Characterization of the A4 cytoplasmic male-sterile lines of sorghum using RFLP of mtDNA

Three sorghum cytoplasmic male sterile lines CSV4 A(V), CSV4 A(G₁) and CSV4 A(M), grouped as A₄, were compared with a milo (A₁) and two other non-milo (A₂ and A₃) cytoplasms for their RFLP patterns of mitochondrial DNA (mtDNA). A 9.7 kb clone from pearl millet mtDNA discriminated each of the three A₄ entries whereas other maize and pearl millet mtDNA clones used could not distinguish this group completely. The molecular differences within the A₄ cytoplasmic group offer some explanation for the inconsistency in the fertility restoration behaviour of these A₄ lines obtained with a definite set of testers in the field. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and characterization of EST-SSRs in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

In recent years, microsatellites have become the most used markers for studying population genetic diversity. The increased availability of the DNA sequences has given the possibility to develop EST-derived SSR markers. A total of 1,927 ESTs of Eleusine coracana available in the NCBI database were mined for SSRs. Di-nucleotides are the most occurring motifs accounting for more than 50% of the repeats, of which GA was the most abundant motif and tetra-nucleotides are the least occurring motifs. Of the 132 markers identified, 30 primer pairs based were synthesized. SSR markers were used for variety discrimination and genetic assessment in 15 finger millet accessions; 20 primers showed polymorphism and 13 primers were identified as having a PIC value above 0.5. On the basis of the distribution of these polymorphic alleles, the 15 accessions were classified into two groups. This study has demonstrated the potential of EST-derived SSR primer pairs in finger millet. These primers will serve as valuable source for further breeding programs.     

Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

RFLP analysis of cytoplasmic male-sterile lines of pigeonpea [Cajanus cajan (L.) Millsp.] developed by interspecific crosses

Total DNA from three putative cytoplasmic male sterile (CMS) progenies derived from crosses between the wild species Cajanus sericeus and the cultivated species Cajanus cajan, five C. cajan, one accession of C. sericeus and two genetic male sterile lines of pigeonpea were compared for their RFLP patterns using maize mitochondrial DNA (mtDNA) specific probes. Three putative cytoplasmic male sterile (CMS) progenies from the multiple cross genome transfer of pigeonpea lines (CMS 7–1, CMS 12–3, and CMS 33–1) showed hybridization patterns identical to that of C. sericeus when DNA was digested with EcoRI and HindIII and probed with maize mtDNA clones. The results suggested that these putative CMS progenies have the mitochondria of the female wild species parent. The hybridization patterns of the three male parental lines used in the development of the CMS progenies were similar in all the restriction enzyme-probe combinations except HindIII-atp6. The genetic male sterile lines, MS Prabhat and QMS 1 differed from each other in their hybridization pattern. The genomic DNA hybridization pattern of HindIII digested DNA from ICPL 87 differed from the other pigeonpea lines when probed with the maize mtDNA clones. The cluster analysis of the hybridization data suggested the occurrence of variation in the mitochondrial genome even among the cultivated species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Validation of molecular markers linked to fertility restorer gene(s) for WA-CMS lines of rice

The present study was carried out with the objective to validate the molecular markers, which have been previously reported to be linked to fertility restorer (Rf) gene(s) for WA-CMS lines of rice. Two mapping populations involving fertility restorer lines for WA-cytoplasm, viz., (i) an F₂ population derived from the cross IR58025A/KMR3R consisting of 347 plants and (ii) a BC₁F₁ population derived from the cross IR62829A/IR10198R//IR62829A consisting of 130 plants were analyzed. Nine SSR and three CAPS markers reported to be linked to Rf genes along with two previously unreported SSR markers were analyzed in the mapping populations. In both the populations studied, the trait of fertility restoration was observed to be under digenic control. Eight SSR markers (RM6100, RM228, RM171, RM216, RM474, RM311, MRG4456 and pRf1&2) showed polymorphism between the parents of the F₂ population, while the SSR markers RM6100 and RM474 showed polymorphism between the parents of both the F₂ and BC₁F₁ populations. Only one CAPS marker, RG146FL/RL was polymorphic between the parents of the BC₁F₁ population. RM6100 was observed to be closely segregating with fertility restoration in both the mapping populations and was located at a distance of ~1.2 cM. The largest phenotypic variation was accounted for the region located between RM311 and RM6100. Using the marker-trait segregation data derived from analysis of both the mapping populations, a local linkage map of the genomic region around Rf-4, a major fertility restoration locus on Chromosome 10 was constructed, and RM6100 was observed to be very close to the gene at a distance of 1.2 cM. The accuracy of the marker RM6100 in predicting fertility restoration was validated in 21 restorers and 18 maintainers. RM6100 amplified the Rf-4 linked allele in a majority of the restorers with a selection accuracy of 94.87%. Through the present study, we have established the usefulness of the marker RM6100 in marker-assisted selection for fertility restoration in segregating populations and identification of restorers while screening rice germplasm for their fertility restoration ability.     

Diversity in selected wild and cultivated species of pigeonpea using RFLP of mtDNA

Diversity in 28 accessions representing 12 species of the genus, Cajanus arranged in 6 sections including 5 accessions of the cultivated species, C. cajan, and 4 species of the genus Rhyncosia available in the germplasm collection at ICRISAT was assessed using RFLP with maize mtDNA probes. Cluster analysis of the Southern blot hybridization data with 3 restriction enzymes – 3 probe combinations placed the genus Rhyncosia in a major group well separated from all the species belonging to the genus Cajanus. Within the genus Cajanus, the 4 accessions of C. platycarpus belonging to section Rhynchosoides formed a separate group in contrast to those in other sections of pigeonpea. In the section, Cajanus all the 5 accessions of C. cajan were grouped together and C. cajanifolius belonging to the same section was in a subgroup by itself closer to the main group. The four accessions of C. scarabaeoides, were together and the other species belonging to section Cantharospermum were in different subgroups. The intra-specific variation was seen even within accessions of certain pigeonpea wild species such as C. scarabaeoides, C. platycarpus, C. acutifolius, and even the cultivated species of C. cajan. This study suggests that RFLP of mtDNA can be used for the diversity analysis of pigeonpea and it gives some indications on the maternal lineage among the species. The variations in the mitochondrial DNA hybridization patterns also suggest the extensive rearrangement of the organelle genome among the Cajanus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic and Phenotypic Diversity in Downy-mildew-resistant Sorghum (Sorghum bicolor (L.) Moench) Germplasm

Genetic and phenotypic diversity among randomly selected 36 downy-mildew-resistant sorghum accessions were assessed, the former using 10 simple sequence repeat (SSR) marker loci and the latter using 20 phenotypic traits. The number of alleles (a _j ) at individual loci varied from five to 14 with an average of 8.8 alleles per locus. Nei's gene diversity (H _j ) varied from 0.59 to 0.92 with an average of 0.81 per locus. High gene diversity and allelic richness were observed in races durra caudatum (H _j = 0.76, a _j = 4.3) and guinea caudatum (H _j = 0.76, a _j = 3.8) and in east Africa (H _j = 0.78, a _j = 7.2). The regions were genetically more differentiated than the races as indicated by Wright's F _st. The pattern of SSR-based clustering of accessions was more in accordance with their geographic proximity than with their racial likeness. This clustering pattern matched little with that obtained from phenotypic traits. The inter-accession genetic distance varied from 0.30 to 1.00 with an average of 0.78. Inter-accession phenotypic distance varied from 0.01 to 0.55 with an average of 0.33. Eleven accession-pairs had phenotypic distance of more than 0.50 and genetic distance of more than 0.70. These could be used as potential parents in a sorghum downy mildew resistance-breeding program.     

Identification of SSR markers which could differentiate blast disease resistance accessions in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

Microsatellites also known as SSR are the class of repetitive DNA sequences present throughout the genome of all eukaryotic organisms. The present study identified SSRs using biotinylated beads capture method. Ten sets of primers were designed based on sequences having at least (CT)10 repeats. A total of 27 accessions having a mix of both African (resistant) and Indian origin (resistant and susceptible) were assessed using 30 microsatellite markers. Amplification products were obtained for all 30 primers studied; 25 out of these primers were found to be polymorphic with 13 primers showing two alleles per locus. The current study identified markers which could differentiate between resistant and susceptible accessions and also segregate accessions based on geographical region. These informative SSR markers can be used in finger millet genetic improvement projects.     

Contribution of Pre- and Post-anthesis Photosynthates to Grains in Pearl Millet under Water Deficit

When rains end early, grain yield productivity of pearl millet ( Pennisetum glaucum [L.] R. Br.), a rainfed cereal crop of arid and semi-arid regions is limited by water deficits during flowering and grainfilling. Reserves of assimilates present at flowering and available for later remobilization to the grains, could act has buffer under water deficit conditions during flowering and grainfilling. The contribution of pre- and post-anthesis assimilates to grain yield under water-deficit conditions during flowering and grainfilling was investigated by ¹⁴C labeling of leaves at boot and 10 days after anthesis of field-grown plants. Water deficit resulted in 20 percent reduction to grain yield in the main tiller. The distribution pattern of labeled carbon at harvest in various plant parts in water deficit and irrigated control treatments was similar. About 25 percent of the pre-anthesis assimilates was translocated to the grain at harvest, while 95 percent of the post-anthesis (current) assimilates was translocated to the grain. At grain maturity, about 60 percent of the pre-anthesis assimilate was distributed in inflorescence structures, and in upper three inter-nodes which were the active sinks at boot stage. Post-anthesis assimilates were directed towards the grain development which was the active sink after flowering. In conclusion, about 25 percent of the pre-anthesis assimilates are translocated to the grain at harvest and there was no additional contribution under water deficit and post-anthesis assimilates were primarily translocated to the developing grain.