Random mutagenesis in vegetatively propagated crops: opportunities, challenges and genome editing prospects.

In order to meet the growing human food and nutrition demand a perpetual process of crop improvement is idealized. It has seen changing trends and varying concepts throughout human history; from simple selection to complex gene-editing. Among these techniques, random mutagenesis has been shown to be a promising technology to achieve desirable genetic gain with less time and minimal efforts. Over the decade, several hundred varieties have been released through random mutagenesis, but the production is falling behind the demand. Several food crops like banana, potato, cassava, sweet potato, apple, citrus, and others are vegetatively propagated. Since such crops are not propagated through seed, genetic improvement through classical breeding is impractical for them. Besides, in the case of polyploids, accomplishment of allelic homozygosity requires a considerable land area, extensive fieldwork with huge manpower, and hefty funding for an extended period of time. Apart from induction, mapping of induced genes to facilitate the knowledge of biological processes has been performed only in a few selected facultative vegetative crops like banana and cassava which can form a segregating population. During the last few decades, there has been a shift in the techniques used for crop improvement. With the introduction of the robust technologies like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) more and more crops are being subjected to gene editing. However, more work needs to be done in case of vegetatively propagated crops.

SSR markers in revealing extent of genetic diversity and phylogenetic relationships among chickpea core collection accessions for Western Himalayas.

BACKGROUND: The exploration of genetic diversity is the key source of germplasm conservation and potential to broaden its genetic base. The globally growing demand for chickpea suggests superior/climate-resilient varieties, which in turn necessitates the germplasm characterization to unravel underlying genetic variation. METHODOLOGY AND RESULTS: A chickpea core collection comprising of diverse 192 accessions which include cultivated Cicer arietinum, and wild C. reticulatum, C. echinospermum, and C. microphyllum species were investigated to analyze their genetic diversity and relationship, by assaying 33 unlinked simple sequence repeat (SSR) markers. The results amplified a total of 323 alleles (Na), ranging from 2 to 8 with an average of 4.25 alleles per locus. Expected heterozygosity (He) differed from 0.46 to 0.86 with an average of 0.68. Polymorphic information content (PIC) ranged from 0.73 to 0.98 with an average of 0.89. Analysis of molecular variance (AMOVA) showed that most of the variation was among individuals (87%). Cluster analysis resulted in the formation of four distinct clusters. Cluster I represented all cultivated and clusters II, III, and IV comprised a heterogeneous group of cultivated and wild chickpea accessions. CONCLUSION: We report considerable diversity and greater resolving power of SSR markers for assessing variability and interrelationship among the chickpea accessions. The chickpea core is expected to be an efficient resource for breeders for broadening the chickpea genetic base and could be useful for selective breeding of desirable traits and in the identification of target genes for genomics-assisted breeding.

Development and validation of a reverse phase HPLC-DAD method for separation, detection & quantification of rutin and quercetin in buckwheat (Fagopyrum spp.).

Buckwheat has tremendous nutraceutical potential owing to its rutin and quercetin content. The aim of this study was to optimise and validate an analytical method for separating and quantifying these two flavonoids from it. Factors, such as range, linearity, precision, accuracy, limit of detection and limit of quantification, were evaluated for the two compounds using high performance liquid chromatography. On the basis of resolution and symmetry, mobile phase consisting of methanol and methanol:water:acetic acid in the ratio of (100:150:5), flow rate 1.3 ml/min and column temperature 30 °C were found to be optimal analytical conditions. Calibration curves exhibited good linearity with correlation coefficient of 0.995 & 0.9907 over the range 60-180 µg/ml & 2-10 µg/ml for rutin and quercetin respectively. LOD and LOQ values for rutin and quercetin were 6.36, 0.58 and 19.28, 1.77 µg/ml respectively. Recovery values of 96-100.8% confirmed that the method was accurate for rutin and quercetin analysis. This validated method was successfully used to analyse rutin and quercetin in leaves and seeds of buckwheat plant.

Development of an efficient in vitro mutagenesis protocol for genetic improvement of saffron (Crocus sativus L.).

Saffron (Crocus sativus L) is a triploid (2n = 3x = 24), sterile geophyte which can only be propagated by means of underground vegetative corms. Since corm multiplication does not induce genome variations, therefore, the entire saffron population is expected to have a similar genetic makeup. Keeping in view the economic importance of the plant and the factors responsible for its low yield, the present investigation has been undertaken to establish an in vitro ethyl methanesulfonate (EMS) mutagenesis protocol followed by characterization of the induced variability in the advanced generations. The present report is limited to standardization of in vitro mutagenesis protocol only. Among the mutagenic treatments tested, concentrations ranging from 0.1 to 0.5% EMS showed a varied survival of explants. Based on various growth parameters, the LD50 was calculated to be 0.3% EMS for 3 h. Among the two types of explants analyzed, the corm explant gave better results for in vitro survival and the growth parameters than callus explant. An average of 57.33 and 92.00 daughter cormlets in all EMS treatments as compared to 47.67 and 57.67 daughter cormlets in control, obtained from callus and corm explants respectively, were transferred to the field. The maximum, average daughter cormlet weight was obtained in control (3.01 g, corm explant) followed by 0.1% EMS (2.8 g, corm explant). In general, the growth parameters showed decreasing trend with an increase in EMS concentration in both the explants. The present study has been a significant achievement in the sense that the first mutagenesis protocol for C. sativus has been standardized.

Genome-wide association analysis to delineate high-quality SNPs for seed micronutrient density in chickpea (Cicer arietinum L.).

Chickpea is the most important nutrient-rich grain legume crop in the world. A diverse core set of 147 chickpea genotypes was genotyped with a Axiom(®)50K CicerSNP array and trait phenotyped in two different environments for four seed micronutrients (Zn, Cu, Fe and Mn). The trait data and high-throughput 50K SNP genotypic data were used for the genome-wide association study (GWAS). The study led to the discovery of genes/QTLs for seed Zn, Cu, Fe and Mn, concentrations in chickpea. The analysis of seed micronutrient data revealed significant differences for all four micronutrient concentrations (P ≤ 0.05). The mean concentrations of seed Zn, Cu, Fe and Mn pooled over the 2 years were 45.9 ppm, 63.8 ppm 146.1 ppm, and 27.0 ppm, respectively. The analysis of results led to the identification of 35 SNPs significantly associated with seed Zn, Cu, Fe and Mn concentrations. Among these 35 marker-trait associations (MTAs), 5 were stable (consistently identified in different environments), 6 were major (explaining more than 15% of the phenotypic variation for an individual trait) and 3 were both major and stable MTAs. A set of 6 MTAs, MTAs (3 for Mn, 2 for Fe, and 1 for Cu) reported by us during the present study have been also reported in the same/almost same genomic regions in earlier studies and therefore declared as validated MTAs. The stable, major and validated MTAs identified during the present study will prove useful in future chickpea molecular breeding programs aimed at enhancing the seed nutrient density of chickpea.

Promoting the accumulation of scopolamine and hyoscyamine in Hyoscyamus niger L. through EMS based mutagenesis.

The overexploitation of medicinal plants is depleting gene pool at an alarming rate. In this scenario inducing the genetic variability through targeted mutations could be beneficial in generating varieties with increased content of active compounds. The present study aimed to develop a reproducible protocol for in vitro multiplication and mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6ß-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway. In vitro raised callus were treated with different concentrations (0.01% - 0.1%) of Ethyl Methane Sulfonate (EMS). Emerging multiple shoots and roots were obtained on the MS media supplemented with cytokinins and auxins. Significant effects on morphological characteristics were observed following exposure to different concentrations of EMS. EMS at a concentration of 0.03% was seen to be effective in enhancing the average shoot and root number from 14.5±0.30 to 22.2 ±0.77 and 7.2±0.12 to 8.8±0.72, respectively. The lethal dose (LD50) dose was calculated at 0.08% EMS. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The transcripts of PMT and H6H were significantly upregulated at 0.03-0.05% EMS compared to control. EMS treated explants showed increased accumulation of scopolamine (0.639 µg/g) and hyoscyamine (0.0344µg/g) compared to untreated.

Cytogenetic effects of three commercially formulated pesticides on somatic and germ cells of Allium cepa.

Cytological effects of Endosri-ES (endosulfan), Nuvan-NU (dichlorvos), and Kvistin-KS (carbendazim) were evaluated on mitotic and meiotic cells of Allium cepa. Test concentrations were chosen by calculating EC50 values of formulated ES, NU, and KS, which turned to be 60, 200, and 500 ppm (parts per million), respectively. Cytological studies were undertaken on root meristem cells of A. cepa using EC50, 1/2 × EC50, and 2 × EC50 of these pesticides for 24 and 48 h. Similarly, a meiotic study was conducted by applying the pesticides at the aforesaid concentrations from seedling to bud stage. A set of onion bulbs exposed to tap water was run parallel for negative control and maleic hydrazide (112.09 ppm) as positive control. During the study period, mitotic index (MI) decreased at all the pesticide concentrations compared to the negative control. Among various chromosomal aberrations, chromatin bridges, breaks, stickiness, laggard, vagrant chromosomes, fragments, C-mitosis, multipolarity, ring chromosome as well as micronuclei were observed in mitotic preparations. In contrast, meiotic aberrations revealed comparatively less frequency of chromosomal aberrations and the most frequent were lagging chromosome, stray bivalents, secondary association, chromatin bridge, disturbed anaphase, and stickiness. Comparative analysis of the pesticides showed that NU was highly toxic to plant cells than KS, while as ES showed intermediate effects between the two. Further, our study revealed that all the three pesticides produce genotoxic effects which can cause health risks to the human populations. Graphical Abstract ᅟ.

Elevated expression of Rad18 regulates melanoma cell proliferation.

The E3 ligase Rad18 is a key regulator for the lesion bypass pathway, which plays an important role in genomic stability. However, the status of Rad18 expression in melanoma is not known. Using melanoma tissue microarray (TMA), we showed that nuclear Rad18 expression was upregulated in primary and metastatic melanoma compared to dysplastic nevi. Rad18 expression was significantly reduced in sun-exposed sites compared to the sun-protected sites. Strong Rad18 expression correlated with worse 5-year patient survival and was an independent prognostic factor for melanoma found in the sun-protected sites. Furthermore, we showed that melanoma cell proliferation and the expression of pAkt and cyclin D1 were reduced upon Rad18 knockdown. We, for the first time, showed that Rad18 is significantly increased in melanoma and predicts the poor outcome for melanoma in the sun-protected sites. Rad18 is involved in the regulation of melanoma cell proliferation, which can be exploited in designing new strategy for melanoma treatment.

The NAD(P)H:Quinone Oxidoreductase 1 induces cell cycle progression and proliferation of melanoma cells.

The oxidoreductase NQO1 plays a prominent role in maintaining the cellular homeostasis. NQO1 is mainly a cytosolic enzyme which catalyzes the metabolism of quinones and is present in almost all tissue types providing protection against different stresses including xenobiotics, oxidants, UV light, and ionizing radiation. This enzyme is overexpressed in many cancerous tissues and its function in carcinogenesis remains unclear. Due to the relative lack of information on the role of NQO1 in melanoma pathogenesis, we attempted to determine the expression and basic function of NQO1 in melanoma cell proliferation. We found that NQO1 is overexpressed in most melanoma cell lines with respect to melanocytes. Furthermore, the expression of this oxidoreductase significantly induces cell cycle progression by upregulating the expression of cyclins A2, B1 and D1, leading to the proliferation of melanoma cells. Our results also indicate that NQO1 is an upstream regulator of NF-kappaB p50, a factor linked to melanoma progression and poor patient prognosis. Interestingly, we found that NQO1 stabilizes the transactivator BCL3, which in turn upregulates NF-kappaB p50. More importantly, our results also indicate that NF-kappaB p50 correlates with the expression of NQO1 and mediates its role in the proliferation of melanoma cells.

Analysis of reactive oxygen species and antioxidant defenses in complex I deficient patients revealed a specific increase in superoxide dismutase activity.

The mechanism of free radical production by complex I deficiency is ill-defined, although it is of significant contemporary interest. This study studied the ROS production and antioxidant defenses in children with mitochondrial NADH dehydrogenase deficiency. ROS production has remained significantly elevated in patients compared to controls. The expression of all antioxidant enzymes significantly increased at mRNA level. However, the enzyme activities did not correlate with high mRNA or protein expression. Only the activity of superoxide dismutase (SOD) was found to correlate with higher mRNA expression in patient derived cell lines. The activities of the enzymes such as glutathione peroxidase (GPx), Catalase (CAT) and glutathione-S-transferase (GST) were significantly reduced in patients (p<0.05 or p<0.01). Glutathione reductase (GR) activity and intracellular glutathione (GSH) levels were not changed. Decreased enzyme activities could be due to post-translational or oxidative modification of ROS scavenging enzymes. The information on the status of ROS and marking the alteration of ROS scavenging enzymes in peripheral lymphocytes or lymphoblast cell lines will provide a better way to design antioxidant therapies for such disorders.

Analysis of mitochondrial DNA sequences in childhood encephalomyopathies reveals new disease-associated variants.

BACKGROUND: Mitochondrial encephalomyopathies are a heterogeneous group of clinical disorders generally caused due to mutations in either mitochondrial DNA (mtDNA) or nuclear genes encoding oxidative phosphorylation (OXPHOS). We analyzed the mtDNA sequences from a group of 23 pediatric patients with clinical and morphological features of mitochondrial encephalopathies and tried to establish a relationship of identified variants with the disease. METHODOLOGY/PRINCIPLE FINDINGS: Complete mitochondrial genomes were amplified by PCR and sequenced by automated DNA sequencing. Sequencing data was analyzed by SeqScape software and also confirmed by BLASTn program. Nucleotide sequences were compared with the revised Cambridge reference sequence (CRS) and sequences present in mitochondrial databases. The data obtained shows that a number of known and novel mtDNA variants were associated with the disease. Most of the non-synonymous variants were heteroplasmic (A4136G, A9194G and T11916A) suggesting their possibility of being pathogenic in nature. Some of the missense variants although homoplasmic were showing changes in highly conserved amino acids (T3394C, T3866C, and G9804A) and were previously identified with diseased conditions. Similarly, two other variants found in tRNA genes (G5783A and C8309T) could alter the secondary structure of Cys-tRNA and Lys-tRNA. Most of the variants occurred in single cases; however, a few occurred in more than one case (e.g. G5783A and A10149T). CONCLUSIONS AND SIGNIFICANCE: The mtDNA variants identified in this study could be the possible cause of mitochondrial encephalomyopathies with childhood onset in the patient group. Our study further strengthens the pathogenic score of known variants previously reported as provisionally pathogenic in mitochondrial diseases. The novel variants found in the present study can be potential candidates for further investigations to establish the relationship between their incidence and role in expressing the disease phenotype. This study will be useful in genetic diagnosis and counseling of mitochondrial diseases in India as well as worldwide.