Comparison of effectiveness and efficiency of electron beam over gamma rays to induce novel mutations in mungbean (Vigna radiata L. Wilczek).

Induced mutation is very useful in mungbean as it is having less natural genetic variation. The present study was conducted to induce variability through induced mutation, compare efficiency and effectiveness of gamma rays with electron beam on the basis of physiological changes in M1 generation; mutation frequency, spectrum of mutant phenotype and efficiency to produce novel mutations in M2 generation. Seeds of mungbean variety TM 96-2 were irradiated with doses of 200, 300, 400 and 500 Gy gamma rays and electron beam. On the basis of M1seedling growth, the effective mutagen dose (Growth Reduction Dose 50 i.e. GR50) was 440 Gy of gamma rays and 470 Gy of electron beam for TM-96-2. In M2 generation, electron beam treatments were found to induce greater frequency of chlorophyll mutations than gamma rays. The frequency of total mutants in electron beam (1.967) was found to be higher than gamma rays (1.343) along with mutation spectrum. The highest mutation spectrum was observed in 200 Gy dose of electron beam followed by 200 Gy gamma rays. Four novel mutants viz., four primary leaves in 400 Gy gamma rays, lanceolated leaves in 200, 300 and 500 Gy electron beam, yellow pod and yellow seed coat colour in 200 Gy treatment of electron beam were identified and isolated. Desirable mutants like early and synchronous maturity, large seed size and long root with drought tolerance were identified and isolated in different doses of both gamma rays and electron beam which were found true breeding in subsequent generations. Mutagenic efficiency of electron beam was higher in 200 and 400 Gy treatment as compared to same doses of gamma rays, while it was less than gamma rays in 300 and 500 Gy treatments. Mutagenic effectiveness was found to be highest in 200 Gy dose of electron beam which was more than twice that of the same dose of gamma rays.

Genetic diversity, allelic variation and marker trait associations in gamma irradiated mutants of rice (Oryza sativa L.).

PURPOSE: Rice is a prime staple crop for more than half of the world population. Improved White Ponni (IWP) is a premium quality grain rice variety that is fetching a good price and is increasingly popular among the consumers of Tamil Nadu. Tall plant stature of IWP makes them susceptible to lodging and medium duration are two undesirable traits in the variety increases yield losses in the field and also productivity. In this context, we aimed to generate a large mutant population of IWP irradiated with various doses of gamma irradiation to recover putative mutants for semi-dwarfism and earliness. MATERIALS AND METHODS: Totally, 34 putative mutants (22 early, 11 semi-dwarf and early and 1 Narrow-leaf dwarf mutant) were phenotyped for nine morphological traits and genotyped using 34 microsatellite markers linked to a trait of interest of earliness and semi-dwarfism. Trait variability, allelic variations, genetic structure and marker-trait associations in gamma-irradiated putative mutants of Improved White Ponni (IWP) rice were investigated in this study. RESULTS: The hierarchical clustering of morphological data produced five clusters with a dissimilarity coefficient of 1.39. A minimum dissimilarity coefficient of 0.23 was observed between the mutants IWPM9 and IWPM20 and a maximum dissimilarity coefficient of 2.55 was observed between IWPM1 and IWPM25. In cluster analysis with molecular marker data, five clusters with a similarity coefficient of 0.67 were observed. The mutant IWPM29 exhibited the most divergence from the wild type at the genotype level. The first principal component explained 50.99% of the total variability and the majority of the traits were contributed positively. The single-marker analysis revealed the strong association of SSR marker RM3912 with the traits plant height, panicle length and number of grains per panicle with an R2 value of 0.235, 0.235 and 0.250 respectively. CONCLUSIONS: The study identified semi-dwarf and short-duration rice mutants of IWP that can be utilized as potential breeding stocks. The trait-linked SSR markers can improve selection cycles in advanced breeding programs.

Morpho-Physiological Traits and Functional Markers Based Molecular Dissection of Heat-Tolerance in Urdbean.

Urdbean (Vigna mungo L. Hepper) is one of the important pulse crops. Its cultivation is not so popular during summer seasons because this crop is unable to withstand excessive heat stress beside lack of humidity in the atmosphere. Therefore, a panel of 97 urdbean diverse genotypes was assessed for yield under stress and non-stress conditions with an aim to identify heat tolerant genotypes. This study identified 8 highly heat tolerant and 35 highly heat sensitive genotypes based on heat susceptibility index. Further, physiological and biochemical traits-based characterization of a group of six highly heat sensitive and seven highly heat tolerant urdbean genotypes showed genotypic variability for leaf nitrogen balance index (NBI), chlorophyll (SPAD), epidermal flavnols, and anthocyanin contents under 42/25°C max/min temperature. Our results showed higher membrane stability index among heat tolerant genotypes compared to sensitive genotypes. Significant differences among genotypes for ETR at different levels of PAR irradiances and PAR × genotypes interactions indicated high photosynthetic ability of a few genotypes under heat stress. Further, the most highly sensitive genotype PKGU-1 showed a decrease in different fluorescence parameters indicating distortion of PS II. Consequently, reduction in the quantum yield of PS II was observed in a sensitive one as compared to a tolerant genotype. Fluorescence kinetics showed the delayed and fast quenching of Fm in highly heat sensitive (PKGU 1) and tolerant (UPU 85-86) genotypes, respectively. Moreover, tolerant genotype (UPU 85-86) had high antioxidant activities explaining their role for scavenging superoxide radicals (ROS) protecting delicate membranes from oxidative damage. Molecular characterization further pinpointed genetic differences between heat tolerant (UPU 85-86) and heat sensitive genotypes (PKGU 1). These findings will contribute to the breeding toward the development of heat tolerant cultivars in urdbean.

Efficiency of electron beam over gamma rays to induce desirable grain-type mutation in rice (Oryza sativa L.).

PURPOSE: Rice is the predominant crop of Tamil Nadu state, India that occupies about 30% of the total cropped area. However, grain type and quality are the critical traits that determine the market value and domestic consumption rice variety. Most of the households of Tamil Nadu, India prefer to consume medium slender to fine grain type of rice. Hence, the present study was conducted to induce medium slender grain type in popular rice variety ADT 37 (Aduthurai 37), a short bold rice variety using gamma rays (GR) and electron beam (EB) mutagens. MATERIALS AND METHODS: Healthy, dried seeds (12.0% moisture content) of ADT 37 rice variety were exposed to various doses of GR (100-500 Gy) and EB (200-600 Gy). The irradiated population were maintained up to M4 generation by plant to progeny row basis to identify stable mutants for grain-type variation. The selected grain-type mutants (medium slender- and slender-type mutants) in M4 generation were characterized for phenotypic and grain quality traits. RESULTS: A high frequency of desirable grain-type variation was observed in EB-irradiated population than gamma-irradiated population. A total of 25 grain-type mutants (long slender and medium slender) were obtained in M4 generation of ADT 37 variety. The morphological characterization and cooking quality assessment of the 'grain-type' mutants revealed that six out of 25 mutants viz., M-3 (Mutant-3), M-5, M-9, M-10, M-13 and M-15 recorded single plant yield of more than 30 g. There was non-significant variation in yield per plant (g) among the mutants and control (parent) due to key changes in grain type and thousand grain weight. CONCLUSION: EB showed higher mutation frequency, mutagenic effectiveness and efficiency than the GR in inducing both chlorophyll and viable mutants. This study revealed that the percentage contribution of the EB was 2.57 times higher than that of GR in obtaining desirable slender and medium slender grain-type mutants. The grain-type mutants obtained in the present study can be either directly released as variety or used as parents in hybridization program of rice crop improvement.

Transcriptome sequencing, de novo assembly, characterisation of wild accession of blackgram (Vigna mungo var. silvestris) as a rich resource for development of molecular markers and validation of SNPs by high resolution melting (HRM) analysis.

BACKGROUND: Blackgram [Vigna mungo (L.) Hepper], is an important legume crop of Asia with limited genomic resources. We report a comprehensive set of genic simple sequence repeat (SSR) and single nucleotide polymorphism (SNPs) markers using Illumina MiSeq sequencing of transcriptome and its application in genetic variation analysis and mapping. RESULTS: Transcriptome sequencing of immature seeds of wild blackgram, V. mungo var. silvestris by Illumina MiSeq technology generated 1.9 × 107 reads, which were assembled into 40,178 transcripts (TCS) with an average length of 446 bp covering 2.97 GB of the genome. A total of 38,753 CDS (Coding sequences) were predicted from 40,178 TCS and 28,984 CDS were annotated through BLASTX and mapped to GO and KEGG database resulting in 140 unique pathways. The tri-nucleotides were most abundant (39.9%) followed by di-nucleotide (30.2%). About 60.3 and 37.6% of SSR motifs were present in the coding sequences (CDS) and untranslated regions (UTRs) respectively. Among SNPs, the most abundant substitution type were transitions (Ts) (61%) followed by transversions (Tv) type (39%), with a Ts/Tv ratio of 1.58. A total of 2306 DEGs were identified by RNA Seq between wild and cultivar and validation was done by quantitative reverse transcription polymerase chain reaction. In this study, we genotyped SNPs with a validation rate of 78.87% by High Resolution Melting (HRM) Assay. CONCLUSION: In the present study, 1621genic-SSR and 1844 SNP markers were developed from immature seed transcriptome sequence of blackgram and 31 genic-SSR markers were used to study genetic variations among different blackgram accessions. Above developed markers contribute towards enriching available genomic resources for blackgram and aid in breeding programmes.

Morphological, Histobiochemical and Molecular Characterisation of Low Lignin Phloem Fibre (llpf) Mutant of Dark Jute (Corchorus olitorius L.).

Lignin is a versatile plant metabolite challenging high-end industrial applications of several plant products including jute. Application of developmental mutant in regulation of lignification in jute may open up door for much awaited jute based diversified products. In the present study, a novel dark jute (Corchorus olitorius L.) mutant with low lignin (7.23%) in phloem fibre being compared to wild-type JRO 204 (13.7%) was identified and characterised. Unique morphological features including undulated stem, petiole and leaf vein distinguished the mutant in gamma ray irradiated mutant population. Histological and biochemical analysis revealed reduced lignification of phloem fibre cells of the plant. RT-PCR analysis demonstrated temporal transcriptional regulation of CCoAMT1 gene in the mutant. The mutant was found an extremely useful model to study phloem fibre developmental biology in the crop besides acting as a donor genetic stock for low lignin containing jute fibre in dark jute improvement programme.

De novo Assembly, Characterization of Immature Seed Transcriptome and Development of Genic-SSR Markers in Black Gram [Vigna mungo (L.) Hepper].

Black gram [V. mungo (L.) Hepper] is an important legume crop extensively grown in south and south-east Asia, where it is a major source of dietary protein for its predominantly vegetarian population. However, lack of genomic information and markers has become a limitation for genetic improvement of this crop. Here, we report the transcriptome sequencing of the immature seeds of black gram cv. TU94-2, by Illumina paired end sequencing technology to generate transcriptome sequences for gene discovery and genic-SSR marker development. A total of 17.2 million paired-end reads were generated and 48,291 transcript contigs (TCS) were assembled with an average length of 443 bp. Based on sequence similarity search, 33,766 TCS showed significant similarity to known proteins. Among these, only 29,564 TCS were annotated with gene ontology (GO) functional categories. A total number of 138 unique KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified, of which majority of TCS are grouped into purine metabolism (678) followed by pyrimidine metabolism (263). A total of 48,291 TCS were searched for SSRs and 1,840 SSRs were identified in 1,572 TCS with an average frequency of one SSR per 11.9 kb. The tri-nucleotide repeats were most abundant (35%) followed by di-nucleotide repeats (32%). PCR primer pairs were successfully designed for 933 SSR loci. Sequences analyses indicate that about 64.4% and 35.6% of the SSR motifs were present in the coding sequences (CDS) and untranslated regions (UTRs) respectively. Tri-nucleotide repeats (57.3%) were preferentially present in the CDS. The rate of successful amplification and polymorphism were investigated using selected primers among 18 black gram accessions. Genic-SSR markers developed from the Illumina paired end sequencing of black gram immature seed transcriptome will provide a valuable resource for genetic diversity, evolution, linkage mapping, comparative genomics and marker-assisted selection in black gram.

Construction of a genetic linkage map of black gram, Vigna mungo (L.) Hepper, based on molecular markers and comparative studies.

A genetic linkage map of black gram, Vigna mungo (L.) Hepper, was constructed with 428 molecular markers using an F9 recombinant inbred population of 104 individuals. The population was derived from an inter-subspecific cross between a black gram cultivar, TU94-2, and a wild genotype, V. mungo var. silvestris. The linkage analysis at a LOD score of 5.0 distributed all 428 markers (254 AFLP, 47 SSR, 86 RAPD, and 41 ISSR) into 11 linkage groups. The map spanned a total distance of 865.1 cM with an average marker density of 2 cM. The largest linkage group spanned 115 cM and the smallest linkage group was of 44.9 cM. The number of markers per linkage group ranged from 11 to 86 and the average distance between markers varied from 1.1 to 5.6 cM. Comparison of the map with other published azuki bean and black gram maps showed high colinearity of markers, with some inversions. The current map is the most saturated map for black gram to date and will provide a useful tool for identification of QTLs and for marker-assisted selection of agronomically important characters in black gram.

A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers.

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the DNA polymorphism in elite blackgram genotypes. A total of 25 random and 16 ISSR primers were used. Amplification of genomic DNA of the 18 genotypes, using RAPD analysis, yielded 104 fragments that could be scored, of which 44 were polymorphic, with an average of 1.8 polymorphic fragments per primer. Number of amplified fragments with random primers ranged from two (OPA-13) to nine (OPK-4) and varied in size from 200 bp to 2,500 bp. Percentage polymorphism ranged from 16.6% (OPK-7) to a maximum of 66.6% (OPE-5, OPH-2, and OPK-8), with an average of 42.7%. The 16 ISSR primers used in the study produced 101 bands across 18 genotypes, of which 55 were polymorphic. The number of amplified bands varied from two (ISSR 858) to ten (ISSR 810), with a size range of 200-2,200 bp. The average numbers of bands per primer and polymorphic bands per primer were 6.3 and 3.4, respectively. Percentage polymorphism ranged from 25% (ISSR 885) to 100% (ISSR 858), with an average percentage polymorphism of 57.5% across all the genotypes. The 3'-anchored primers based on poly(GA) and poly(AG) motifs produced high average polymorphisms of 54.98% and 58.32%, respectively. ISSR markers were more efficient than the RAPD assay, as they detected 57.4% polymorphic DNA markers in Vigna mungo as compared to 42.7% for RAPD markers. The Mantel test between the two Jaccard's similarity matrices gave r=0.32, showing low correlation between RAPD- and ISSR-based similarities. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in ISSR and combined data of RAPD and ISSR.