Identification of Parental Lines and Hybrid using Molecular Techniques in CGMS based Chilli (Capsicum annuum L.) Hybrid UARChH42 (JCH42)

Background: Molecular markers are the landmarks on DNA that identifies a particular sequence of base pairs coding for a character. SCAR (Sequenced Characterized Amplified Region) markers are proving to be more effective in identification of genotypes as they are PCR based co-dominant markers. In view of plant variety registration under PPV and FRA, 2001 molecular characterization/ identification of the variety is essential to ascertain the trueness of the variety, hence present studies have been planned and executed. Methods: In present investigation CGMS based chilli hybrid UARChH42 and its parental lines were identified using molecular techniques. A line, B line, R line and hybrid seedlings were used for DNA extraction and characterized on the basis of polymorphism with respect to sterility or fertility by using SCAR markers. Result: P1 and P2 and coxII-SCAR which are sterility specific markers could amplify A line and hybrid showing a definite band. CRF-SCAR 870 is a fertility specific marker amplified R line and hybrid. Also CMS-SCAR 130 and CMS-SCAR 130/140 were able to identified A line, B line, R line and hybrid. Two of the markers viz. orf-456-SCAR atp6-SCAR and were not able to specify any case of parental lines or hybrid identification. Hybridity of UARChH42 (JCH42) chilli hybrid was determined by any similarity in the banding pattern with any of its parent. This study would help in the fulfillment of the requirements of protection of plant varieties and farmers’ rights authority (PPVFRA), New Delhi for registration purpose.

Combination Breeding and Marker-Assisted Selection to Develop Late Blight Resistant Potato Cultivars

(1) Background: Although resistance to pathogens and pests has been researched in many potato cultivars and breeding lines with DNA markers, there is scarce evidence as to the efficiency of the marker-assisted selection (MAS) for these traits when applied at the early stages of breeding. A goal of this study was to estimate the potential of affordable DNA markers to track resistance genes that are effective against the pathogen Phytophthora infestans (Rpi genes), as a practical breeding tool on a progeny of 68 clones derived from a cross between the cultivar Sudarynya and the hybrid 13/11-09. (2) Methods: this population was studied for four years to elucidate the distribution of late blight (LB) resistance and other agronomical desirable or simple to phenotype traits such as tuber and flower pigmentation, yield capacity and structure. LB resistance was phenotypically evaluated following natural and artificial infection and the presence/absence of nine Rpi genes was assessed with 11 sequence-characterized amplified region (SCAR) markers. To validate this analysis, the profile of Rpi genes in the 13/11-09 parent was established using diagnostic resistance gene enrichment sequencing (dRenSeq) as a gold standard. (3) Results: at the early stages of a breeding program, when screening the segregation of F1 offspring, MAS can halve the workload and selected SCAR markers for Rpi-genes provide useful tools.

Combination Breeding and Marker-Assisted Selection to Develop Late Blight Resistant Potato Cultivars

(1) Background: Although resistance to pathogens and pests has been researched in many potato cultivars and breeding lines with DNA markers, there is scarce evidence as to the efficiency of the marker-assisted selection (MAS) for these traits when applied at the early stages of breeding. A goal of this study was to estimate the potential of affordable DNA markers to track Rpi disease resistance genes, that are effective against the pathogen Phytophthora infestans, as a practical breeding tool on a progeny of 68 clones derived from a cross between the cultivar Sudarynya and 13/11-09. (2) Methods: this population was studied for four years to elucidate the distribution of LB resistance and other agronomical desirable or simple to phenotype traits such as tuber and flower pigmentation, capacity and structure of yield. LB resistance was phenotypically determined through natural and artificial infection and the presence/absence of nine Rpi genes was assessed via 11 sequence-characterized amplified region (SCAR) markers. To aid this analysis, the profile of Rpi genes in the 13/11-09 parent was established using diagnostic resistance gene enrichment sequencing (dRenSeq) as a gold standard. (3) Results: at the early stages of a breeding program, MAS can halve the workload when screening the segregation of F1 offspring and selected SCAR markers for Rpi-genes provide useful tools.

Distribution of Puccinia striiformis f. sp. tritici races and virulence in wheat growing regions of Kenya from 1970 TO 2014

Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a major threat to wheat (Triticum spp.) production worldwide. The objective of this study was to determine the virulence of Pst races prevalent in the main wheat growing regions of Kenya, which includes Mt. Kenya, Eastern Kenya, and the Rift Valley (Central, Southern, and Northern Rift). Fifty Pst isolates collected from 1970 to 1992 and from 2009 to 2014 were virulence phenotyped using stripe rust differential sets, and 45 isolates were genotyped with sequence characterized amplified region (SCAR) markers to differentiate among the isolates and identify aggressive strains PstS1 and PstS2. Virulence corresponding to stripe rust resistance genes Yr1, Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, Yr27 and the seedling resistance in genotype Avocet S were detected. Ten races were detected in the Pst samples obtained from 1970 to 1992, and three additional races were detected from 2009 to 2014, with a single race being detected in both periods. The SCAR markers detected both Pst1 and Pst2 strains in the collection. Increasing Pst virulence was found in the Kenyan Pst population, and that diverse Pst race groups dominated different wheat growing regions. Moreover, recent Pst races in east Africa indicated possible migration of some race groups into Kenya from other regions. This study is important in understanding Pst evolution and virulence diversity and useful in breeding wheat cultivars with effective resistance to stripe rust. Keywords: pathogenicity, Puccinia f. sp. tritici stripe (yellow) rust, Triticum aestivum

Genetic diversity analysis of Chinese Leishmania isolates and development of L. donovani complex-specific markers by RAPD

Background Leishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and classification of Chinese Leishmania has not been completely clarified to date, especially within the Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers have been performed. More analytic methods and data are still needed. Random amplified polymorphic DNA (RAPD) technology can sensitively identify slight intraspecific differences, and it is a powerful tool to seek species-specific markers. This work attempted to identify Chinese Leishmania isolates from diverse geographic regions at the genomic level. Meanwhile, specific markers of the L. donovani complex were also developed by RAPD. Methods RAPD was applied to 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into a data matrix, based on which genetic similarity was calculated, and a UPGMA dendrogram was constructed to analyse the genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of the L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified region (SCAR) markers, which were validated preliminarily in 17 available Leishmania strains in this study and analysed by bioinformatics. Results The cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, the strains of the L. donovani complex clearly divided into two clades, and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of the L. donovani complex, i.e., 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on 17 available Leishmania strains in this study. Through bioinformatic analyses, Marker 1-AD17 may have more specificity for PCR detection of VL, and Marker 3-O13 has the potential to encode a protein. Conclusions The RAPD results verified that the undescribed Leishmania species causing visceral leishmaniasis (VL) in China was a unique clade distinguished from L. donovani and revealed that there was genetic differentiation among Chinese L. donovani. The identification of L. donovani-specific markers may help to provide a foundation for future research attempting to develop new specific diagnostic markers of VL and identify specific gene functions.

Production and Molecular Identification of Interspecific Hybrids between Phaius mishmensis (Lindl. and Paxton) Rchb. f. and Phaius tankervilliae (Banks) Blume

This study aimed at assessing the hybridization feasibility and evaluating genetic fidelity of the hybrid seedlings originated from Phaius mishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume. Intra- and interspecific hybridization between Phaius mishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume were examined to establish the primary hybrid, observe their cross ability and identify the F1 hybrids using sequence-characterized amplified region (SCAR) markers. Self-incompatibility and cross ability of P. mishmensis and P. tankervilliae were tested before starting the breeding program. Results showed that they were self-compatible orchids. The interspecific hybridization between P. mishmensis and P. tankervilliae was achieved with the highest pod setting (80%), seed germination percentage (94.8%) and the rate of protocorm development into mature seedlings (stage 6) (10.6%), but the smallest size of embryo with width 46.5 μm, length 67.3 μm was also observed when P. mishmensis was taken as the female parent. A comparative study on leaf morphology and anatomy of plantlets regenerated from intra- and interspecific hybrids of P. mishmensis and P. tankervilliae showed a transitional character to the parental species. Herein, the presence of interspecific hybrids between P. mishmensis and P. tankervilliae, as well as their reciprocal cross, was verified using Pmis524 SCAR markers developed by the decamer primer.

Development of an ISSR based SCAR marker to identify small cardamom Malabar (prostrate panicle) variety (Elettaria cardamomum Maton.)

Small cardamom (Elettaria cardamomum Maton) a perennial monocot, exhibits an array of variation in nature, mainly due to cross pollination. Based on the nature of the panicle orientation, cardamom is broadly grouped into three main ‘cultivated types’ – Malabar, Mysore and Vazhuka having prostrate, erect and semi-erect panicles respectively. These morphologically discriminative markers manifest itself during panicle emergence as is only possible. Among the three varieties Malabar variety is relatively superior with respect to different qualitative and quantitative characteristics. The objective of the present study was to develop and characterize molecular markers for enabling differentiation of Malabar variety at juvenile stage. One accession specific ISSR marker generated by UBC 866 was selected which consistently amplified an intact, distinct, ̴1500bp band specifically in individuals of Malabar variety, which was therefore cloned, sequenced and characterized. Ten primers were designed from the sequences for converting them to SCAR markers. The developed SCAR markers were tested for variety specificity and one primer pair (SBBT4F/SBBT3R) was validated using small cardamom accessions belonging to Malabar variety from different geographic locations and varieties with erect panicles as well as hybrids. The findings suggest that the SCAR marker is promising in identifying cardamom varieties having prostrate panicle (Malabar) and therefore is expected to make significant contributions in selection of F1 hybrids during breeding programmes.