Heat-Stress-Induced Changes in Physio-Biochemical Parameters of Mustard Cultivars and Their Role in Heat Stress Tolerance at the Seedling Stage.

In the era of global warming, heat stress, particularly at the seedling stage, is a major problem that affects the production and productivity of crops such as mustard that are grown in cooler climates. Nineteen mustard cultivars were exposed to contrasting temperature regimes-20 °C, 30 °C, 40 °C and a variable range of 25-40 °C-and evaluated for changes in physiological and biochemical parameters at the seedling stage to study their role in heat-stress tolerance. Exposure to heat stress showed detrimental effects on seedling growth as revealed by reduced vigor indices, survival percentages, antioxidant activity and proline content. The cultivars were grouped into tolerant, moderately tolerant and susceptible based on the survival percentage and biochemical parameters. All the conventional and three single-zero cultivars were found to be tolerant and moderately tolerant, respectively, while double-zero cultivars were reckoned to be susceptible except for two cultivars. Significant increases in proline content and catalase and peroxidase activities were found associated with thermo-tolerant cultivars. More efficient antioxidant system activity and proline accumulation were noticed in conventional along with three single-zero (PM-21, PM-22, PM-30) and two double-zero (JC-21, JC-33) cultivars that might have provided better protection to them under heat stress than the remaining one single- and nine double-zero cultivars. Tolerant cultivars also resulted in significantly higher values of most of the yield attributing traits. Heat-stress-tolerant cultivars could easily be selected based on the survival percentage, proline and antioxidants at the seedling stage and included as efficient cultivars in breeding programs.

Seed coat colour of Indian mustard [Brassica juncea (L.) Czern. and Coss.] is associated with Bju.TT8 homologs identifiable by targeted functional markers.

Seed coat colour is an important trait in Indian mustard. Breeding for seed coat colour needs precise knowledge of mode of inheritance and markers linked to it. The present study was focussed on genetics and development of functional markers for seed coat colour. F1s (direct and reciprocal) and F2 populations were developed by crossing two contrasting parents for seed coat colour (DRMRIJ-31, brown seeded and RLC-3, yellow seeded). Phenotypic results have shown that the seed coat colour trait was under the influence of maternal effect and controlled by digenic-duplicate gene action. Further, Bju.TT8 homologs of both parents (DRMRIJ-31 and RLC-3) were cloned and sequenced. Sequencing results of Bju.TT8 homologs revealed that in RLC-3, gene Bju.ATT8 had an insertion of 1279bp in the 7th exon; whereas, gene Bju.BTT8 had an SNP (C→T) in the 7th exon. These two mutations were found to be associated with yellow seed coat colour. Using sequence information, functional markers were developed for both Bju.TT8 homologs, validated on F2 population and were found highly reliable with no recombination between the markers and the phenotype. Further, these markers were subjected to a germplasm assembly of Indian mustard, and their allelic combination for the seed coat colour genes has been elucidated. The comparative genomics of TT8 genes revealed high degree of similarity between and across the Brassica species, and the respective diploid progenitors in tetraploid Brassica species are the possible donors of TT8 homologs. This study will help in the marker-assisted breeding for seed coat colour, and aid in understanding seed coat colour genetics more precisely.

Deciphering the Genetic Inheritance of Tocopherols in Indian Mustard (Brassica juncea L. Czern and Coss).

Tocopherol is vital for the nutritional value and stability of Indian mustard (Brassica juncea L. Czern and Coss) oil; nonetheless, the lack of information on genetic control is hampering its improvement. In this study, six populations (P1, P2, F1, F2, BC1P1, and BC1P2) of RLC3 × NPJ203 were evaluated in a family block design to evaluate the inheritance pattern, gene effects, and various other genetic parameters of tocopherol content (α, γ, and total), using generation mean analysis. The comparison of direct and reciprocal crosses indicated that the tocopherol content was not influenced by maternal inheritance. Negative directional heterosis showed that ATC, GTC, and TTC are governed by recessive genes. Potence ratio and degree of dominance highlighted an over-dominance type of gene interaction for GTC and TTC, whereas ATC was governed by epistatic interactions. Furthermore, the six-parameter model revealed a duplicate gene action for α-tocopherol content. Broad and narrow sense heritability coupled with genetic advances were high.

Genetic diversity and population structure in onion (Allium cepa L.) accessions based on morphological and molecular approaches.

Bulb onion is cultivated throughout the world for consumption as vegetable and processed products. Although having high global demand and economic significance, information about genetic diversity and genomic resources is limited. This study investigated the variability of 96 accessions representing seventeen countries. Out of 145 SSR markers, 62 SSRs amplified and 15 SSRs gave consistent polymorphic bands. Fifty three alleles were detected with an average of 3.533 alleles per locus. PIC value ranged from 0.219 (ACM463) to 0.715 (ACM091). Structure and cluster analysis grouped the onion accessions into two clusters. Discriminant analysis of principal components, a tool that maximizes variation between groups while minimizing that within groups, assorted accessions into five clusters. Analysis of molecular variance revealed maximum variation within the populations than among the populations. Highest genetic differentiation (FST = 0.11045; p < 0.001) was observed between Europe and Japan populations whereas the lowest genetic differentiation (FST = 0.05714; p < 0.001) was recorded between India and Japan. Principal component analysis of morphological traits suggested two principal components cumulatively accounting for 74.4% of the total variance. First component (PC1) was positively and strongly correlated with bulbing whereas second component (PC2) had leaf colour with the highest coefficient. Clustering was not on the basis of bulb colour, bulb formation, or flowering but on the basis of geographical origin. Based on clustering, crossing of distantly related accessions can provide an insight about the hybrid vigour of these diverse accessions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01101-3.

Agro-morphological and molecular diversity in different maturity groups of Indian cauliflower (Brassica oleracea var. botrytis L.).

The present study analysed the molecular and agro-morphological diversity in a set of 92 diverse cauliflower genotypes and two each of cabbage and broccoli. Field evaluation of the genotypes was done in randomized block design (RBD) at two locations (i.e. IARI, New Delhi and ICAR-RC-NEH Region, Barapani) during Rabi2019-20. Genotypes showed variation for all the eight observed traits at both locations and, the differences in early and snowball groups were distinct. Pusa Meghna, DC-33-8, Pusa Kartiki and CC-14 were earliest for curd initiation. Genotypes showed higher values for curd traits at Delhi. Molecular diversity was detected with 90 polymorphic simple sequence repeats (SSR). Number of alleles ranged from 1 to 9 with mean value of 2.16 and the highest polymorphic information content (PIC) value was observed for primer BoGMS0742 (0.68) with a mean value of 0.18. Cluster analysis using agro-morphological traits substantiated classification of the genotypes for maturity groups. However, SSR analysis revealed four clusters and with a composite pattern of genotype distribution. STRUCTURE analysis also supported the admixture and four subpopulations. The studyindicates for introgression of genetic fragments across the maturity groups, thereby, potential for use in further genetic improvement and heterosis breeding.

Understanding population structure and detection of QTLs for curding-related traits in Indian cauliflower by genotyping by sequencing analysis.

Curd initiation and development are complex traits and highly responsive for different temperature ranges in cauliflower. The present study was aimed to identify QTLs for eight traits associated with curding behaviour in diverse germplasm of Indian cauliflower. For this, 92 genotypes of cauliflower and 2 each of tropical broccoli and cabbage were genotyped through genotyping by sequencing (GBS). It generated ≈302 million reads (9.1226E + 10 bp) and identified 35,381 SNPs, maximum from chromosome 3 (4735) with a mean value of 3981.1 SNPs. Ts/Tv ratio was 1.74, suggesting transition bias. STRUCTURE analysis revealed delta value of K = 4 and four subpopulations and prominence of population admixture. In total, 121 significant SNPs were detected for eight traits, 38 for Delhi (North Indian plain) and 83 for Barapani (North-East India). Twelve QTLs were detected for traits associated with regulation of curd formation and development, five of which were for marketable curd length, curd width, days to 50% curd harvest and marketable curd weight from Delhi region and seven for curd length, curd width, days to 50% curd harvest, gross plant weight, leaf length, marketable/net curd weight and number of leaves per plant for Barapani area of North East India. The SNPs identified will be useful for development of markers for curding-related traits and their use in breeding varieties with wider curding plasticity.

Exploitation of Novel Bt ICPs for the Management of Helicoverpa armigera (Hübner) in Cotton (Gossypium hirsutum L.): A Transgenic Approach.

Cotton is a commercial crop of global importance. The major threat challenging the productivity in cotton has been the lepidopteron insect pest Helicoverpa armigera or cotton bollworm which voraciously feeds on various plant parts. Biotechnological interventions to manage this herbivore have been a universally inevitable option. The advent of plant genetic engineering and exploitation of Bacillus thuringiensis (Bt) insecticidal crystal proteins (ICPs) marked the beginning of plant protection in cotton through transgenic technology. Despite phenomenal success and widespread acceptance, the fear of resistance development in insects has been a perennial concern. To address this issue, alternate strategies like introgression of a combination of cry protein genes and protein-engineered chimeric toxin genes came into practice. The utility of chimeric toxins produced by domain swapping, rearrangement of domains, and other strategies aid in toxins emerging with broad spectrum efficacy that facilitate the avoidance of resistance in insects toward cry toxins. The present study demonstrates the utility of two Bt ICPs, cry1AcF (produced by domain swapping) and cry2Aa (produced by codon modification) in transgenic cotton for the mitigation of H. armigera. Transgenics were developed in cotton cv. Pusa 8-6 by the exploitation of an apical meristem-targeted in planta transformation protocol. Stringent trait efficacy-based selective screening of T1 and T2 generation transgenic plants enabled the identification of plants resistant to H. armigera upon deliberate challenging. Evaluation of shortlisted events in T3 generation identified a total of nine superior transgenic events with both the genes (six with cry1AcF and three with cry2Aa). The transgenic plants depicted 80-100% larval mortality of H. armigera and 10-30% leaf damage. Molecular characterization of the shortlisted transgenics demonstrated stable integration, inheritance and expression of transgenes. The study is the first of its kind to utilise a non-tissue culture-based transformation strategy for the development of stable transgenics in cotton harbouring two novel genes, cry1AcF and cry2Aa for insect resistance. The identified transgenic events can be potential options toward the exploitation of unique cry genes for the management of the polyphagous insect pest H. armigera.

Unraveling the Relationship Between Seed Yield and Yield-Related Traits in a Diversity Panel of Brassica juncea Using Multi-Traits Mixed Model.

The response to selection in any crop improvement program depends on the degree of variance and heritability. The objective of the current study was to explain variance and heritability components in Indian mustard Brassica juncea (L). Czern & Coss to recognize promising genotypes for effective breeding. Two hundred and eighty-nine diverse accessions of Indian mustard belonging to four continents were analyzed for yield and yield-related traits (20 traits) over two seasons (2017-2018 and 2018-2019) using an alpha lattice design. The genetic variance was found to be significant (P ≤ 0.01) for the individual and under pooled analysis for all of the evaluated traits, demonstrating the presence of significant genetic variability in the diversity panel, which bids greater opportunities for utilizing these traits in future breeding programs. High heritability combined with high genetic advance as percent of mean and genotypic coefficient of variation was observed for flowering traits, plant height traits, seed size, and seed yield/plant; hence, a better genetic gain is expected upon the selection of these traits over subsequent generations. Both correlation and stepwise regression analysis indicated that the main shoot length, biological yield, total seed yield, plant height up to the first primary branch, seed size, total siliqua count, days to flowering initiation, plant height at maturity, siliquae on the main shoot, main shoot length, and siliqua length were the most significant contributory traits for seed yield/plant. Also, promising genotypes were identified among the diversity panel, which can be utilized as a donor to improve Indian mustard further. These results indicated a greater scope for improving seed yield per plant directly through a selection of genotypes having the parsimonious combination of these nine traits.

Development and validation of functional CAPS markers for the FAE genes in Brassica juncea and their use in marker-assisted selection.

Low erucic acid is a major breeding target to improve the edible oil quality in Brassica juncea. The single nucleotide polymorphism (SNP) in fatty acid elongase 1 (FAE1.1 and FAE1.2) gene was exploited to expedite the breeding program. The paralogs of FAE1 gene were sequenced from low erucic acid genotype Pusa Mustard 30 and SNPs were identified through homologous alignment with sequence downloaded from NCBI GenBank. Two SNPs in FAE1.1 at position 591 and 1265 and one in FAE1.2 at 237 were found polymorphic among low and high erucic acid genotypes. These SNPs either create or change the recognition site of restriction enzymes. Transition of a single nucleotide at position 591 and 1265 in FAE1.1, and at position 237 in FAE1.2, leads to a change in the recognition site of Hpy99I, BglII and MnlI restriction enzymes, respectively. Two CAPS markers for FAE1.1 and one for FAE1.2 were developed to differentiate low and high erucic acid genotypes. The efficiency of these CAPS markers was found 100 per cent when validated in Brassica juncea, and B. nigra genotypes and used in back-cross breeding. These CAPS markers will facilitate in marker-assisted selection for improvement of oil quality in Brassica juncea.

Lack of genetic diversity of a critically endangered important medicinal plant Chlorophytum borivilianum in Central India revealed by AFLP markers.

Chlorophytum borivilianum is a traditional medicinal plant distributed throughout the tropics and subtropics. In the present investigation, AFLP analysis was used to assess the genetic similarity among 34 accessions. Nine primer sets of AFLP amplified 612 fragments, of which 246 fragments were found to be polymorphic. The average number of polymorphic bands per AFLP primer pair was 27.33. The amplified fragments ranged from 50 base pairs to 600 base pairs. Significant correlation was observed between total number of amplified fragments and polymorphic bands (p > 0.05) per primers. Cluster analysis based on AFLP data revealed limited genetic variation within the thirty four accessions collected from various parts of Central Indian forests.

Recombination suppression at the dominant Rhg1/Rfs2 locus underlying soybean resistance to the cyst nematode.

Host resistance to "yellow dwarf" or "moonlight" disease cause by any population (Hg type) of Heterodera glycines I., the soybean cyst nematode (SCN), requires a functional allele at rhg1. The host resistance encoded appears to mimic an apoptotic response in the giant cells formed at the nematode feeding site about 24-48 h after nematode feeding commences. Little is known about how the host response to infection is mediated but a linked set of 3 genes has been identified within the rhg1 locus. This study aimed to identify the role of the genes within the locus that includes a receptor-like kinase (RLK), a laccase and an ion antiporter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles, gene-based markers, and a new Hg type 0 and new recombination events. A syntenic gene cluster on Lg B1 was found. The effectiveness of SNP probes from the RLK for distinguishing homolog sequence variants on LgB1 from alleles at the rhg1 locus on LgG was shown. The resistant allele of the rhg1 locus was shown to be dominant in NILs. None of the recombination events were within the cluster of the three candidate genes. Finally, rhg1 was shown to reduce the plant root development. A model for rhg1 as a dominant multi-gene resistance locus based on the developmental control was inferred.

The nematode resistance allele at the rhg1 locus alters the proteome and primary metabolism of soybean roots.

Heterodera glycines, the soybean cyst nematode (SCN), causes the most damaging chronic disease of soybean (Glycine max). Host resistance requires the resistance allele at rhg1. Resistance destroys the giant cells created in the plant's roots by the nematodes about 24 to 48 h after commencement of feeding. In addition, 4 to 8 d later, a systemic acquired resistance develops that discourages later infestations. The molecular mechanisms that control the rhg1-mediated resistance response appear to be multigenic and complex, as judged by transcript abundance changes, even in near isogenic lines (NILs). This study aimed to focus on key posttranscriptional changes by identifying proteins and metabolites that were increased in abundance in both resistant and susceptible NILs. Comparisons were made among NILs 10 d after SCN infestation and without SCN infestation. Two-dimensional gel electrophoresis resolved more than 1,000 protein spots on each gel. Only 30 protein spots with a significant (P < 0.05) difference in abundance of 1.5-fold or more were found among the four treatments. The proteins in these spots were picked, trypsin digested, and analyzed using quadrupole time-of-flight tandem mass spectrometry. Protein identifications could be made for 24 of the 30 spots. Four spots contained two proteins, so that 28 distinct proteins were identified. The proteins were grouped into six functional categories. Metabolite analysis by gas chromatography-mass spectrometry identified 131 metabolites, among which 58 were altered by one or more treatment; 28 were involved in primary metabolism. Taken together, the data showed that 17 pathways were altered by the rhg1 alleles. Pathways altered were associated with systemic acquired resistance-like responses, including xenobiotic, phytoalexin, ascorbate, and inositol metabolism, as well as primary metabolisms like amino acid synthesis and glycolysis. The pathways impacted by the rhg1 allelic state and SCN infestation agreed with transcript abundance analyses but identified a smaller set of key proteins. Six of the proteins lay within the same small region of the interactome identifying a key set of 159 interacting proteins involved in transcriptional control, nuclear localization, and protein degradation. Finally, two proteins (glucose-6-phosphate isomerase [EC 5.3.1.9] and isoflavone reductase [EC 1.3.1.45]) and two metabolites (maltose and an unknown) differed in resistant and susceptible NILs without SCN infestation and may form the basis of a new assay for the selection of resistance to SCN in soybean.

Re-annotation of the physical map of Glycine max for polyploid-like regions by BAC end sequence driven whole genome shotgun read assembly.

BACKGROUND: Many of the world's most important food crops have either polyploid genomes or homeologous regions derived from segmental shuffling following polyploid formation. The soybean (Glycine max) genome has been shown to be composed of approximately four thousand short interspersed homeologous regions with 1, 2 or 4 copies per haploid genome by RFLP analysis, microsatellite anchors to BACs and by contigs formed from BAC fingerprints. Despite these similar regions,, the genome has been sequenced by whole genome shotgun sequence (WGS). Here the aim was to use BAC end sequences (BES) derived from three minimum tile paths (MTP) to examine the extent and homogeneity of polyploid-like regions within contigs and the extent of correlation between the polyploid-like regions inferred from fingerprinting and the polyploid-like sequences inferred from WGS matches. RESULTS: Results show that when sequence divergence was 1-10%, the copy number of homeologous regions could be identified from sequence variation in WGS reads overlapping BES. Homeolog sequence variants (HSVs) were single nucleotide polymorphisms (SNPs; 89%) and single nucleotide indels (SNIs 10%). Larger indels were rare but present (1%). Simulations that had predicted fingerprints of homeologous regions could be separated when divergence exceeded 2% were shown to be false. We show that a 5-10% sequence divergence is necessary to separate homeologs by fingerprinting. BES compared to WGS traces showed polyploid-like regions with less than 1% sequence divergence exist at 2.3% of the locations assayed. CONCLUSION: The use of HSVs like SNPs and SNIs to characterize BACs wil improve contig building methods. The implications for bioinformatic and functional annotation of polyploid and paleopolyploid genomes show that a combined approach of BAC fingerprint based physical maps, WGS sequence and HSV-based partitioning of BAC clones from homeologous regions to separate contigs will allow reliable de-convolution and positioning of sequence scaffolds (see BES_scaffolds section of SoyGD). This approach will assist genome annotation for paleopolyploid and true polyploid genomes such as soybean and many important cereal and fruit crops.