K. V. Prabhu

Molecular fingerprinting of hybrids and assessment of genetic purity of hybrid seeds in rice using microsatellite markers

Microsatellite markers were used for fingerprinting of hybrids, assessing variation within parental lines and testing the genetic purity of hybrid seed lot in rice. Ten sequence tagged microsatellite sites (STMS) markers were employed for fingerprinting 11 rice hybrids and their parental lines. Nine STMS markers were found polymorphic across the hybrids and produced unique fingerprint for the 11 hybrids. A set of four markers (RM 206, RM 216, RM 258 and RM 263) differentiated all the hybrids from each other, which can be used as referral markers for unambiguous identification and protection of these hybrids. Cluster analysis based on Jaccards similarity coefficient using UPGMA grouped the hybrids into three clusters. Within the cluster all the hybrids shared a common cytoplasmic male sterile line as female parent. The genetic similarity between the hybrids ranged from 0.33 to 0.92 with an average similarity index of 0.63. The analysis of plant-to-plant variation within the parental lines of the hybrid Pusa RH 10, using informative markers indicated residual heterozygosity at two marker loci. This highlights the importance of STMS markers in maintaining the genetic purity of the parental lines. The unique value of the restorer gene linked marker for testing the genetic purity of hybrid seeds is demonstrated for the first time.

Molecular markers linked to white rust resistance in mustard Brassica juncea

Abstract White rust, caused by Albugo candida (Pers.) Kuntze, is an economically important disease of Brassica juncea (L.) Czern. and Coss mustard, particularly in India. The most efficient and cost-effective way of protecting mustard plants from white rust disease is through genetic resistance. The objective of this study was to identify RAPD markers for white rust resistance in an F1-derived doubled-haploid (DH) population originating from a cross between white rust-susceptible and white rust-resistant breeding lines of B. juncea from the canola-quality B. juncea breeding project of the Agriculture and Agri-Food Canada-Saskatoon Research Centre. The DH population was used to screen for RAPD markers associated with white rust resistance/susceptibility using bulked segregant analysis. Two markers, WR2 and WR3, linked to white rust resistance, flanked the resistance locus Ac21 and were highly effective in identifying the presence or absence of the resistance gene in the DH population. These two markers were shown to be specific to the Russian source of white rust resistance utilized in this project. It is concluded that the availability of these RAPD markers will enhance the breeding for white rust resistance in B. juncea.

Molecular mapping of Aegilops speltoides derived leaf rust resistance gene Lr28 in wheat

In a segregating homozygous F2 population of bread wheat involving a leaf rust resistance gene Lr28 derived from Aegilops speltoides, six randomly amplified polymorphic DNA (RAPD) markers, three each in coupling and repulsion phase were identified as linked to Lr28, mapped to a region spanning 32 cM including the locus. The F2 and F3 populations were studied in the phytotron challenged with the most virulent pathotype 77-5 of leaf rust. A coupling phase linked RAPD marker S464721 and a repulsion phase linked RAPD marker S326550 flanked the gene Lr28 by a distance of 2.4± 0.016 cM on either side. The flanking markers genetically worked as co-dominant markers when analyzed together after separate amplification in the F2 population by distinguishing the homozygotes from the heterozygotes and increased the efficiency of marker assisted selection by reducing the false positives and negatives. One of the three RAPD markers, S421640 was converted to locus specific SCAR marker SCS421640 which was further truncated by designing primers internal from both ends of the original RAPD amplicon to eliminate a non-specific amplification of nearly same size. The truncated polymorphic sequence characterized amplified region marker (TPSCAR) SCS421570 was 70 bp smaller, but resulted in a single band polymorphism specific to Lr28 resistance. The TPSCAR marker was validated for its specificity to the gene Lr28 in nine different genetic backgrounds and on 43 of the 50 Lr genes of both native and alien origin, suggesting the utility of the SCAR markers in pyramiding leaf rust resistance genes in wheat.

QTL analysis of novel genomic regions associated with yield and yield related traits in new plant type based recombinant inbred lines of rice (Oryza sativa L.)

BackgroundRice is staple food for more than half of the world’s population including two billion Asians, who obtain 60-70% of their energy intake from rice and its derivatives. To meet the growing demand from human population, rice varieties with higher yield potential and greater yield stability need to be developed. The favourable alleles for yield and yield contributing traits are distributed among two subspecies i.e., indica and japonica of cultivated rice (Oryza sativa L.). Identification of novel favourable alleles in indica/japonica will pave way to marker-assisted mobilization of these alleles in to a genetic background to break genetic barriers to yield.ResultsA new plant type (NPT) based mapping population of 310 recombinant inbred lines (RILs) was used to map novel genomic regions and QTL hotspots influencing yield and eleven yield component traits. We identified major quantitative trait loci (QTLs) for days to 50% flowering (R2 = 25%, LOD = 14.3), panicles per plant (R2 = 19%, LOD = 9.74), flag leaf length (R2 = 22%, LOD = 3.05), flag leaf width (R2 = 53%, LOD = 46.5), spikelets per panicle (R2 = 16%, LOD = 13.8), filled grains per panicle (R2 = 22%, LOD = 15.3), percent spikelet sterility (R2 = 18%, LOD = 14.24), thousand grain weight (R2 = 25%, LOD = 12.9) and spikelet setting density (R2 = 23%, LOD = 15) expressing over two or more locations by using composite interval mapping. The phenotypic variation (R2) ranged from 8 to 53% for eleven QTLs expressing across all three locations. 19 novel QTLs were contributed by the NPT parent, Pusa1266. 15 QTL hotpots on eight chromosomes were identified for the correlated traits. Six epistatic QTLs effecting five traits at two locations were identified. A marker interval (RM3276-RM5709) on chromosome 4 harboring major QTLs for four traits was identified.ConclusionsThe present study reveals that favourable alleles for yield and yield contributing traits were distributed among two subspecies of rice and QTLs were co-localized in different genomic regions. QTL hotspots will be useful for understanding the common genetic control mechanism of the co-localized traits and selection for beneficial allele at these loci will result in a cumulative increase in yield due to the integrative positive effect of various QTLs. The information generated in the present study will be useful to fine map and to identify the genes underlying major robust QTLs and to transfer all favourable QTLs to one genetic background to break genetic barriers to yield for sustained food security.

Development and validation of SCAR markers co-segregating with an Agropyron elongatum derived leaf rust resistance gene Lr24 in wheat

SummaryAn Agropyron elongatum-derived leaf rust resistance gene Lr24 located on chromosome 3DL of wheat was tagged with six random amplified polymorphic DNA (RAPD) markers which co-segregated with the gene. The markers were identified in homozygous resistant F2 plants taken from a population segregating for leaf rust resistance generated from a cross between two near-isogenic lines (NILs) differing only for Lr24. Phenotyping was done by inoculating the plants with pathotype 77-5 of Puccinia triticina. To enable gene-specific selection, three RAPD markers (S1302609, S1326615 and OPAB-1388) were successfully converted to polymorphic sequence characterized amplified region (SCAR) markers, amplifying only the critical DNA fragments co-segregating with Lr24. The SCAR markers were validated for specificity to the gene Lr24 in wheat NILs possessing Lr24 in 10 additional genetic backgrounds including the Thatcher NIL, but not to 43 Thatcher NILs possessing designated leaf rust resistance genes other than Lr24. This indicated the potential usefulness of these SCAR markers in marker assisted selection (MAS) and for pyramiding leaf rust resistance genes in wheat.

Molecular breeding for the development of multiple disease resistance in Basmati rice

Marker assisted backcross breeding for combining three resistance genes (xa13 and Xa21 for Bacterial Blight, Pi54 for blast) and a major QTL (qSBR11-1 for resistance to Sheath blight) in Basmati rice.

Transcriptome-wide analysis of WRKY transcription factors in wheat and their leaf rust responsive expression profiling

AbstractWRKY, a plant-specific transcription factor family, has important roles in pathogen defense, abiotic cues and phytohormone signaling, yet little is known about their roles and molecular mechanism of function in response to rust diseases in wheat. We identified 100 TaWRKY sequences using wheat Expressed Sequence Tag database of which 22 WRKY sequences were novel. Identified proteins were characterized based on their zinc finger motifs and phylogenetic analysis clustered them into six clades consisting of class IIc and class III WRKY proteins. Functional annotation revealed major functions in metabolic and cellular processes in control plants; whereas response to stimuli, signaling and defense in pathogen inoculated plants, their major molecular function being binding to DNA. Tag-based expression analysis of the identified genes revealed differential expression between mock and Puccinia triticina inoculated wheat near isogenic lines. Gene expression was also performed with six rust-related microarray experiments at Gene Expression Omnibus database. TaWRKY10, 15, 17 and 56 were common in both tag-based and microarray-based differential expression analysis and could be representing rust specific WRKY genes. The obtained results will bestow insight into the functional characterization of WRKY transcription factors responsive to leaf rust pathogenesis that can be used as candidate genes in molecular breeding programs to improve biotic stress tolerance in wheat.

Genomic Selection in the Era of Next Generation Sequencing for Complex Traits in Plant Breeding

Genomic selection (GS) is a promising approach exploiting molecular genetic markers to design novel breeding programs and to develop new markers-based models for genetic evaluation. In plant breeding, it provides opportunities to increase genetic gain of complex traits per unit time and cost. The cost-benefit balance was an important consideration for GS to work in crop plants. Availability of genome-wide high-throughput, cost-effective and flexible markers, having low ascertainment bias, suitable for large population size as well for both model and non-model crop species with or without the reference genome sequence was the most important factor for its successful and effective implementation in crop species. These factors were the major limitations to earlier marker systems viz., SSR and array-based, and was unimaginable before the availability of next-generation sequencing (NGS) technologies which have provided novel SNP genotyping platforms especially the genotyping by sequencing. These marker technologies have changed the entire scenario of marker applications and made the use of GS a routine work for crop improvement in both model and non-model crop species. The NGS-based genotyping have increased genomic-estimated breeding value prediction accuracies over other established marker platform in cereals and other crop species, and made the dream of GS true in crop breeding. But to harness the true benefits from GS, these marker technologies will be combined with high-throughput phenotyping for achieving the valuable genetic gain from complex traits. Moreover, the continuous decline in sequencing cost will make the WGS feasible and cost effective for GS in near future. Till that time matures the targeted sequencing seems to be more cost-effective option for large scale marker discovery and GS, particularly in case of large and un-decoded genomes.

Molecular Mapping of a Gene for Fertility Restoration of Wild Abortive (WA) Cytoplasmic Male Sterility using a Basmati Rice Restorer Line

The inheritance and molecular mapping of a fertility restorer gene in basmati quality restorer line PRR-78 was carried out using an F2 mapping population from the cross IR58025A X PRR-78 employing microsatellite markers. Dominant monogenic control of fertility restoration was observed in the F2, and further confirmed by test cross data. Out of 44 sequence tagged microsatellite (STMS) markers used in the bulked segregant analysis (BSA), four differentiated the fertile bulk from the sterile bulk as well as the two parental lines from each other. One of these markers, RM258 located on chromosome 10, was found linked to the restorer gene at a distance of9.5 cM. Considering the RM258 location, additional STMS (RM171 and RM294A) and sequence tagged site (STS) primers derived from restriction fragment length polymorphic (RFLP) clones (G2155 and C1361) linked to fertility restorer gene(s) in other populations, were also used to find out a marker more tightly linked to the restorer gene. However, of these, RM171, RM294A and G2155 based primers amplified monomorphic fragments between parental lines and no amplification was observed with C1361. Cleaved amplified polymorphic sequence (CAPS) analysis of non-polymorphic STMS and STS markers and random amplified polymorphic DNA (RAPD) analysis using five random primers reportedly linked to restorer gene in other populations, also failed to differentiate the two parents. While, the marker RM258 is being used in the restorer breeding to identify putative restorer lines, search for additional tightly linked markers is underway.

De Novo Assembled Wheat Transcriptomes Delineate Differentially Expressed Host Genes in Response to Leaf Rust Infection

Pathogens like Puccinia triticina, the causal organism for leaf rust, extensively damages wheat production. The interaction at molecular level between wheat and the pathogen is complex and less explored. The pathogen induced response was characterized using mock- or pathogen inoculated near-isogenic wheat lines (with or without seedling leaf rust resistance gene Lr28). Four Serial Analysis of Gene Expression libraries were prepared from mock- and pathogen inoculated plants and were subjected to Sequencing by Oligonucleotide Ligation and Detection, which generated a total of 165,767,777 reads, each 35 bases long. The reads were processed and multiple k-mers were attempted for de novo transcript assembly; 22 k-mers showed the best results. Altogether 21,345 contigs were generated and functionally characterized by gene ontology annotation, mining for transcription factors and resistance genes. Expression analysis among the four libraries showed extensive alterations in the transcriptome in response to pathogen infection, reflecting reorganizations in major biological processes and metabolic pathways. Role of auxin in determining pathogenesis in susceptible and resistant lines were imperative. The qPCR expression study of four LRR-RLK (Leucine-rich repeat receptor-like protein kinases) genes showed higher expression at 24 hrs after inoculation with pathogen. In summary, the conceptual model of induced resistance in wheat contributes insights on defense responses and imparts knowledge of Puccinia triticina-induced defense transcripts in wheat plants.