gray cottony growth, dull white/greenish black, and white--on the basis of their growth pattern. The frequency of the black suppressed type was maximum (45.63%), whereas the white isolate displayed lowest frequency (6.96%) in the natural population. Twenty RAPD (random amplified polymorphic DNA) primers were used to observe the variability among the identified groups of B. sorokininana. From each group, eight random isolates were investigated. A total of 143 bands were amplified, out of which 107 (74.83%) were polymorphic and 36 (25.17%) were monomorphic. On an average, the total numbers of bands generated per primer were 7.15, of which 5.35 and 1.80 were polymorphic and monomorphic, respectively. Dendrograms based on molecular polymorphism unveiled a considerable amount of diversity among the isolates. Specific DNA bands were identified for selected isolates. The distinct markers appeared to be potential enough to be employed as genetic fingerprints for future strain identification and classification. The study indicated that the RAPD primers provide an easy, rapid, and simple technique for the preliminary assessment of genetic diversity among the fungal isolates.     

Molecular Diversity in some Ghanaian Cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> L. (Walp)] Accessions

Cowpea [Vigna unguiculata L. (Walp)] is grown mainly for its protein-rich grains and is consumed in various forms in sub-Saharan Africa. Average grain yield in farmers’ fields is generally low due to a number of biotic and abiotic stresses. One hundred and six cowpea accessions from Ghana, which had previously been evaluated for seedling drought tolerance, were used for this study. This paper attempts to use three multi-locus PCR-based molecular markers; simple sequence repeats (SSR), inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphisms (REMAP), to analyse genetic diversity in the cowpea accessions. Analysis of the polymorphic bands data indicated that 101 alleles were amplified among 121 cowpea genotypes (83.4%) from 16 SSR primer pairs out of a total of 30 SSR primer pairs. Likewisely, a total of 66 (54.5%) polymorphic bands were obtained from IRAP and a total of 114 (94.2%) highly polymorphic bands obtained from REMAP analysis. The outcome indicated the highly polymorphic nature of the DNA markers, as small groups of these molecular markers were found to be able to identify each of the accessions used. Microsatellite markers (SSRs) and retrotransposon-based markers, like IRAP and REMAP, were found to be highly polymorphic and informative, suggesting that genomic fingerprinting has a major role in characterizing populations.     

Assessing Genetic Diversity in <Emphasis Type="Italic">Olea europaea</Emphasis> L. Using ISSR and SSR Markers

Olea europaea L. is one of the most economically important crops in the Mediterranean area, and known for having large genetic variability. In order to assess the genetic diversity, DNA from 41 olive cultivars, present in the protected denomination of origin (PDO) region of Trás-os-Montes, was screened using inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. Eleven ISSR primers amplified 135 reproducible bands of which 108 were polymorphic. The percentage of polymorphic bands detected by ISSR was 79%. The highest number of polymorphic bands was obtained by the use of primers UBC807 (15) and UBC809 (16). A total of 67 alleles were detected by six SSR primers, with an average of 11 alleles per primer. The number of alleles per locus ranged from five (ssrOeUaDCA05) to 15 (ssrOeUaDCA03). The observed heterozygosity ranged from 0.219 (ssrOeUaDCA05) to 0.900 (ssrOeUaDCA04), while the expected heterozygosity varied between 0.426 (ssrOeUaDCA05) and 0.887 (ssrOeUaDCA03). The polymorphism information content (PIC) values ranged from 0.392 (ssrOeUaDCA05) to 0.863 (ssrOeUaDCA03). The collection of primers selected gave a reasonable number of amplification products for the genetic diversity analysis. Based on the results, the genetic diversity among 41 olive cultivars is discussed. This study reveals the great importance of guaranteeing the differentiation of olive cultivars and their application for certification purposes.     

Study of genetic variation in sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) using agro-morphological traits and <Emphasis Type="Italic">ISSR</Emphasis> markers

This research was conducted to study the genetic variation among eighteen genotypes of sesame (Sesamum indicum L.) collected from various agro-climatic regions of Iran along with six exotic genotypes from the Asian countries using both agro-morphological and ISSR marker traits. The results showed significant differences among genotypes for all agro-morphological traits and a relatively high genetic coefficient of variation observed for number of fruiting branches per plant, capsules per plant, plant height and seed yield per plant. Cluster analysis based on these traits grouped the genotypes into five separate clusters. Larger interthan intra cluster distances implies the presence of higher genetic variability between the genotypes of different groups. Genotypes of two clusters with a good amount of genetic divergence and desirable agronomic traits were detected as promising genotypes for hybridization programs. The 13 ISSR primers chosen for molecular analysis revealed 170 bands, of which 130 (76.47%) were polymorphic. The generated dendrogram based on ISSR profiles divided the genotypes into seven groups. A principal coordinate analysis confirmed the results of clustering. The agro-morphological traits and ISSR markers reflected different aspects of genetic variation among the genotypes as revealed by a non significant cophenetic correlation in the Mantel test. Therefore the complementary application of both types of information is recommended to maximize the efficiency of sesame breeding programs. The discordance among diversity patterns and geographical distribution of genotypes found in this investigation implies that the parental lines for hybridization should be selected based on genetic diversity rather than the geographical distribution.     

Neutral and functional marker based genetic diversity in kodo millet (<Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L.)

Kodo millet (Paspalum scrobiculatum L.) is known for its high nutritive value, dietary fiber, antioxidant activity, as well as for drought tolerance. It is primarily grown as a grain in India and in Africa it is either cultivated or harvested in wild. Neutral—ISSR (inter simple sequence repeat) as well as functional—SCoT (start codon targeted) and SRAP (sequence-related amplified polymorphism) markers were employed for genetic diversity studies in 96 accessions of kodo millet collected from diverse regions of India. The genetic diversity parameters like average bands per primer, Polymorphic information content, Nei’s gene diversity and Shannon’s information index of 11.22, 9.69; 0.12, 0.11; 0.15 ± 0.14, 0.13 ± 0.13 and 0.26 ± 0.21, 0.22 ± 0.19 was observed with neutral and functional markers respectively. Neutral markers were showing higher values as compared to functional markers for the genetic diversity parameters as discussed. Structure based analysis placed all the accessions into four sub-groups not strictly according to their geographical locations. The accessions from Bihar followed by Karnataka were showing high diversity based on both the marker systems useful for designing exploration, conservation and germplasm enrichment strategies. Further, the set of diverse accessions selected based on these markers would serve as potential sources of unique alleles and may be exploited in future for enhancement and utilization of kodo millet germplasm. Usage of African gene pool and wild species for broadening the genetic base of Indian kodo millet was also suggested based on the present studies.     

Development and Characterization of SSR Markers to Study Genetic Diversity and Population Structure of Horsegram Germplasm (<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis>)

Development of genomic resources in any crop is the pre-requisite for the construction of linkage map and implementation of molecular breeding strategies to develop superior cultivars. Large number of molecular markers are required to enrich the scanty information available in horsegram (Macrotyloma uniflorum).We employed the next-generation Illumina sequencing platform to develop a large number of microsatellite markers in this species. Of the total 23,305 potential SSRs motifs, 5755 primers were designed. Of these, 1425, 1310, 856, 1276, and 888 were of di-, tri-, tetra-, penta-, and hexa-nucleotide repeats respectively. Thirty polymorphic SSR primers and 24 morphological traits were used in 360 horsegram accessions to detect the genetic diversity and population structure. Thirty primers amplified 170 polymorphic alleles with an average of 5.6 alleles per primer having size 80 to 380 bp. The polymorphism information content (PIC) ranged from 0.15 to 0.76 with an average of 0.50, suggesting that SSR markers used in the study were polymorphic and suitable for characterization of horsegram germplasm. Dendrogram-based on Jaccard’s similarity coefficient and neighbor-joining tree grouped the horsegram accessions into two major clusters. Similarly, STRUCTURE analysis assigned genotypes into two gene pools namely Himalayan origin and Southern India. Diversity analysis based on 24 agro-morphological traits also suggested the presence of high level of diversity among the accessions.     

Analysis of genetic diversity among <Emphasis Type="Italic">Swertia chirayita</Emphasis> genotypes

Inter simple sequence repeat (ISSR) markers were used to analyse genetic diversity of Swertia chirayita genotypes collected from the temperate Himalayas of India. Allied species of Swertia chirayita were used in the study as outliers. Nineteen UBC primers generated a total of 315 ISSR bands, revealing 98.7 % polymorphism among the genotypes assayed. This was reduced to 42.5 % when the outliers were excluded. The results revealed a high genetic diversity within the genotypes.     

Genetic diversity of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. in Azerbaijan

To examine the genetic diversity of Vitis vinifera L., growing in the region near the Caspian Sea of Azerbaijan Republic, nuclear genomes of 31 cultivated and 34 wild grapevine accessions were studied at population and individual levels using five ISSR primers. In total, 51 fragments were amplified, of which 45 were found to be polymorphic. A high level of polymorphism was revealed (the mean PPF and PIC values constituted 87.69% and 0.94, respectively). High values of the EMR, MI, and RP indices showed the effectiveness of the application of ISSR primers and the possibility of their use in further investigations in this direction. Cluster analysis based on Nei’s genetic distance values showed that all genotypes could be grouped into seven main clusters. Furthermore, no differences between the wild and cultivated grape wine accessions were revealed. For instance, there was no distinct distribution of the accessions according to their geographical localization. On the basis of the PIC values, the group of cultivars from Absheron Peninsula was distinguished by the highest polymorphism level (PIC = 0.36). Natural populations from the Guba and Shabran regions were characterized by a relatively low polymorphism level (PIC = 0.31 and PIC = 0.28, respectively), and a wild population from Nabran demonstrated the lowest polymorphism level (PIC = 0.25). The data obtained confirmed paleontological and historical data of different periods and provided the supposition that Azerbaijan was the center of diversity of V. vinifera L. In addition, our data indicate that Azerbaijan grape landraces originated from local wild forms.     

Development of Dof (DNA binding with one finger) transcription factor gene-specific primers through data mining as a functional marker and their use for genetic diversity study in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) germplasm

The genetic diversity among Hordeum vulgare L. species were assessed based on PCR amplification pattern derived from 75 set of Dof domain and Dof genes specific primers. Multiple bands showing variability in terms of both number and sizes of bands ranging from 0.1 to 3.0 kbp were observed. Out of a total of 2449 bands, 2328 polymorphic and 121 monomorphic bands were obtained and the percentage of polymorphism ranged from 70.27 to 100%. A very high degree of polymorphism was observed with all the primers except HvDof3, HvDof4, HvDof10, HvDof16, HvDof18, HvDof18, HvDof24, Dof4, Dof11, Dof13, Dof15, Dof16, Dof19, Dof20, Dof21, Dof22, Dof23, Dof28, dof38, sbDof23 and sbDof24 primers. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. According to results, the genetic resources and diversity in barley germplasm of H. vulgare were rich. The number of polymorphic fragments per primer detected ranged from 11 to 56 bands with an average of 32.65 bands. Average polymorphic information content (PIC) was 0.81 in overall Dof domain and gene specific primers. HvDof 39 showed the highest PIC (0.99) which can be a good candidate primer to verify genetic diversity in H. vulgare. The unweighted pair-group method of the arithmetic average and principal coordinate analysis showed a clear distinction among the genotypes and the genotypes divided into three clusters in the dendrogram results. A model-based structure analysis revealed the presence of three groups. The study showed that genetic variation and population structure are determined among the species of H. vulgare collected from different geographical origins.     

High efficiency and reliability of inter-simple sequence repeats (ISSR) markers for evaluation of genetic diversity in Brazilian cultivated <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. accessions

Jatropha curcas L. is found in all tropical regions and has garnered lot of attention for its potential as a source of biodiesel. As J. curcas is a plant that is still in the process of being domesticated, interest in improving its agronomic traits has increased in an attempt to select more productive varieties, aiming at sustainable utilization of this plant for biodiesel production. Therefore, the study of genetic diversity in different accessions of J. curcas in Brazil constitutes a necessary first step in genetic programs designed to improve this species. In this study we have used ISSR markers to assess the genetic variability of 332 accessions from eight states in Brazil that produce J. curcas seeds for commercialization. Seven ISSR primers amplified a total of 21,253 bands, of which 19,472 bands (91%) showed polymorphism. Among the polymorphic bands 275 rare bands were identified (present in fewer than 15% of the accessions). Polymorphic information content (PIC), marker index (MI) and resolving power (RP) averaged 0.26, 17.86 and 19.87 per primer, respectively, showing the high efficiency and reliability of the markers used. ISSR markers analyses as number of polymorphic loci, genetic diversity and accession relationships through UPGMA-phenogram and MDS showed that Brazilian accessions are closely related but have a higher level of genetic diversity than accessions from other countries, and the accessions from Natal (RN) are the most diverse, having high value as a source of genetic diversity for breeding programs of J. curcas in the world.     

Identification of Genetic Diversity Among Cultivars of <Emphasis Type="Italic">Phyllostachys violascens</Emphasis> Using ISSR,SRAP and AFLP Markers

Bamboo is one of the most important forest resources with a strong carbon fixation capability. To utilize genetic resource of Phyllostachys violascens, ISSR (inter-simple sequence repeat), SRAP (sequence-related amplified polymorphism), and AFLP (amplified fragment length polymorphism) techniques were used for the first time for the assessment of genetic diversity within its different cultivars. A total of 209 (136 polymorphic), 222 (152 polymorphic), and 434 (253 polymorphic) bands were detected using 15 ISSR primers, 15 primer combinations of SRAP, and 15 primer combinations of AFLP, respectively. The mean genetic similarity of Ph. violascens was 0.872, 0.867 or 0.871 for the ISSR, SRAP and AFLP analyses, respectively. Based on genetic diversity, all the cultivars of Ph. violascens could be divided into four groups, which are reflected by their morphologies. Our data demonstrated that all three methods are useful in the identification of genetic diversity in Ph.violascens, but AFLP is the most efficient.     

ISSR Analysis of Variability of Cultivated Form and Varieties of Pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L.) from Azerbaijan

The article presents the results of a study of genetic polymorphism for the first time carried out on pomegranate varieties and forms of Azerbaijan origin using molecular markers. In total, 102 PCR fragments were identified, of which 80 were polymorphic. The high level of polymorphism (75.5%) and the rich genetic diversity were identified among the studied pomegranate collection. As a result of data analysis and on the basis of the values of the basic parameters (PIC, EMR, MI, RP, MRP) determining informativeness of markers, all 14 ISSR primers were suitable for genotyping pomegranate accessions. The most effective markers (UBC808, UBC811, UBC834, and UBC840) were identified among the set of primers tested. A dendrogram was constructed on the basis of the data obtained, which made it possible to group genotypes into 16 major clusters. The genetic similarity index ranged from 0.032 to 0.94. The study of the genetic relationship of different pomegranate varieties confirms the effectiveness of the ISSR method, which makes it possible to determine the level of genetic diversity, as well as to establish the relationship among the studied pomegranate accessions.     

Development of expressed sequence tag-derived microsatellite markers for <Emphasis Type="Italic">Saccharina</Emphasis> (<Emphasis Type="Italic">Laminaria</Emphasis>) <Emphasis Type="Italic">japonica</Emphasis>

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by PCR-RAPD based fingerprinting

Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.     

Exploration of genetic diversity among Xinjiang<Emphasis Type="Italic">Triticum</Emphasis> and<Emphasis Type="Italic">Triticum polonicum</Emphasis> by AFLP markers

Seventy-two Xinjiang Triticum and Triticum polonicum accessions were subjected to AFLP analyses to discuss the origin of Triticum petropavlovskyi. A total of 91 putative loci were produced by four primer combinations. Among them 56 loci were polymorphic, which is equivalent to 61.53 % of the total number of putative loci. Genetic diversity among 11 T. petropavlovskyi accessions was narrow due to the lowest number (32) of polymorphic loci among the wheat species. Forty four polymorphic loci were found in T. aestivum and T. compactum, whereas the highest polymorphism was observed in T. polonicum. On the basis of the UPGMA clustering and PCO grouping and genetic similarity estimates from the AFLPs, we noted that T. petropavlovskyi was more closely related to the Chinese accessions of T. polonicum than to T. polonicum from other countries. Two accessions of T. aestivum were grouped with T. petropavlovskyi in the UPGMA clustering. Both of them were similar to T. petropavlovskyi in respect of spike structure, i.e. the presence of awn, glume awn and also the presence of leaf pubescence. Six loci, which were commonly absent in Chinese T. polonicum, were also absent in almost all of the T. petropavlovskyi accessions. Findings of this study reduced the probability of an independent allopolyploidization event in the origin of T. petropavlovskyi and indicated a greater degree of gene flow between T. aestivum and T. polonicum leading to T. petropavlovskyi. It is most likely that the P-gene of T. petropavlovskyi hexaploid wheat was introduced from T. polonicum to T. aestivum via a spontaneous introgression or breeding effort.     

The origin of cultivated <Emphasis Type="Italic">Coffea arabica</Emphasis> L. varieties revealed by AFLP and SSR markers

Molecular markers were used to assess polymorphism between and within the genetic bases of coffee (i.e. Typica and Bourbon) spread from Yemen since the early 18th century that have given rise to most arabica cultivars grown world-wide. Eleven Coffea arabica accessions derived from the disseminated bases were evaluated by amplified fragment length polymorphism (AFLP) using 37 primer combinations and simple-sequence repeats (SSRs) produced by six microsatellites. Four cultivars growing in Yemen and 11 subspontaneous accessions collected in the primary centre of diversity of the species were included in the study in order to define their relationship with the accessions derived from the genetic bases of cultivars. One hundred and seven AFLP markers were used to calculate genetic distances and construct a dendrogram. The accessions derived from the disseminated bases were grouped separately, according to their genetic origin, and were distinguished from the subspontaneous accessions. The Yemen cultivars were classified with the Typica-derived accessions. Except for one AFLP marker, all AFLP and SSR markers present in the cultivated accessions were also detected in the subspontaneous accessions. Polymorphism among the subspontaneous accessions was much higher than among the cultivated accessions. It was very low within the genetic bases, confirming the historical documentation on their dissemination. The results enabled a discussion of the genetic diversity reductions that successively occurred during the dissemination of C. arabica from its primary centre of diversity.     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

     

Analysis of the genetic diversity of physic nut, <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. accessions using RAPD markers

A sum of 48 accessions of physic nut, Jatropha curcas L. were analyzed to determine the genetic diversity and association between geographical origin using RAPD-PCR markers. Eight primers generated a total of 92 fragments with an average of 11.5 amplicons per primer. Polymorphism percentages of J. curcas accessions for Selangor, Kelantan, and Terengganu states were 80.4, 50.0, and 58.7%, respectively, with an average of 63.04%. Jaccard’s genetic similarity co-efficient indicated the high level of genetic variation among the accessions which ranged between 0.06 and 0.81. According to UPGMA dendrogram, 48 J. curcas accessions were grouped into four major clusters at coefficient level 0.3 and accessions from same and near states or regions were found to be grouped together according to their geographical origin. Coefficient of genetic differentiation (G_st) value of J. curcas revealed that it is an outcrossing species.