Genetic analysis of the variation for mineral accumulation in the leaves and seeds of natural germplasm of Brassica rapa L. (AA) and the its derived forms extracted from an allotetraploid B.juncea L.(AABB).

Brassica rapa L. (2n = 20; AA) is a vegetable and oilseed crop that is grown all over the world. Its leaves, shoots, and seeds store significant amounts of minerals. We used inductively coupled plasma-optical emission spectroscopy (ICP-OES) to determine the concentrations of eleven minerals in the leaves and seeds of 195 advanced generation inbred lines, of which 92 represented natural (NR) B. rapa and the remaining 103 were derived (DR) from a set of mother genotypes originally extracted from an allotetraploid B. juncea (2n = 36; AABB). The inbred lines differed for the composition of leaf and seed minerals. Leaf concentrations of N, K, Zn, and Se were higher in the DR subpanel as compared to NR subpanel, along with high seed accumulations of K and Se. DArT genotyping and genome wide association mapping led to the identification of SNPs associated with leaf and seed mineral compositions. Chromosomes A03, A05, and A10 harboured the most associated loci. Annotations of the regions adjacent to respective GWAS peaks allowed prediction of genes known for acquisition, transport, and accumulation of minerals and heavy metal detoxification. Transcriptome analysis revealed differential expression patterns of the predicted candidates, with most genes either down-regulated in derived genotypes relative to natural forms or their expression being comparable between the two. General downregulation may be a consequence of extracting B. rapa from allotetraploid B. juncea through genome resection. Some of the identified SNPs may be used as DNA markers for breeding programmes designed to modify the leaf and seed mineral compositions.

Genome wide association analyses to understand genetic basis of flowering and plant height under three levels of nitrogen application in Brassica juncea (L.) Czern & Coss.

Timely transition to flowering, maturity and plant height are important for agronomic adaptation and productivity of Indian mustard (B. juncea), which is a major edible oilseed crop of low input ecologies in Indian subcontinent. Breeding manipulation for these traits is difficult because of the involvement of multiple interacting genetic and environmental factors. Here, we report a genetic analysis of these traits using a population comprising 92 diverse genotypes of mustard. These genotypes were evaluated under deficient (N75), normal (N100) or excess (N125) conditions of nitrogen (N) application. Lower N availability induced early flowering and maturity in most genotypes, while high N conditions delayed both. A genotyping-by-sequencing approach helped to identify 406,888 SNP markers and undertake genome wide association studies (GWAS). 282 significant marker-trait associations (MTA's) were identified. We detected strong interactions between GWAS loci and nitrogen levels. Though some trait associated SNPs were detected repeatedly across fertility gradients, majority were identified under deficient or normal levels of N applications. Annotation of the genomic region (s) within ± 50 kb of the peak SNPs facilitated prediction of 30 candidate genes belonging to light perception, circadian, floral meristem identity, flowering regulation, gibberellic acid pathways and plant development. These included over one copy each of AGL24, AP1, FVE, FRI, GID1A and GNC. FLC and CO were predicted on chromosomes A02 and B08 respectively. CDF1, CO, FLC, AGL24, GNC and FAF2 appeared to influence the variation for plant height. Our findings may help in improving phenotypic plasticity of mustard across fertility gradients through marker-assisted breeding strategies.

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

KEY MESSAGE: Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard. Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (No without added N and N100 added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

Physical mapping of introgressed chromosome fragment carrying the fertility restoring (Rfo) gene for Ogura CMS in Brassica juncea L. Czern & Coss.

KEY MESSAGE: Rfo is located on a radish chromosome fragment (~ 108 Kb), which is seated in the middle of a pretty large C genome translocation at the distal region of chromosome A09 of B. juncea. Ogura cytoplasmic male sterility (CMS) is used to produce hybrids in Indian mustard (Brassica juncea L.). Fertility restorers for this CMS were developed by cross-hybridizing B. juncea (AABB; 2n = 36) with B. napus (AACC; 2n = 38) carrying radish Rfo gene. This hybrid production system is normally stable, but many commercial mustard hybrids show male sterile contaminants. We aimed to identify linkage drag associated with Rfo by comparing hybridity levels of 295 handmade CMS x Rfo crosses. Although Rfo was stably inherited, hybridity was < 85 percent in several combinations. Genome re-sequencing of five fertility restorers, mapping sequencing reads to B. juncea reference and synteny analysis with Raphanus sativus D81Rfo genomic region (AJ550021.2) helped to detect ~ 108 Kb of radish chromosome (R) fragment substitution in all fertility restorers. This radish segment substitution was itself located amidst a large C genome translocation on the terminal region of chromosome A09 of B. juncea. The size of alien segment substitution varied from 11.3 (NTCN-R9) to 22.0 Mb (NAJR-102B-R). We also developed an in silico SSR map for chromosome A09 and identified many homoeologous A to the C genome exchanges in the introgressed region. A to the R genome exchanges were rare. Annotation of the substituted fragment showed the gain of many novel genes from R and C genomes and the loss of B. juncea genes from the corresponding region. We have developed a KASPar marker for marker-aided transfer of Rfo and testing hybridity levels in seed production lots.

Genome wide association mapping and candidate gene analysis for pod shatter resistance in Brassica juncea and its progenitor species.

We investigated phenotypic variations for pod shattering, pod length and number of seeds per pod in large germplasm collections of Brassica juncea (2n = 36; AABB) and its progenitor species, B. rapa (2n = 20; AA) and B. nigra (2n = 16; BB). Pod shatter resistance was measured as energy required for rupturing a mature dry pod, with a specially fabricated pendulum machine. Rupture energy (RE) ranged from 3.3 to 11.0 mJ in B. juncea. MCP 633, NR 3350 and Albeli required maximum energy to shatter a pod. It ranged from 2.5 to 7.8 mJ for B. rapa with an average of 5.5 mJ. B. nigra possessed easy to rupture pods. Correlation analysis showed strong associations among these traits in B. juncea and B. rapa. Genome wide association studies were conducted with select sets of B. juncea and B. rapa germplasm lines. Significant and annotated associations predict the role of FRUITFULL, MANNASE7, and NAC secondary wall thickening promoting factor (NST2) in the genetic regulation of shatter resistance in B. juncea. NST2 and SHP1 appeared important for pod length and seeds per pod in B. rapa. Candidate gene based association mapping also confirmed the role of SHP1 and NST2 in regulating pod shattering and related pod traits in B. rapa and B. juncea. Footprints of selection were detected in SHP1, SHP2 (B. rapa, B. nigra and B. juncea), RPL (B. rapa) and NAC (B. juncea). Our results provide insights into the genetic architecture of three pod traits. The identified genes are relevant to improving and securing crop productivity of mustard crop.

Molecular and genetic analysis of defensive responses of Brassica juncea - B. fruticulosa introgression lines to Sclerotinia infection.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a major disease of crop brassicas, with inadequate variation for resistance in primary gene pools. We utilized a wild Brassicaceae species with excellent resistance against stem rot to develop a set of B. juncea - B. fruticulosa introgression lines (ILs). These were assessed for resistance using a highly reproducible stem inoculation technique against a virulent pathogen isolate. Over 40% of ILs showed higher levels of resistance. IL-43, IL-175, IL-215, IL-223 and IL-277 were most resistant ILs over three crop seasons. Sequence reads (21x) from the three most diverse ILs were then used to create B. juncea pseudomolecules, by replacing SNPs of reference B. juncea with those of re-sequenced ILs. Genotyping by sequencing (GBS) was also carried out for 88 ILs. Resultant sequence tags were then mapped on to the B. juncea pseudomolecules, and SNP genotypes prepared for each IL. Genome wide association studies helped to map resistance responses to stem rot. A total of 13 significant loci were identified on seven B. juncea chromosomes (A01, A03, A04, A05, A08, A09 and B05). Annotation of the genomic region around identified SNPs allowed identification of 20 candidate genes belonging to major disease resistance protein families, including TIR-NBS-LRR class, Chitinase, Malectin/receptor-like protein kinase, defensin-like (DEFL), desulfoglucosinolate sulfotransferase protein and lipoxygenase. A majority of the significant SNPs could be validated using whole genome sequences (21x) from five advanced generation lines being bred for Sclerotinia resistance as compared to three susceptible B. juncea germplasm lines. Our findings not only provide critical new understanding of the defensive pathway of B. fruticulosa resistance, but will also enable development of marker candidates for assisted transfer of introgressed resistant loci in to agronomically superior cultivars of crop Brassica.

Detection of First Marker Trait Associations for Resistance Against Sclerotinia sclerotiorum in Brassica juncea-Erucastrum cardaminoides Introgression Lines.

A set of 96 Brassica juncea-Erucastrum cardaminoides introgression lines (ILs) were developed with genomic regions associated with Sclerotinia stem rot (Sclerotinia sclerotiorum) resistance from a wild Brassicaceous species E. cardaminoides. ILs were assessed for their resistance responses to stem inoculation with S. sclerotiorum, over three crop seasons (season I, 2011/2012; II, 2014/2015; III, 2016-2017). Initially, ILs were genotyped with transferable SSR markers and subsequently through genotyping by sequencing. SSR based association mapping identified six marker loci associated to resistance in both A and B genomes. Subsequent genome-wide association analysis (GWAS) of 84 ILs recognized a large number of SNPs associated to resistance, in chromosomes A03, A06, and B03. Chromosomes A03 and A06 harbored the maximum number of resistance related SNPs. Annotation of linked genomic regions highlighted an array of resistance mechanisms in terms of signal transduction pathways, hypersensitive responses and production of anti-fungal proteins and metabolites. Of major importance was the clustering of SNPs, encoding multiple resistance genes on small regions spanning approximately 885 kb region on chromosome A03 and 74 kb on B03. Five SNPs on chromosome A03 (6,390,210-381) were associated with LRR-RLK (receptor like kinases) genes that encode LRR-protein kinase family proteins. Genetic factors associated with pathogen-associated molecular patterns (PAMPs) and effector-triggered immunity (ETI) were predicted on chromosome A03, exhibiting 11 SNPs (6,274,763-994). These belonged to three R-Genes encoding TIR-NBS-LRR proteins. Marker trait associations (MTAs) identified will facilitate marker assisted introgression of these critical resistances, into new cultivars of B. juncea initially and, subsequently, into other crop Brassica species.

Profiling of Intra- and Extracellular Enzymes Involved in Fructification of the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

Fruiting bodies of Ganoderma lucidum have been widely used as a source of potent nutraceutical products. However, the key proteins involved in fructifying G. lucidum, to our knowledge, have not yet been reported. We evaluated the protein profile of fruiting and nonfruiting G. lucidum strains at various developmental stages: mycelia, spawn running, pinning, and fruiting body. Four strains of G. lucidum (GL-I to GL-IV) were grown in both liquid medium (mushroom minimal medium broth) and bags of wheat straw, after which the biomass and fruiting bodies were harvested. Enzyme studies revealed enhanced intracellular and extracellular enzymatic activities during the spawn run stage compared with that during mycelial growth in broth. The esterase and peroxidase activities increased significantly during the pinning of the fruiting cultures, thus indicating their positive role in fructification. Fourier transform infrared spectroscopy of proteins at 3 stages of cultivation-spawn run, pin head formation, and fruiting-exhibited the presence of hydrophobic amino acids and an ordered protein structure in fruiting strains (GL-I and GL-II), indicating the presence of hydrophobin proteins and their role in mushroom fructification. However, basic and aromatic amino acids predominated in the nonfruiting strain GL-IV, and an unordered protein structure was present, which indicate the positive role of hydrophobic amino acids and hydrophobin proteins in mushroom fructification.

High-Performance Liquid Chromatography Studies to Estimate Ergosterol Content at Different Developmental Stages of the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

Ganoderma lucidum has been widely used as a source of potent nutraceutical products. This study was planned to identify and characterize the role of ergosterol in the developmental process of G. lucidum. Four strains of G. lucidum (GL-I-IV) showed a gradual increase in biomass (from 25.52 to 31.72 g) after 3 weeks of growth in mushroom complete medium broth, with maximum biomass observed for strain GL-III. Upon cultivation of G. lucidum strains on wheat straw supplemented with 5% wheat bran, maximum biological efficiency was recorded for the GL-I strain (31.23%), followed by GL-II (26.73%); the number of fruiting bodies were 927 and 693, each weighing 33.7 and 38.6 g, respectively. The amount of ergosterol in the Ganoderma test strains varied among the strains and at different developmental stages, namely, the vegetative mycelium, spawn run, pinhead, and fruiting body phases. The maximum ergosterol content was produced by the GL-I strain during the vegetative mycelium (4601 p.g/g) and reproductive fruiting body (7009 p.g/g) stages. However, strain GL-IV followed by strain GL-II exhibited maximum ergosterol content in the spawn run stage. The ergosterol content was better for GL-II at the pinhead stage. This report indicates that ergosterol content varies among the test strains. Moreover, it increases with each stage of the cultivation process, that is, from spawn run to pinhead to and fruiting body formation.