Genomic characterization of Puccinia triticina using molecular marker technology / Caracterização genômica de Puccinia triticina usando tecnologia de marcadores moleculares

Leaf rust, caused by Puccinia triticina, is the most common rust disease of wheat. The fungus is an obligate parasite capable of producing infectious urediniospores. To study the genetic structure of the leaf rust population 20 RAPD primers were evaluated on 15 isolates samples collected in Pakistan. A total of 105 RAPD fragments were amplified with an average of 7 fragments per primer. The number of amplified fragments varied from 1 to 12. GL Decamer L-07 and GL Decamer L-01 amplified the highest number of bands (twelve) and primer GL Decamer A-03 amplified the lowest number of bands i.e one. Results showed that almost all investigated isolates were genetically different that confirms high genetic diversity within the leaf rust population. Rust spores can follow the migration pattern in short and long distances to neighbor areas. Results indicated that the greatest variability was revealed by 74.9% of genetic differentiation within leaf rust populations. These results suggested that each population was not completely identical and high gene flow has occurred among the leaf rust population of different areas. The highest differentiation and genetic distance among the Pakistani leaf rust populations were detected between the leaf rust population in NARC isolate (NARC-4) and AARI-11and the highest similarity was observed between NARC isolates (NARC-4) and (NARC-5). The present study showed the leaf rust population in Pakistan is highly dynamic and variable.

A ferrugem da folha, causada por Puccinia triticina, é a ferrugem mais comum do trigo. O fungo é um parasita obrigatório, capaz de produzir urediniósporos infecciosos. Para estudar a estrutura genética da população de ferrugem da folha, 20 primers RAPD foram avaliados em 15 amostras de isolados coletadas no Paquistão. Um total de 105 fragmentos RAPD foram amplificados com uma média de 7 fragmentos por primer. O número de fragmentos amplificados variou de 1 a 12. GL Decamer L-07 e GL Decamer L-01 amplificaram o maior número de bandas (doze), e o primer GL Decamer A-03 amplificou o menor número de bandas, ou seja, um. Os resultados mostraram que quase todos os isolados investigados eram geneticamente diferentes, o que confirma a alta diversidade genética na população de ferrugem da folha. Os esporos de ferrugem podem seguir o padrão de migração em distâncias curtas e longas para áreas vizinhas. Os resultados indicaram que a maior variabilidade foi revelada por 74,9% da diferenciação genética nas populações de ferrugem. Esses resultados sugeriram que cada população não era completamente idêntica e um alto fluxo gênico ocorreu entre a população de ferrugem da folha de diferentes áreas. A maior diferenciação e distância genética entre as populações de ferrugem da folha do Paquistão foram detectadas entre a população de ferrugem da folha no isolado NARC (NARC-4) e AARI-11 e a maior similaridade foi observada entre os isolados NARC (NARC-4) e (NARC-5). O presente estudo mostrou que a população de ferrugem da folha no Paquistão é altamente dinâmica e variável.

Genomic characterization of Puccinia triticina using molecular marker technology.

Leaf rust, caused by Puccinia triticina, is the most common rust disease of wheat. The fungus is an obligate parasite capable of producing infectious urediniospores. To study the genetic structure of the leaf rust population 20 RAPD primers were evaluated on 15 isolates samples collected in Pakistan. A total of 105 RAPD fragments were amplified with an average of 7 fragments per primer. The number of amplified fragments varied from 1 to 12. GL Decamer L-07 and GL Decamer L-01 amplified the highest number of bands (twelve) and primer GL Decamer A-03 amplified the lowest number of bands i.e one. Results showed that almost all investigated isolates were genetically different that confirms high genetic diversity within the leaf rust population. Rust spores can follow the migration pattern in short and long distances to neighbor areas. Results indicated that the greatest variability was revealed by 74.9% of genetic differentiation within leaf rust populations. These results suggested that each population was not completely identical and high gene flow has occurred among the leaf rust population of different areas. The highest differentiation and genetic distance among the Pakistani leaf rust populations were detected between the leaf rust population in NARC isolate (NARC-4) and AARI-11and the highest similarity was observed between NARC isolates (NARC-4) and (NARC-5). The present study showed the leaf rust population in Pakistan is highly dynamic and variable.

Application of SCAR markers linked with mungbean yellow mosaic virus disease-resistance gene in Pakistan mungbean germplasm.

Mungbean yellow mosaic virus disease (MYMVD) caused by single-stranded DNA begomovirus is the most prominent threat to the mungbean crop in Pakistan. Mungbean genotypes consisting of 127 varieties/lines were screened for MYMVD under natural field conditions. No genotypes were found to be immune or highly resistant against MYMVD. Genotypes showing resistant and tolerant responses in field screening trials were screened using sequence characterized amplified region (SCAR) markers linked with the MYMVD-resistance gene. SCAR markers amplified the desired band only in the resistant and tolerant genotypes, while no amplification was observed in susceptible genotypes. SCAR markers will be useful for future breeding and varietal developmental programs and mungbean genotypes can be screened on the molecular level.

Identification and authentication of Rosa species through development of species-specific SCAR marker(s).

Roses (Rosa indica) belong to one of the most crucial groups of plants in the floriculture industry. Rosa species have special fragrances of interest to the perfume and pharmaceutical industries. The genetic diversity of plants based on morphological characteristics is difficult to measure under natural conditions due to the influence of environmental factors, which is why a reliable fingerprinting method was developed to overcome this problem. The development of molecular markers will enable the identification of Rosa species. In the present study, randomly amplified polymorphic DNA (RAPD) analysis was done on four Rosa species, Rosa gruss-an-teplitz (Surkha), Rosa bourboniana, Rosa centifolia, and Rosa damascena. A polymorphic RAPD fragment of 391 bp was detected in R. bourboniana, which was cloned, purified, sequenced, and used to design a pair of species-specific sequence-characterized amplified region (SCAR) primers (forward and reverse). These SCAR primers were used to amplify the specific regions of the rose genome. These PCR amplifications with specific primers are less sensitive to reaction conditions, and due to their high reproducibility, these species-specific SCAR primers can be used for marker-assisted selection and identification of Rosa species.

Molecular characterization of urdbean (Vigna mungo) germplasm related to resistance against urdbean leaf crinkle virus.

Urdbean (Vigna mungo) is an important pulse crop grown worldwide. Urdbean leaf crinkle virus (ULCV) is a pathogen of urdbean found in Pakistan that causes huge losses in yield. Forty urdbean varieties/lines were screened against the virus under field conditions during spring season 2009. None of the lines appeared to be highly resistant or resistant. On the basis of a 0-5 disease rating scale and disease severity index, genotypes varied significantly in their reaction to ULCV. Four lines (M-6206, IAM-382-15, IAM-133, and Mash-1) were moderately resistant, eight were rated as moderately susceptible, and 21 as susceptible; the remaining seven lines were highly susceptible. RAPD analyses revealed an extensive amount of variation, which could be used for cultivar identification. Genetic differentiation among urdbean genotypes was similar to the field screening data. The varieties 6065-3 and 6206 were highly susceptible and moderately resistant, respectively, to ULCV under field conditions, confirmed by the RAPD analysis. These varieties were the most diverse varieties in the similarity matrix (67.2%), while the varieties IAM-382-9 and 07M003 were the most similar (98.4%). This information will help in the recognition of available resistant germplasms that can resist this disease and will be utilized for urdbean improvement in Pakistan.

Elucidation of thermotolerance diversity in cotton (Gossypium hirsutum L.) using physio-molecular approaches.

Cotton (Gossypium hirsutum) is an important cash crop, but high temperature during its growing season is one of the major factors that limit its productivity. This problem compels plant breeders to breed for heat tolerance, which can help to overcome this challenge. It is very important to make a comprehensive screening of heat-tolerant genotypes so that only the best are chosen. Here we report the combined use of several techniques that can help breeders to screen their germplasm. Twelve cultivated cotton genotypes were evaluated for thermotolerance, using assays that included electrolyte leakage, chlorophyll accumulation and protein profiling, as well as RAPDs to assess genetic diversity. Two genotypes (B-557 and NIAB-78) showed tolerant behavior in three thermotolerance assays. RAPD analysis results showed maximum similarity in a range of 86.7-66.7% between the genotypes MNH-554 and CIM-443. We conclude that combined use should be made of relative electrolyte leakage, chlorophyll stability and differential display with SDS-PAGE to aid in screening for stress tolerance. RAPD-based diversity analysis will further help to improve the efficiency of breeding programs.

Estimation of genetic distance based on RAPDs between 11 cotton accessions varying in heat tolerance.

The genetic distance of 11 cotton genotypes varying in heat tolerance was studied using RAPD markers. Fifty-three random decamer primers were used for the estimation of genetic distance. Among the 53 RAPD primers, which were custom synthesized by GeneLink Inc., UK, 32 were polymorphic and 21 were monomorphic. The 32 polymorphic primers produced 273 fragments, with a mean of 8.3 fragments per primer. The number of polymorphic bands produced in the 11 cotton accessions ranged from 1 to 31. Primer GLC-20 produced 31 polymorphic bands, while two primers, GLB-5 and GLC-12, produced one polymorphic band each. A range of 88.89 to 42.48% genetic similarity was observed among the 11 cotton accessions. The highest genetic similarity was observed between FH-945 and BH-160 (88.89%), whereas the lowest value was found between NIAB-801/2 and FH-945 (42.48%). Unique amplification profiles were produced by most of the cultivars; the differences were sufficient to distinguish them from other genotypes. This confirms the efficacy of RAPD markers for the identification of plant genotypes. An accumulative analysis of amplified products generated by RAPDs was sufficient to assess the genetic diversity among the genotypes. This information should be helpful for formulating breeding and genome mapping programs.

Genetic diversity of chickpea (Cicer arietinum L.) germplasm in Pakistan as revealed by RAPD analysis.

Genetic diversity analysis of chickpea germplasm can provide practical information for the selection of parental material and thus assist in planning breeding strategies. Chickpea seed is a good source of carbohydrates and proteins, constituting 80% of the total dry seed weight. Released cultivars and advanced lines of 30 chickpea genotypes were subjected to RAPD analysis for assessment of genetic diversity. We used 16 RAPD primers. Amplification of genomic DNA of the 30 genotypes yielded 62 fragments that could be scored. The number of amplification products produced per primer varied from two to four, with a mean of three bands. The total number of bands amplified by 16 anchored primers varied from 16 to 34. The primer GLK-15 produced the largest number (N = 4) of fragments, whereas primers GLK-19 and GLD-19 produced the smallest number (N = 1) of fragments. The single band produced by the GTGTGCCCCA primer in the PB-2000 and 07005 genotypes may be attributed to temperature tolerance phenotypes.

Biodiversity in the sorghum (Sorghum bicolor L. Moench) germplasm of Pakistan.

Sorghum ranks fifth in worldwide economic importance among cereal crops and is one of the most important summer annual grasses of Pakistan. As it is a very diverse crop, sorghum genetic fingerprinting requires an efficient marker system. We estimated genetic divergence among 29 sorghum (Sorghum bicolor) genotypes, including approved varieties and local and exotic lines collected from different ecological regions of Pakistan, using random amplified polymorphic DNA (RAPD) markers. A total of 125 RAPD loci, with an average of 66 loci per genotype, were used to calculate genetic divergence among these genotypes, of which 119 were polymorphic, showing 95% overall polymorphism. Genetic similarity ranged from 0.36 to 0.92, indicating a relatively broad genetic base. RAPD analysis revealed maximum similarity between the Indian III and K-A-113 sorghum genotypes (both exotic lines), while the F-601 and F-606 were observed to be the most diverse genotypes. Mean band frequency revealed by these RAPD primers ranged from 0.17 to 0.56, with an average of 0.36. The data presented here support the findings that RAPDs can be effectively used for studying genetic diversity in sorghum.

Estimation of genetic distance between 10 maize accessions with varying response to different levels of soil moisture.

Ten maize accessions (NC-9, A50-2, M-14, B-42, NC-3, T-7, N-48-1, B-34, USSR, and WFTMS) were studied to estimate the genetic distance on molecular level by random amplified polymorphic DNA. These accessions were selected on the basis of their variable responses against different levels of moisture. Twenty-five primers were used to test genetic diversity, of which 14 were observed to be polymorphic. Ninety-three loci were amplified; among these, 77 showed polymorphism and the other 16 were monomorphic. Primers A-13 and C-02 gave the most polymorphic bands, while primers A-01 and C-06 gave the fewest polymorphic bands. The genetic similarities of the 10 maize accessions ranged from 82.8 to 54.8%. Accessions USSR and WFTMS showed greatest similarity, and accessions M-14 and B-42 were found more dissimilar than the other accessions. On the basis of cluster analysis, these 10 accessions were classified in two major groups, A and B, and than further divided into sub-groups. The cluster analysis showed that accessions in the same group as well as in the sub-groups were similar in their physical and morphological characters, since the characters are controlled genetically.

Persistent infection of Strongyloides venezuelensis and normal expulsion of Nippostrongylus brasiliensis in Mongolian gerbils, Meriones unguiculatus, with reference to the cellular responses in the intestinal mucosa.

The kinetics of daily faecal egg count, worm burdens, and intestinal cellular responses were examined in Mongolian gerbils after infection with either Strongyloides venezuelensis or Nippostrongylus brasiliensis alone, or concurrently with both parasites. The results show that, both in individual and concurrent infections, S. venezuelensis infection persisted for over 10 weeks and elicited a gradual increase in number of mast cells in the jejunal mucosa. On the other hand, N. brasiliensis worms were expelled by 3 weeks in association with goblet cell hyperplasia. These results suggest that effector/regulator cells involved in worm expulsion are different and highly selective depending on the genus of intestinal helminths.