Development and mapping of Simple Sequence Repeat markers for pearl millet from data mining of Expressed Sequence Tags

<p>Abstract</p> <p>Background</p> <p>Pearl millet [<it>Pennisetum glaucum </it>(L.) R. Br.] is a staple food and fodder crop of marginal agricultural lands of sub-Saharan Africa and the Indian subcontinent. It is also a summer forage crop in the southern USA, Australia and Latin America, and is the preferred mulch in Brazilian no-till soybean production systems. Use of molecular marker technology for pearl millet genetic improvement has been limited. Progress is hampered by insufficient numbers of PCR-compatible co-dominant markers that can be used readily in applied breeding programmes. Therefore, we sought to develop additional SSR markers for the pearl millet research community.</p> <p>Results</p> <p>A set of new pearl millet SSR markers were developed using available sequence information from 3520 expressed sequence tags (ESTs). After clustering, unigene sequences (2175 singlets and 317 contigs) were searched for the presence of SSRs. We detected 164 sequences containing SSRs (at least 14 bases in length), with a density of one per 1.75 kb of EST sequence. Di-nucleotide repeats were the most abundant followed by tri-nucleotide repeats. Ninety primer pairs were designed and tested for their ability to detect polymorphism across a panel of 11 pairs of pearl millet mapping population parental lines. Clear amplification products were obtained for 58 primer pairs. Of these, 15 were monomorphic across the panel. A subset of 21 polymorphic EST-SSRs and 6 recently developed genomic SSR markers were mapped using existing mapping populations. Linkage map positions of these EST-SSR were compared by homology search with mapped rice genomic sequences on the basis of pearl millet-rice synteny. Most new EST-SSR markers mapped to distal regions of linkage groups, often to previous gaps in these linkage maps. These new EST-SSRs are now are used by ICRISAT in pearl millet diversity assessment and marker-aided breeding programs.</p> <p>Conclusion</p> <p>This study has demonstrated the potential of EST-derived SSR primer pairs in pearl millet. As reported for other crops, EST-derived SSRs provide a cost-saving marker development option in pearl millet. Resources developed in this study have added a sizeable number of useful SSRs to the existing repertoire of circa 100 genomic SSRs that were previously available to pearl millet researchers.</p

QTL and QTL × Environment Effects on Agronomic and Nitrogen Acquisition Traits in Rice

Agricultural environments deteriorate due to excess nitrogen application. Breeding for low nitrogen responsive genotypes can reduce soil nitrogen input. Rice genotypes respond variably to soil available nitrogen. The present study attempted quantification of genotype × nitrogen level interaction and mapping of quantitative trait loci (QTLs) associated with nitrogen use efficiency (NUE) and other associated agronomic traits. Twelve parameters were observed across a set of 82 double haploid (DH) lines derived from IR64/Azucena. Three nitrogen regimes namely, native (0 kg/ha; no nitrogen applied), optimum (100 kg/ha) and high (200 kg/ha) replicated thrice were the environments. The parents and DH lines were significantly varying for all traits under different nitrogen regimes. All traits except plant height recorded significant genotype×environment interaction. Individual plant yield was positively correlated with nitrogen use efficiency and nitrogen uptake. Sixteen QTLs were detected by composite interval mapping. Eleven QTLs showed significant QTL×environment interactions. On chromosome 3, seven QTLs were detected associated with nitrogen use, plant yield and associated traits. A QTL region between markers RZ678, RZ574 and RZ284 was associated with nitrogen use and yield. This chromosomal region was enriched with expressed gene sequences of known key nitrogen assimilation genes

Influence of season, age and breed on prevalence of haemoprotozoan diseases in cattle of Tamil Nadu, India

Aim: To assess the prevalence of haemoprotozoan diseases in cross-bred and indigenous cattle in relation to season, age and breed in Western part of Tamil Nadu, India. Materials and Methods: A total of 2637 blood smears were screened for haemoprotozoan diseases and samples were received from the college hospital and veterinary dispensaries in Western part of Tamil Nadu, India. Blood smears were stained using Giemsa's technique and examined under oil immersion. Results: Microscopic examination of blood smears revealed an overall prevalence of 16.64 %; of which theileriosis was 13 %, followed by anaplasmosis 2.64 % and then babesiosis 1.0%. Among the haemoprotozoan diseases, the prevalence of theileriosis was significantly (p<0.05) high during summer (14.4%), followed by moderate in monsoon (13.8%) and less in fair (11.5%) seasons. However, there was no significant seasonal influence on the prevalence of babesiosis and anaplasmosis. The data on influence of breed revealed that there was a significantly (p<0.05) high prevalence of haemoprotozoan diseases in Holstein Friesian (HF) and Jersey cross breeds than indigenous breed and the occurrence of these haemoprotozoan diseases was found to be high among the age groups of 2-7 years in cross-bred animals and below 2 years in indigenous animals. Conclusion: The present study suggests that Western part of Tamil Nadu is highly endemic for theileriosis and occurrence of the disease was high during summer. Cross-bred animals aged 2-7 years are highly susceptible to these haemoprotozoan diseases than indigenous animals

Perspectives of Neer-Nirakkuri (colour of the urine) and diagnosis according to Saint Therar

The bioactive compounds and pH of the urine are the reason for dip and spike of its surface appearance. The colour variations relate on its primary and secondary metabolites due to the presence of bacterial reaction. The urine analysis may show a light on serious medical condition lurking unnoticed by us. The time to evolve the nature of the urine with its thousands of unique chemicals drained by the kidneys is the future scenario for cost effective diagnosis. According to Siddha philosophy urine is made up of appu bhutham (water element) like blood, semen, fat and bone marrow. The food stuffs are pancha bhutha (five elements) in nature which nourish all ezhu udarkattugal (the seven body constituents) for the functioning of subtle principles vali (vaadham), azhal (pittham) and iyam (kabham). The functional change of seven body constituents due to the derangement of intrinsic and extrinsic factors like diet, work, environmental change etc. cause vitiation mukkutram viz. vali, azhal and iyam making them deranged to develop various diseases. &nbsp;Siddha literatures explain five different colours of urine. Every colour has further variants. Yellow colour urine has six variants, red colour urine has four variants, green colour urine has five variants, black colour urine has four variants and white colour urine has two variants. Colour of the urine and its interpretation is only the beginning to assess the severity of problem alerting an individual that something is wrong in metabolism or renal function. The colour of the urine is further correlated with food already had. The taste, sediments, smell, froth, mass are the various factors certainly considered for further evaluation. The colour of the urine and its relation with humour disorder and five elements would explore scope for future research cost effectively. Keywords: Colour, Neerkuri, Nirakkuri, Therayar, Siddha marutthuva sudar, mukkutram, panchabhutha

Virulence Diversity in North Indian Isolates of Sclerospora Graminicola, the pearl millet Downy Mildew Pathogen

On-farm surveys were conducted in the Uttar Pradesh (India) during the two rainy seasons 2007 and 2008 to monitor pearl millet (Pennisetum glaucum) downy mildew incidence. Twenty-one isolates of Sclerospora graminicola, the pearl millet downy mildew pathogen, were collected from different hybrid cultivars. These isolates were established on seedlings of the highly susceptible line 7042S grown in the greenhouse and were characterized for their virulence diversity using a set of seven host differential lines. Quantitative differences in virulence among pathogen isolates were determined by calculating virulence index (percent disease incidence × latent period-1). Results were submitted to cluster analysis using the Average Linkage method to determine similarity among pathogen isolates. The two highly virulent isolates, Sg 492 from Aligarh and Sg 510 from Badaun, representing geographically diverse locations were selected for use in greenhouse screening of pearl millet breeding lines

SSR allelic diversity in relation to morphological traits and resistance to grain mould in sorghum

Allelic variation at 46 simple sequence repeat (SSR) marker loci well distributed across the sorghum genome was used to assess genetic diversity among 92 sorghum lines, 74 resistant and 18 susceptible to grain mould. Of the 46 SSR markers, 44 were polymorphic, with the number of alleles ranging from 2 to 20 with an average of 7.55 alleles per locus. Genetic diversity among the sorghum lines was high as indicated by polymorphic information content (PIC) and gene diversity values. PIC values of polymorphic SSR markers ranged from 0.16 to 0.90, with an average of 0.54. Gene diversity among the sorghum lines varied from 0.16 to 0.91, with an average score of 0.58 per SSR marker. AMOVA indicated that 12% of the total variation observed among the sorghum lines was accounted for between grain mould resistant and susceptible types. Diversity based on six morphological traits and grain mould scores indicated major roles of panicle type and glumes coverage, followed by grain colour, in clustering of the lines. Seven grain mould resistant/ susceptible pairs with dissimilarity indices >0.50, but with similar flowering time, plant height, and panicle type/ inflorescence within each pair, were selected for use in developing recombinant inbred line mapping populations to identify genomic regions (and quantitative trait loci) associated with sorghum grain mould resistance

In silico mapping of important genes and markers available in the public domain for efficient sorghum breeding

Crop genome sequencing projects generate massive amounts of genomic sequence information, and the utilization of this information in applied crop improvement programs has been augmented by the availability of sophisticated bioinformatics tools. Here, we present the possible direct utilization of sequence data from a sorghum genome sequencing project in applied crop breeding programs. Based on sequence homology, we aligned all publicly available simple sequence repeat markers on a sequencebased physical map for sorghum. Linking this physical map with already existing linkage map(s) provides better options for applied molecular breeding programs. When a new set of markers is made available, the new markers can be first aligned on a sequence-based physical map, and those located near the quantitative trait locus (QTL) can be identified from this map, thereby reducing the number of markers to be tested in order to identify polymorphic flanking markers for the QTL for any given donor 9 recurrent parent combination. Polymorphic markers that are expected (on the basis of their position on the sequence-based physical map) to be closely linked to the target can be used for foreground selection in marker-assisted breeding. This map facilitates the identification of a set of markers representing the entire genome, which would provide better resolution in diversity analyses and further linkage disequilibrium mapping. Filling the gaps in existing linkage maps and fine mapping can be achieved more efficiently by targeting the specific genomic regions of interest. It also opens up new exciting opportunities for comparative mapping and for the development of new genomic resources in related crops, both of which are lagging behind in the current genomic revolution. This paper also presents a number of examples of potential applications of sequence-based physical map for sorghum

Identification and Characterization of Toxigenic Fusaria Associated with Sorghum Grain Mold Complex in India

Fusarium species are dominant within the sorghum grain mold complex. Some species of Fusarium involved in grain mold complex produce mycotoxins, such as fumonisins. An attempt was made to identify Fusarium spp. associated with grain mold complex in major sorghum-growing areas in India through AFLP-based grouping of the isolates and to further confirm the species by sequencing part of alpha-Elongation factor gene and comparing the sequences with that available in the NCBI database. The dendrogram generated from the AFLP data clustered the isolates into 5 groups. Five species of Fusarium-F. proliferatum, F. thapsinum, F. equiseti, F. andiyazi and F. sacchari were identified based on sequence similarity of alpha-Elongation factor gene of the test isolates with those in the NCBI database. Fusarium thapsinum was identified as predominant species in Fusarium-grain mold complex in India and F. proliferatum as highly toxigenic for fumonisins production. Analysis of molecular variance (AMOVA) revealed 54% of the variation in the AFLP patterns of 63 isolates was due to the differences between Fusarium species, and 46% was due to differences between the strains within a species

Assessment of genetic diversity in the sorghum reference set using EST-SSR markers

Selection and use of genetically diverse genotypes are key factors in any crop breeding program to develop cultivars with a broad genetic base. Molecular markers play a major role in selecting diverse genotypes. In the present study, a reference set representing a wide range of sorghum genetic diversity was screened with 40 EST-SSR markers to validate both the use of these markers for genetic structure analyses and the population structure of this set. Grouping of accessions is identical in distance-based and model-based clustering methods. Genotypes were grouped primarily based on race within the geographic origins. Accessions derived from the African continent contributed 88.6 % of alleles confirming the African origin of sorghum. In total, 360 alleles were detected in the reference set with an average of 9 alleles per marker. The average PIC value was 0.5230 with a range of 0.1379–0.9483. Sub-race, guinea margaritiferum (Gma) from West Africa formed a separate cluster in close proximity to wild accessions suggesting that the Gma group represents an independent domestication event. Guineas from India and Western Africa formed two distinct clusters. Accessions belongs to the kafir race formed the most homogeneous group as observed in earlier studies. This analysis suggests that the EST-SSR markers used in the present study have greater discriminating power than the genomic SSRs. Genetic variance within the subpopulations was very high (71.7 %) suggesting that the germplasm lines included in the set are more diverse. Thus, this reference set representing the global germplasm is an ideal material for the breeding community, serving as a community resource for trait-specific allele mining as well as genome-wide association mapping

Evaluation of genetic diversity in Magnaporthe grisea populations adapted to finger millet using simple sequence repeats (SSRs) markers

Finger millet blast caused by Magnaporthe grisea (anamorph: Pyricularia grisea) is a great threat to finger millet production worldwide. Genetic diversity and population structure of 72 M. grisea isolates collected from finger millet (56), foxtail millet (6), pearl millet (7) and rice (3) from major crop growing areas in India was studied using 24 SSR markers. None of the SSRs detected polymorphism in the M. grisea isolates from pearl millet. Seventeen SSR markers were polymorphic in the 65 non pearl millet isolates and detected 105 alleles, of which one was rare, 83 common, 9 frequent and 12 most frequent. A model-based population structure analysis of the genomic data identified two distinct populations with varying levels of ancestral admixtures among the 65 M. grisea isolates. Analysis of molecular variance (AMOVA) indicated that 52% of the total variation among the isolates used in this study was due to differences between the pathogen populations adapted to different hosts, 42% was due to differences in the isolates from the same host, and the remaining 6% due to heterozygosity within isolates. High genetic variability present in M. grisea isolates calls for the continuous monitoring of M. grisea populations anticipating blast resistance breakdown in finger millet cultivars grown in India