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.

Resynthesis of Brassica napus through hybridization between B. juncea and B. carinata.

KEY MESSAGE: First report for the resynthesis of Brassica napus by recombining A and C genome from B. juncea and B. carinata , respectively. Also documents B genome introgressions in resynthesized B. napus. Resynthesis of Brassica napus (AACC) was achieved by hybridizing Brassica juncea (AABB) with Brassica carinata (BBCC). This was facilitated by spontaneous chromosome doubling in the F1 hybrid (ABBC) to yield octaploid (AABBBBCC), elimination of extra B genome chromosomes in the resulting octaploid and in subsequent selfed generations, aided with directed selection for fertile plants having B. napus morphology. Twenty-five plants with varying degrees of resemblance to natural B. napus were identified from 17 A5 progenies and assayed for cytogenetic stability and genetic diversity. Majority of these plants, except six (2n = 38) were hyperploids (2n = 40-56). The six plants with 2n = 38 were designated as derived B. napus types. These showed an expected meiotic configuration of 19II at metaphase-I, with 19-19 distribution at anaphase-I. Genotyping based on A and C genome specific primers confirmed genetic identity of six derived (2n = 38) B. napus plants with natural types whereas genotyping with B genome specific primers indicated introgression of B genome segments. This was also confirmed by genomic in situ hybridization (GISH). Strong signals of B genome probe were detected, proving hitherto unreported genetic exchanges between B and A/C chromosomes. These introgressions possibly occurred en route five generations of selfing. Derived plants yielded fertile hybrids in crosses with natural B. napus var. GSC 6. The selfed derived plants as evaluated in A6 plant to progeny rows were morphologically similar to natural B. napus, and meiotically stable. Agronomic assessment of these progenies revealed variation for key morpho-physiological traits. Of special interest were the progenies with plants having oil content exceeding 47% as against about 39-41% in existing cultivars.

Molecular-cytogenetic characterization of C-genome chromosome substitution lines in Brassica juncea (L.) Czern and Coss.

KEY MESSAGE: C genome chromosome substitution lines of B. juncea constitute a key genetic resource for increased genetic diversity and hybrid performance. C genome chromosome substitution lines were found in the progenies of derived B. juncea (2n = 36; AABB), synthesized through hybridization between B. napus and B. carinata. These were originally recognized based on the morphology and genomic in situ hybridization. Genotyping using the Brassica Illumina 60K Infinium SNP array confirmed the presence of C genome chromosomes in a large number of derived B. juncea genotypes. Three whole chromosome substitutions and 13 major C genome fragment substitutions were identified. Fragment substitutions were primarily terminal, but intercalary substitution(s) involving chromosome C1 and C2 were identified in three genotypes. The size of substituted C genome fragments varied from 0.04 Mbp (C1) to 64.85 Mbp (C3). In terms of proportions, these ranged from 0.10 % (C1) to 100 % (C1, C3 and C7) of the substituted chromosome. SSR genotyping with B genome specific primers suggested that substituting C genome chromosome(s) are likely to have replaced B genome chromosome(s). C1 was the most common substituting chromosome while substituted B chromosome seemed random. Study of the phenotypic traits underlined the importance of the substitution lines (especially of chromosome C1) for conferring superior trait performance (main shoot length and pods on the main shoot). High heterosis was also indicated in hybrid combinations of substitution lines with natural B. juncea. These substitution genotypes constituted a valuable resource for targeted gene transfer and QTL identification.

Polyphyletic origin of Brassica juncea with B. rapa and B. nigra (Brassicaceae) participating as cytoplasm donor parents in independent hybridization events.

• Premise of the study: Brassica juncea is a major source of edible oil in the Indian subcontinent and northern China. It is also used as a root and leaf vegetable in China and as a condiment in Europe and America. There is a long-standing view that B. juncea originated from multiple hybridization events between B. rapa and B. nigra and that hybridizations were always unidirectional with B. rapa as the cytoplasmic donor. These conclusions were, however, centered primarily on nuclear markers.• Methods: Two hundred forty-six accessions of B. juncea, B. rapa, and B. nigra were genotyped using chloroplast and nuclear simple sequence repeat (SSR) markers.• Key results: A structure analysis assigned B. juncea germplasm (122) into three major groups based on plasmotype variation. The bulk of Indian B. juncea genotypes were grouped along with Chinese and Australian accessions. This plasmotype was absent in sampled accessions of B. rapa (97), B. nigra (27), and other wild crucifers (10). The second group of B. juncea included East European genotypes and four accessions from India. It showed unambiguous homology with the predominant B. nigra plasmotype. The neighbor joining tree produced seven subgroups, arranged into two broad lineages. The first lineage included Indian, Australian, and Chinese B. juncea genotypes; it was associated with wild species belonging to the "rapa" lineage. Nuclear SSR marker-based analyses were largely supportive of results from chloroplast SSR analyses.• Conclusions: Based on these results, we provide the first report that B. juncea originated several times with both B. rapa and B. nigra as cytoplasmic donors in separate hybridization events.

Cytogenetic and Molecular Characterization of B-Genome Introgression Lines of Brassica napus L.

Brassica napus introgression lines (ILs), having B-genome segments from B. carinata, were assessed genetically for extent of introgression and phenotypically for siliqua shatter resistance. Introgression lines had 7-9% higher DNA content, were meiotically stable, and had almost normal pollen fertility/seed set. Segment introgressions were confirmed by fluorescent genomic in situ hybridization (fl-GISH), SSR analyses, and SNP studies. Genotyping with 48 B-genome specific SSRs detected substitutions from B3, B4, B6, and B7 chromosomes on 39 of the 69 ILs whereas SNP genotyping detected a total of 23 B-segments (≥3 Mb) from B4, B6, and B7 introgressed into 10 of the 19 (C1, C2, C3, C5, C6, C8, C9, A3, A9, A10) chromosomes in 17 ILs. The size of substitutions varied from 3.0 Mb on chromosome A9 (IL59) to 42.44 Mb on chromosome C2 (IL54), ranging from 7 to 83% of the recipient chromosome. Average siliqua strength in ILs was observed to be higher than that of B. napus parents (2.2-6.0 vs. 1.9-4.0 mJ) while siliqua strength in some of the lines was almost equal to that of the donor parent B. carinata (6.0 vs.7.2 mJ). These ILs, with large chunks of substituted B-genome, can prove to be a useful prebreeding resource for germplasm enhancement in B. napus, especially for siliqua shatter resistance.

Heterosis as investigated in terms of polyploidy and genetic diversity using designed Brassica juncea amphiploid and its progenitor diploid species.

Fixed heterosis resulting from favorable interactions between the genes on their homoeologous genomes in an allopolyploid is considered analogous to classical heterosis accruing from interactions between homologous chromosomes in heterozygous plants of a diploid species. It has been hypothesized that fixed heterosis may be one of the causes of low classical heterosis in allopolyploids. We used Indian mustard (Brassica juncea, 2n = 36; AABB) as a model system to analyze this hypothesis due to ease of its resynthesis from its diploid progenitors, B. rapa (2n = 20; AA) and B. nigra (2n = 16; BB). Both forms of heterosis were investigated in terms of ploidy level, gene action and genetic diversity. To facilitate this, eleven B. juncea genotypes were resynthesized by hybridizing ten near inbred lines of B. rapa and nine of B. nigra. Three half diallel combinations involving resynthesized B. juncea (11×11) and the corresponding progenitor genotypes of B. rapa (10×10) and B. nigra (9×9) were evaluated. Genetic diversity was estimated based on DNA polymorphism generated by SSR primers. Heterosis and genetic diversity in parental diploid species appeared not to predict heterosis and genetic diversity at alloploid level. There was also no association between combining ability, genetic diversity and heterosis across ploidy. Though a large proportion (0.47) of combinations showed positive values, the average fixed heterosis was low for seed yield but high for biomass yield. The genetic diversity was a significant contributor to fixed heterosis for biomass yield, due possibly to adaptive advantage it may confer on de novo alloploids during evolution. Good general/specific combiners at diploid level did not necessarily produce good general/specific combiners at amphiploid level. It was also concluded that polyploidy impacts classical heterosis indirectly due to the negative association between fixed heterosis and classical heterosis.

Generation of B. nigra-B. rapa chromosome addition stocks: cytology and microsatellite markers (SSRs) based characterization.

To improve Brassica nigra, the B-genome donor for Brassica juncea through selective introgression of useful variation from A-genome chromosomes, B. nigra-B. rapa chromosome addition stocks were successfully synthesized for the first time. Resynthesized B. juncea was used as B-genome donor species and A-genome addition stocks were developed by hybridizing sesquidiploid plant (ABB) as female and using B. nigra as the male parent. Various cycles of backcrossing and/or selfing were utilized to isolate plants carrying addition of three A-genome chromosomes in the background of B. nigra. These chromosome addition stocks were characterized by chromosome counts, pollen and seed fertility and chromosome specific microsatellite (SSRs) markers. The chromosome number in different backcross/self generations ranged between 2n=26 and 2n=19 with relatively high frequency of univalents (8-10I) at in meiotic configurations observed, suggesting the role of preferential transmission of A-genome chromosomes. SSRs analysis revealed that B. rapa chromosomes 3 and 4 were the first to get eliminated followed by chromosome 10. Remaining chromosomes were maintained till BC(1)F(4). However, second cycle of backcrossing (BC(2)) led to the elimination of chromosome numbers 1 and 2. BC(2)F(2) plants carried the chromosome numbers 6, 7, 8 and 9. Generation BC(3) having plants with 2n=19 carried chromosome numbers 6, 7 and 8. It is possible that chromosomes 6, 7 and 8 had higher transmission frequency and these were better tolerated by the B. nigra genome.

A microsatellite (SSR) based linkage map of Brassica rapa.

In the present study we describe the construction of a genetic linkage map for the Brassica rapa (AA) genome that will act as a key resource in undertaking future structural and functional genomic studies in B. rapa. A F(2) mapping population consisting of 48 F(2) individual plants developed following hybridization of 2 inbred lines Bathari mandi and IC 331817 was used to construct the map. The map comprises 53 SSR markers derived from 3 different public domain resources. Nine linkage groups along with a small subgroup were identified and designated as R(1)-R(9) through alignment and orientation using SSR markers in common with existing B. rapa reference linkage maps. The total length of the genetic linkage map was 354.6 cm with an average interval of 6.6 cm between adjacent loci. The length of linkage groups ranged from 28.0 cm to 44.2 cm for R(6) and R(1A), respectively. The number variability of markers in the 9 linkage groups ranged from 3 for R(6) to 10 for R(1). Of the 53 SSR markers assigned to the linkage groups, only 5 (9.4%) showed deviation from the expected segregation ratio. The development of this map is vital to the genome integration and genetic information and will enable the international research community to share resources and data for the improvement of B. rapa and other cultivated Brassica species.