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Intron Polymorphism (IP) markers were used to unravel the genetic variation and relationship among 26 genotypes representing six cultivated Brassica species described in the classical U triangle. One hundred and twenty-five Arabidopsis thaliana-derived IP markers were assayed and 90 to 100% cross-transferability was observed in the six Brassica species suggesting that IP markers were highly conserved during the evolution of different Brassica species. The number of alleles observed in species at each locus ranged from one to ten with an average of 2.89 alleles per primer pair and there was no consensus between the number of alleles amplified in diploid and tetraploid species. The size range of amplified alleles was 120-1250bp, which reflects enormous deletions/insertions in different alleles. In B. juncea, 100% cross-transferability had been obtained and 121 IP markers resulted in polymorphic amplicons with PIC value of 0.04 to 0.48. The dendrogram divided all the 26 genotypes into two groups composed of B. napus/B. rapa/B. oleracea and B. carinata/B. nigra/B. juncea. A-genome present in B. juncea and B. napus/B. rapa seems distinct from each other and hence provides a great opportunity for generating diversity through resynthesizing amphidiploids from different available sources of Agenome. The A and B genomes are more similar in comparison to C genome in tetra-diploid species.The evolutionary relationship established between various Brassica species would support in formulating suitable breeding approaches for widening the genetic base of Brassica amphidiploids by exploiting the genetic diversity found in diploid progenitor gene pools.

Crop plants have an innate capacity to acclimatize and survive myriad stresses in field conditions. This acclimatization to stress enhances crop stand in field and productivity of plant. Inter alia field crops withstand drought stress (hydropenia) by inducing synthesis or accumulation of osmolytes such as (i) proline and other amino acids, (ii) glycine betaine (GB), (iii) soluble carbohydrates, and (iv) reactive oxygen species (ROS) scavenging system as intrinsic drought antagonizing molecules. Precise in vivo induction of osmolytes and their effect on ROS scavenging system in flax/linseed has not been elucidated. The investigation was carried out to identify a tolerant and susceptible cultivar of flax from a core collection of 53 core accessions and evaluate the role of compatible osmolytes in Linum usitatissimum under hydropenia. We screened eight morphometrically diverse flax genotypes in field under irrigated and un-irrigated condition and classified them as tolerant and susceptible genotypes. Further, we examined the effect of ex-foliar glycine betaine application - a signature molecule involved in drought tolerance, on selected tolerant and susceptible varieties. Our results showed stimulatory impact of glycine betaine on accumulation of ROS scavenging antioxidants, total soluble protein and on its own accumulation. While the ex-foliar application had no inhibitory effect on the growth of plants; accumulation of free proline, amino acids and carbohydrates are inhibited par se in flax. Our findings reveal, flax is a non-accumulator of glycine betaine and exogenous application of glycine betaine enhances its own levels during drought stress.

Codon usage bias (CUB) phenomenon varies with the species and even within the genes of the same species, where few codons are preferred more frequently than their other synonymous codons. It also categorizes the differences between species. Nucleotide compositional analysis reveals the molecular mechanisms of genes and the evolutionary relationship of a gene in dissimilar plant species. In the present study, three orthologous sequences of each FAE1 (FAE1.1, FAE1.2, and FAE1.3) and FAD2 (FAD2.1, FAD2.2, and FAD2.3) genes, from six Brassica species were accessed using the GenBank database. Further, CUB-related parameters such as nucleotide composition (AT and GC content), relative synonymous codon usage (RSCU), the effective number of codons (ENC), frequency of optimal codons (Fop), relative codon usage bias (RCBS), neutrality plot (GC12 vs. GC3), parity rule-2 [(A3/(A3 + T3) vs. (G3/(G3 + C3)], and correspondence analysis (COA) were analyzed to compare codon bias in U’s triangle Brassica species. The FAE1 genes were AT-biased and FAD2 genes were GC-biased across the studied Brassica species. RSCU values indicated that both the genes had moderate codon usage frequency for selected amino acids. The evolutionary study confirmed that codon usage preference is similar within the species grouped into the same cluster for FAE1; however, B. nigra performed differently for FAD2.2 orthologue. The high ENC value, low Fop, and RSCU value highlighted that FAE1 and FAD2 genes had a low level of gene expression and moderate preference for codon usage across the Brassicas. In addition, neutrality plot, parity rule, and correspondence analysis revealed that natural selection pressure had significantly contributed to CUB for FAE1 genes, whereas mutation and selection pressure occurred for FAD2 genes. This study would help to decode codon optimization, improve the level of expression of exogenous genes, and transgenic engineering to increase fatty acid profiling for the betterment of seed oil in Brassica species.

Canola-type genotypes in Indian mustard (Brassica juncea) are a new kind of quality resource developed for their low levels of erucic acid (

White rust [Albugo candida (Pers. Ex. Lev) Kuntze] is an important oomycetes disease of Indian mustard [Brassica juncea (L.) Czern & Coss] that causes a drastic reduction in seed yield and quality when the conditions are favorable. A set of 25 Indian mustard genotypes were screened against A. candida Delhi isolate (Ac-Dli) under both controlled and natural epiphytotic conditions. Out of 25, only six genotypes (Pusa Karishma, PDZ-3, Heera, BEC-144, BIO YSR, and Donskaja) were found highly resistant in both environments. To study the inheritance of resistance, four resistant genotypes (BEC-144, BIO YSR, Pusa Karishma, and Donskaja) were crossed with three susceptible genotypes (PM-24, Pusa Vijay, and MSTWR-17-15) in a definite design. The parents, F1, F2, and BC1F1 (F1 backcrossed with susceptible parent) generations were screened under both artificially controlled and natural epiphytotic conditions and the results indicated that the resistance in BIO YSR, BEC-144, and Pusa Karishma is governed by a single dominant gene, but more than one dominant gene is responsible for resistance in Donskaja. A test of an allelism conducted on the F2 population derived by crossing resistant (BEC-144) × resistant (BIO YSR) genotypes indicated that the gene imparting resistance to Ac-Dli isolate in the resistant parents BEC-144 and BIO YSR is the same and they are allelic to each other. Therefore, in broader terms, the information generated in the present study will be useful in Indian mustard breeding programs for the development of durable resistant cultivars.

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.

Conventional plant breeding amalgamates the genetic diversity present within a crop species to develop combinations of alleles that results in desirable traits. Epigenetic diversity offers additional sources of variation within a species that could be captured for crop improvement. Heritable chromatin modifications resulting in altered gene expression without any changes in the underlying DNA sequence is epigenetics. Developmental cues and environmental signals remodel the chromatin structure, thus affecting various processes, including flowering time, imprinting, floral development, biotic, and abiotic stress responses in plants. Chromatin remodeling through histone tail posttranslational modifications, DNA methylation, and ATP-dependent nucleosome reorganization help regulate gene expression. Understanding the sources of epigenetic variation and the stability of newly formed epigenetic variants over generations is essential to fully use its potential to improve crops. The rapid progress of high-throughput technologies has led to the deciphering of the full methylomes, at single-base resolution, of the model plant Arabidopsis and crop plants such as rice. The comparison of different epigenomes can offer valuable information for the development of biomarkers paving the way for plant epibreeding. In this chapter, we summarize the progress in plant epigenetics, epigenomics, and the newly emerging field of epibreeding to expose the plant breeders, to this new and fast-evolving epigenetic research field, the molecular mechanisms involved, and the methodologies used.

Based on C (wild) to T (mutant) transition at amino acid position 1432 bp of lpa1-1 gene, two dominant markers each specific to wild type (LPA1) and mutant (lpa1-1) allele were developed and validated across seven F2 populations. Joint segregation of these markers behaved in co-dominant fashion, clearly distinguishing heterozygote from two other homozygote genotypes. Full length sequence alignment between wild type (LPA2) and mutant (lpa2-1) allele revealed one transition mutation (A to G) and a co-dominant CAPS marker was developed which differentiated all three types of segregants across seven F2 populations. Across populations, segregants with lpa1-1/lpa1-1 (1.77 mg/g) and lpa2-1/lpa2-1 (1.85 mg/g) possessed significantly lower phytic acid compared to LPA1/LPA1 (2.58 mg/g) and LPA2/LPA2 (2.53 mg/g). Inorganic phosphorus was however higher in recessive homozygotes (lpa1-1/lpa1-1: 0.77 mg/g, lpa2-1/lpa2-1: 0.53 mg/g) than the dominant homozygotes (LPA1/LPA1: 0.33 mg/g, LPA2/LPA2: 0.19 mg/g). Overall, homozygous segregants of lpa1-1 and lpa2-1 showed 31% and 27% reduction of phytic acid, respectively. Analysis of phytate and inorganic phosphorous in the maize kernel in these segregating populations confirmed co-segregation of trait and markers specific to lpa1-1 and lpa2-1. This is the first report of the development of breeder-friendly gene-based markers for lpa1-1 and lpa2-1; and it holds great significance for maize biofortification.

Based on C (wild) to T (mutant) transition at amino acid position 1432 bp of lpa1-1 gene, two dominant markers each specific to wild type (LPA1) and mutant (lpa1-1) allele were developed and validated across seven F(2) populations. Joint segregation of these markers behaved in co-dominant fashion, clearly distinguishing heterozygote from two other homozygote genotypes. Full length sequence alignment between wild type (LPA2) and mutant (lpa2-1) allele revealed one transition mutation (A to G) and a co-dominant CAPS marker was developed which differentiated all three types of segregants across seven F(2) populations. Across populations, segregants with lpa1-1/lpa1-1 (1.77 mg/g) and lpa2-1/lpa2-1 (1.85 mg/g) possessed significantly lower phytic acid compared to LPA1/LPA1 (2.58 mg/g) and LPA2/LPA2 (2.53 mg/g). Inorganic phosphorus was however higher in recessive homozygotes (lpa1-1/lpa1-1: 0.77 mg/g, lpa2-1/lpa2-1: 0.53 mg/g) than the dominant homozygotes (LPA1/LPA1: 0.33 mg/g, LPA2/LPA2: 0.19 mg/g). Overall, homozygous segregants of lpa1-1 and lpa2-1 showed 31% and 27% reduction of phytic acid, respectively. Analysis of phytate and inorganic phosphorous in the maize kernel in these segregating populations confirmed co-segregation of trait and markers specific to lpa1-1 and lpa2-1. This is the first report of the development of breeder-friendly gene-based markers for lpa1-1 and lpa2-1; and it holds great significance for maize biofortification. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-2113-x) contains supplementary material, which is available to authorized users.

FAE1, the gene encoding fatty acid elongase, has been implicated to play a decisive role in the synthesis of erucic acid. Allelic variations in this gene, such as single nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and insertions of transposable element (TE), are predominantly responsible for variability in erucic acid content in the seed, among the Brassica genotypes. Brassica juncea being an amphidiploid, contains two paralogs, FAE1.1 and FAE1.2. Although the coding DNA sequences (CDS) of FAE gene is highly conserved (> 95% identical), the promoter region is found to be polymorphic. The sequence variability in the promoter region of FAE1 across different Brassica spp. suggests their likely involvement in the regulation of the gene expression. This study was aimed to evaluate polymorphism in the upstream region of FAE1.1 and FAE1.2 genes, across the low erucic acid (LEA) and high erucic acid (HEA) cultivars, and to develop molecular markers based on this variability. The upstream regions of FAE were sequenced from LEA cultivar “Pusa Mustard 30” and HEA cultivar “Pusa Bold.” A 28-bp deletion in the promoter of FAE1.1 and a 340-bp insertion of a transposon-like element in the FAE1.2 gene promoter were discovered in LEA genotype. Markers based on the sequence variability in the promoter regions of FAE1.1 and FAE1.2 were found to completely co-segregate with the seed erucic acid content. These markers can be effectively used in marker-assisted selection for development of low erucic acid cultivars in B. juncea.

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.

High-quality maize oil, having low level of saturated fatty acids, is highly suitable for human consumption. It is considered to be better than most of other edible oils due to its fatty acid composition and stability during storage and cooking. There is about 3–4 % oil content in maize kernel. However, more than 6–7 % oil is reported in high-oil corn genotypes. High-oil lines, in general, have reduced yields. Large numbers of genes/QTLs were reported to control this trait, thus, making it difficult to improve. A combination of conventional breeding methods, marker-assisted selection and transgenic approach would help in developing high-yielding genotypes with enhanced oil content in maize.

Six earlier reported markers closely linked to low glucosinolate QTLs of Brassica juncea, spread across ‘A’ genome (A2, A3 and A9) were validated in a recombinant inbred line (RIL) population of a cross between Pusa Mustard-21 (low erucic acid) and EC-597325 (double low) genotypes, to utilize them in marker-assisted selection (MAS). Of them, four markers viz., GER 1 amplified alleles of 650 bp and of 950 bp, GER 5 amplified 310 bp and 350 bp, At5gAJ67 amplified 500 bp and 450 bp and Myb28 amplified alleles of size 900 bp and 920 bp in EC597325 and Pusa Mustard-21, respectively and therefore differentiated low and high glucosinolate parents. These four polymorphic markers were then used to genotype the phenotyped RIL population consisting 608 plants. Marker-trait association was tested for goodness of fit using 2 test. Of the four markers, GER1 and GER5 showed higher phenotypic variance (R2 value) compared to the others, indicating their significance in determination of glucosinolates and prospects for use in MAS for development of Indian mustard genotypes with low glucosinolates content.