Jogeswar Panigrahi

Marker-Assisted Breeding for Stress Resistance in Crop Plants

Molecular markers have extensively been used for tagging and mapping of genes and QTLs conferring resistance to biotic and abiotic stresses. These tools have also been used for screening of germplasms, fingerprinting, and marker-assisted breeding in crop systems. This chapter presents an overview on the basic concepts of molecular mapping and marker-assisted breeding and its most widely used applications in crop improvement programs, viz., marker-assisted backcross breeding, gene introgression, gene pyramiding, and marker-assisted selection at an early generation, with emphasis on stress-related traits and examples from several crops. We have also discussed some quantitative aspects of marker-based introgression, backcross breeding, and gene pyramiding programs. We have also added a note on breeding by design and genomic selection as tools for future breeding endeavors aiming at introgression of stress resistance into high-yielding cultivars. Harnessing the full potential of marker-aided breeding for improvement of stress resistance in crop systems will require a multidisciplinary approach and integrated knowledge of the molecular and physiological processes influencing the stress-related traits. Hence, marker-aided breeding for stress resistance in the post-genomic era poses great challenge for molecular breeders to realize the target objectives.

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

Abstract This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl−1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA3), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO4·7H2O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.

Identification of inter-specific hybrid between Cajanus cajan (L.) and C. cajanifolius (H.) using cyto-morphological and DNA markers

Cajanus cajanifolius, the putative progenitor of domesticated C. cajan, has many agro-economic traits those need to be introgressed into the cultivars of pigeonpea to widen its genetic base. In this study interspecific F1 hybrids, involving C. cajan (ICPL 87119) and C. cajanifolius (ICPW 31), were raised, and their response to bruchid infestation also assessed. Subsequently, the hybrids were characterized using the morphological, cytological and DNA markers based attributes. The F1 hybrids showed resistance to bruchid infestation caused by Callosbrochus maculatus and C. chinensis. The morphological traits of F1 hybrids with respect to their parents revealed that these were intermediate between the parents with predominance of the characteristics of both the parents. The pollen mother cells (PMC) obtained from the F1 hybrids showed regular meiosis involving 11 bivalents in majority of the cases which affirmed the genetic homology between these two species. But, heteromorphism was noticed for two bivalents during diakinesis. DNA marker (SCoT and SSR) analysis also revealed polymorphic fragments between the parents and their inheritance to the putative F1 hybrid. In conclusion, the simultaneous use of cyto-morphological analysis and DNA marker based genotyping demonstrated the genetic divergence between the parental genotypes with contrasting response to bruchid infestation and the hybridity of the F1 plants with accuracy.

Analysis of Signaling Pathways During Heavy Metal Toxicity: A Functional Genomics Perspective

Abiotic stresses have major limiting factors for plant growth and crop productivity. Plants have different mechanisms to maintain the physiological concentrations of essential metal ions and to minimize exposure to non-essential heavy metals. Some mechanisms are ubiquitous because they are also required for general metal homeostasis, and they minimize the damage caused by high concentrations of heavy metals in plants by detoxification, thereby conferring tolerance to heavy metal stress. Metals in the cell are addressed using a range of storage and detoxification strategies, including metal transport, chelating, trafficking, and sequestration into the vacuole. A large number of genes encoding MAPK pathway components have a major role in cell proliferation and hormone action as well as in stress signaling. Germin-like protein genes were developed by various stresses including metal stress. Functional genomics (integrating genome sequencing, transcriptomics, proteomics, metabolomics, ionomics, and phenomics) allows large-scale gene function analysis with high-throughput technology and incorporates interaction of gene products at cellular and organism level.

RFLP mapping of loci controlling self-incompatibility in Brassica campestris and their comparative mapping with B. napus and B. oleracea

RFLP analysis of a cDNA probe SLG6, governing self incompatibility (SI) in Brassica oleracea, using a recombinant inbred population of Brassica campestris followed by genetic linkage analysis led to the detection of two marker loci, SLG6a and SLG6b controlling SI. SLG6a was mapped in linkage group (LG) 9 and was flanked by the RFLP markers ec4f10 (6.4 cM) and wg5b9 (4.2 cM). SLG6b positioned in LG 2 and was flanked by the RFLP markers wg2d11 (9.9 cM) and ec4e7 (26.9 cM). These results indicated the scope of marker-aided introgression of these genes into self-compatible genotypes for production of SI lines suitable for hybridization in B. campestris. Comparative mapping of LG 9 containing SLG6b with corresponding linkage groups of B. oleracea (BO 2) and B. napus (BN 16) led to the detection of small homologous regions with SLG6 locus linked with another RFLP locus. This evidenced for homology of the SLG genes across Brassica species and possibility of using any single cloned SLG gene for development of SI lines in any Brassica species.

Estimation of genetic diversity among 34 genotypes in the genus Cajanus with contrasting host response to the pod borer and its allied pests

The genetic divergence among 34 genotypes belonging to 12 species of genus Cajanus were carried out using plant pest interaction and DNA marker analysis. Principal component analysis based on average percentage of pod damage caused by pod borer, plume moth, and blue butterfly in the field conditions, and growth of their larva and pupa on an artificial diet in vitro dispersed these genotypes into four coordinates evincing high genetic divergence as expected. DNA marker analysis using 11 pairs of SSR and nine ISSR primers showed higher polymorphism at the species level, and these primers exhibited variation with regard to average band informativeness, resolving power, and PIC value. No single primer was able to distinguish between all the 34 genotypes of Cajanus but nine species specific amplified fragments were generated by five ISSR primers. The pairwise Jaccard’s similarity coefficient and Nei’s genetic distance values revealed a higher level of inter-specific genetic variation in the genus Cajanus. The clustering of genotypes based on Jaccard’s similarity coefficient vis-a-vis Nei’s genetic distance agreed with the sectional classification of the genus Cajanus. Seven cultivars of C. cajan and the genotypes of their wild progenitor C. cajanifolius remained in one cluster, whereas accessions of C. platycarpus and C. scarabaeoides were out grouped. The rest of the genotypes belonging to nine species of Cajanus formed another cluster. The principal coordinate analysis also supported this clustering pattern. Moreover, these findings have good many implications for future breeding endeavors aimed at the introgression of pod borer resistance alleles.

Genetic linkage mapping of loci conferring resistance to Blue butterfly (Lampides boeticus L.) and Plume moth (Exelastis atomosa Wals.) on chromosome 2 (CcLG02) in Pigeonpea

Abstract Inheritance of host resistance to blue butterfly (PBB1) and plume moth (PPM1) in interspecific mapping populations (F2, F3 and BC1) derived from a cross involving Cajanus cajan (cv. ICP-26) × Cajanus scarabaeoides (acc. ICPW-94) appeared to be under monogenic control either by a single major gene or a cluster of tightly linked genes. Bulked segregant analysis using 237 [85 simple sequence repeats (SSR), 143 random amplified polymorphic DNA (RAPD) and nine inter simple sequence repeats (ISSR)] parental polymorphic primers led to the identification of 43 markers that distinguished the resistant and susceptible bulks alike to parents, and which were also segregating among F2 progenies. Linkage analysis of these markers along with interaction phenotype score for both traits generated a linkage group consisting of 11 markers (two SSR, seven RAPD and two ISSR) and two trait loci (PBB1 and PPM1). This linkage group distributed over 133.9 cM with an average marker interval of 10.3 cM. The PBB1 and PPM1 loci were linked to each other by 11.2 cM (rf 0.110), and were flanked by ISSR marker UBC8722000 (15.9 cM), and RAPD marker OPA09910 (15.3 cM), respectively. On the basis of sequence homology of linked marker OPA09910 these two loci were assigned to chromosome 2 (CcLG02). Composite interval mapping led to the detection of two major quantitative trait loci (qPBB2.1 and qPPM2.1) controlling blue butterfly and plume moth resistance, respectively and the quantitative trait locus peaks coinciding with PBB1 and PPM1 loci on the map.

Recent progress in biotechnological interventions in Piper species: A review

Medicinal plants have tremendous potential for the development of new drugs molecule effective against various diseases. Genus Piper is the largest genus in family Piperaceae and indigenous to India with one of the most widely used spice in the world. Biotechnological innovations in Piper species have lead to the development of plant-derived molecules which exhibit a promising effect in therapeutics. The present paper reviews the genetic diversity analysis of major Piper species through DNA markers, to establish in vitro protocol for mass multiplication of elite clone of different species, phytochemical compounds extracted in various solvent systems in Piper species and comparative analysis of active compounds associated with Piper species through HPTLC analysis as well as testing their antimicrobial activities. The activity has been tested against bacteria like E. coli, Micrococcus luteus, Streptomyces epidermidis, etc. and fungi like Aspergillus niger, A. flavus and Rhizoctonia solani using agar well diffusion method. The review also highlights the use of in vitro strategy in genetic manipulation of transgenic traits using specific promoters to accomplish high tissue specific protein production and overcome biotic stresses.

Addition of Restriction Fragment Length Polymorphism Markers to the Genetic Linkage Map of Brassica rapa L. (syn. campestris)

Genetic linkage analysis of 151 restriction fragment length polymorphism (RFLP) loci, that included eight new loci, detected by the six probes in the present study, and four trait loci including seed colour, leaf pubescence, resistance to white rust caused by Albugo candida race-2 (AC-2) and race-7 (AC-7) employing the MAPMAKER/EXP 3.0 programme led to the development of 10 linkage groups (LGs) spanning over 44.4 centiMorgan (cM) to 130.4 cM containing 9 to 22 loci and two short LGs with two or three marker loci in Brassica rapa. The enriched map covers 993.1 cM of B. rapa genome with an average marker interval of 6.41. Eight new RFLP loci occupied new map positions on five linkage groups, LG 2, 3, 6, 8 and 9. Addition of these RFLP loci led to appreciable changes in the corresponding linkage groups and resulted in an increase of the total map length by 102.8 cM and of the marker interval by 0.35 cM. Interval mapping by using the computer programme MAPMAKER/ QTL 1.1 for scanning the genetic map led to the detection of one major quantitative trait locus (QTL) in LG 4 and one minor QTL in LG 8 governing resistance to AC-7. Both QTLs contributed 7.89 to the interaction phenotype (IP) score with 96.3% genetic variation. The multi-locus model suggested additive gene action with 96.8% genetic variation.