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; 2
n
= 36) with
B. napus
(AACC; 2
n
= 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
D81
Rfo
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Key message
Genome wide association studies enabled prediction of many candidate genes for flowering, maturity and plant height under differing day-length conditions. Some genes were envisaged only ...from derived
B. rapa
.
Flowering and plant height are the key life history traits. These are crucial for adaptation and productivity. Current investigations aimed to examine genotypic differences governing days to flowering, maturity and plant height under contrasting day-length conditions; and identify genomic regions governing the observed phenotypic variations. An association panel comprising 195 inbred lines, representing natural (NR) and derived (DR) forms of
Brassica rapa
(AA; 2
n
= 20), was evaluated at two sowing dates and two locations, representing different day-length regimes. Derived
B. rapa
is a unique pre-breeding material extracted from
B. juncea
(AABB; 2
n
= 36). Population structure analysis, using DArT genotypes established derived
B. rapa
as a genetic resource distinct from natural
B. rapa
. Genome wide association studies facilitated detection of many trait associated SNPs. Chromosomes A03, A05 and A09 harboured majority of these. Functional annotation of the associated SNPs and surrounding genome space(s) helped to predict 43 candidate genes. Many of these were predicted under specific day-length conditions. Important among these were the genes encoding floral meristem identity (
SPL3
,
SPL15
,
AP3
,
BAM2
), photoperiodic responses (
COL2
,
AGL18
,
SPT
,
NF-YC4
), gibberellic acid biosynthesis (
GA1
) and regulation of flowering (
EBS
). Some of the predicted genes were detected for DR subpanel alone. Genes controlling hormones, auxins and gibberellins appeared important for the regulation of plant height. Many of the significant SNPs were located on chromosomes harbouring previously reported QTLs and candidate genes. The identified loci may be used for marker-assisted selection after due validation.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Key message
Heavy doses of gamma irradiation can reduce linkage drag by disrupting large sized alien translocations and promoting exchanges between crop and wild genomes.
Resistance to mustard aphid ...(
Lipaphis erysimi
) infestation was significantly improved in
Brassica juncea
through
B. juncea
-
B. fruticulosa
introgression. However, linkage drag caused by introgressed chromatin fragments has so far prevented the deployment of this resistance source in commercial cultivars. We investigated the patterns of donor chromatin segment substitutions in the introgression lines (ILs) through genomic in situ hybridization (GISH) coupled with
B. juncea
chromosome-specific oligonucleotide probes. These allowed identification of large chromosome translocations from
B. fruticulosa
in the terminal regions of chromosomes A05, B02, B03 and B04 in three founder ILs (AD-64, 101 and 104). Only AD-101 carried an additional translocation at the sub-terminal to intercalary position in both homologues of chromosome A01. We validated these translocations with a reciprocal blast hit analysis using shotgun sequencing of three ILs and species-specific contigs/scaffolds (kb sized) from a de novo assembly of
B. fruticulosa
. Alien segment substitution on chromosome A05 could not be validated. Current studies also endeavoured to break linkage drag by exposing seeds to a heavy dose (200kR) of gamma radiation. Reduction in the size of introgressed chromatin fragments was observed in many M
3
plants. There was a complete loss of the alien chromosome fragment in one instance. A few M
3
plants with novel patterns of chromosome segment substitutions displayed improved agronomic performance coupled with resistance to mustard aphid. SNPs in such genomic spaces should aid the development of markers to track introgressed DNA and allow application in plant breeding.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Sclerotinia stem rot (Sclerotinia sclerotiorum) is a major disease of Brassica oilseeds. As suitable donors to develop resistant cultivars are not available in crop Brassicas, we introgressed ...resistance from a wild Brassicaceae species, B. fruticulosa. We produced 206 B. juncea-B. fruticulosa introgression lines (ILs). These were assessed for pollen grain fertility, genome size variations and resistance responses to Sclerotinia following stem inoculations under disease-conducive conditions. Of these, 115 ILs showing normal fertility and genome size were selected for cytogenetic characterization using florescent genomic in situ hybridization (Fl-GISH). B. fruticulosa segment substitutions were indicated in 28 ILs. These were predominantly terminal and located on B-genome chromosomes. A final set of 93 highly fertile and euploid (2n = 36) ILs were repeat-evaluated for their resistance responses during 2014-15. They were also genotyped with 202 transferable and 60 candidate gene SSRs. Association mapping allowed detection of ten significant marker trait associations (MTAs) after Bonferroni correction. These were: CNU-m157-2, RA2G05, CNU-m353-3, CNU-m442-5, ACMP00454-2, ACMP00454-3, EIN2-3-1, M641-1, Na10D09-1 and Na10D11-1. This is the first time such a molecular mapping technique has been deployed with introgression lines carrying genomic segments from B. fruticulosa, and the first to show that they possess high levels of resistance against S. sclerotiorum.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Lipaphis erysimi
is a key pest of rapeseed-mustard in Indian subcontinent. Although chemical control is the basis of its management, the unsustainability of this approach has accelerated global ...research efforts to find alternate solutions. Host plant resistance is one among these. A set of introgression lines were developed using
Brassica fruticulosa
previously found to be resistant to
L. erysimi
. Rigorous screening over the years led to the identification of 3 introgression lines (I
8
, I
79
, and I
82
) for field resistance to aphids. We evaluated these introgression lines under field and laboratory conditions along with
B. fruticulosa
(resistant parent),
B. juncea
var. PBR-210 (susceptible parent) to elucidate the mechanism of resistance. Significantly a smaller number of aphids settled on circular leaf discs of
B. fruticulosa
, I
8
and I
82
compared to that on PBR-210 after 24 and 48 h of release. A similar trend was observed in free choice field experiment with significantly less aphid colonization on
B. fruticulosa
, I
8
, I
79
and I
82
compared to PBR-210 indicating lower aphid preference for these genotypes. Further, no choice experiments revealed significant negative effects of these genotypes on aphid demographic parameters (nymphal survival, development period, fecundity and longevity). Tolerance may not be a mechanism of resistance as aphid population failed to develop on these genotypes. Thus, resistance in these introgression lines may be attributed to a synergistic combination of antixenosis and antibiosis mechanisms.
The lack of resistance to
Lipaphis erysimi
in cultivated Brassicas makes caused this pest highly devastating resulting in significant loss of rapeseed-mustard productivity in India.
B. fruticulosa
, ...a wild crucifer is known as an excellent source of resistance to
L. erysimi
. Therefore, we planned to assess defense associated biochemical alterations and molecular components of
B. juncea
–
B. fruticulosa
ILs to mustard aphid. Phenotypic assessment of ILs on the basis of aphid population per plant (APP) categorized genotypes into resistant (7.15–18.50 APP), moderately susceptible (42.29–53.33 APP) and susceptible (70.00–77.07 APP) genotypes. Mustard aphid infested minimally
B. fruticulosa
(0.80 APP) among tested genotypes. The maximum increase in catalase (CAT) activity was determined in
B. fruticulosa
and resistant ILs after 48 h (2.03 and 1.76-fold, respectively) and one week (2.98 and 1.79-fold, respectively) of mustard aphid infestation. The strong induction of
CAT2
transcripts (19.25-fold) and CAT activity (5.88-fold) along with low aphid count in resistant IL, Ad
4
-64 (13.85 APP) suggested the pivotal role of CAT in resistance to mustard aphid. Guaiacol peroxidase (GPX) was significantly decreased following pest infestation at both infestation stages. The ascorbate content was highest in resistant IL, ADV-6RD (2.14-fold) after one week of aphid infestation. H
2
O
2
content rapidly increased in
B. juncea
–
B. fruticulosa
derived lines after 48 h of aphid infestation. The negative and significant association between APP and CAT (− 0.56** and − 0.48*, respectively), glutathione (− 0.43* and − 0.40*, respectively), H
2
O
2
(− 0.57** and − 0.43*, respectively) at both 48 h and one week infestation stages signified their role in deterring mustard aphid infestation. The positive and significant association between total sugars (0.33* at 7 DPI), reducing sugars (0.33* at 7 DPI), sucrose (0.36** at 48 h) and APP indicated that higher the sugars content, higher will be mustard aphid infestation in
B. juncea
derived ILs. The information being generated and key candidates (
CAT2
, ascorbate and H
2
O
2
) being identified may help in effective deployment of
B. fruticulosa
resistance in mustard breeding.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
A set of 96
-
introgression lines (ILs) were developed with genomic regions associated with
stem rot (
) resistance from a wild
species
. ILs were assessed for their resistance responses to stem ...inoculation with
, 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
initially and, subsequently, into other crop
species.
Key message
We report first-time synthesis of a stable
Brassica
allohexaploid. It may evolve into a new species and also advance our understanding of pairing regulation and genome evolution in ...complex allopolyploids.
Crop Brassicas include both monogenomic and digenomic species. A trigenomic
Brassica
(AABBCC) is not known to exist in nature. Past attempts to synthesize a stable allohexaploid were not successful due to aberrant meiosis and very high proportion of aneuploid plants in the selfed progenies. We report the development of a stable allohexaploid
Brassica
(2
n
= 54; AABBCC). Genomic in situ hybridization confirmed the complete assemblage of three genomes. Only allohexaploids involving
B. rapa
cv. R01 (2
n
= 20; AA) as pollinator with a set of
B. carinata
(2
n
= 34; BBCC) were stable. These exhibited a high proportion (0.78–0.94) of pollen mother cells with normal meiosis and an excellent hexaploid ratio (0.80–0.94) in the selfed progenies. Stability of two allohexaploid combinations was demonstrated from H
1
to H
4
generations at two very diverse locations in India. Graphical genotyping of allohexaploids allowed detection of chromosome fragment exchanges among three genomes. These were much smaller for meiotically stable allohexaploids as compared to unstable ones. The putative hexaploids were morphologically closer to the female donor,
B. carinata
, for leaf morphology, inflorescence structure and flower shape. The newly formed allohexaploid may also provide unique opportunities to investigate the immediate genetic and genomic consequences of a
Brassica
allohexaploid with three resident genomes.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Understanding the stability of photosynthetic pigments is crucial for developing crop cultivars with high productivity and resilience to the environmental stresses. This study leveraged GGE biplot, ...WAASB, and MTSI indices to assess the stability of content and composition of photosynthetic pigments in leaves and siliques of 286 Brassica juncea (L.) Czern. genotypes across three environments. The GGE biplot analysis identified NRCQR-9901 as the best genotype in terms of chlorophyll 'a' under conditions of high irradiance and long days (E1). For chlorophyll 'b' and total chlorophyll, NC-533728 performed the best. AJ-2 and NPJ-208 had the maximum total carotenoids levels in leaves. RLC-2 was characterized by maximum values for chlorophyll a, chlorophyll b, and total chlorophyll in the siliques. The low irradiance, short days, and moderate to high temperatures (E2) seemed perfect for the synthesis of photosynthetic pigments. NPJ-182 shows the maximum concentrations of chlorophyll 'a', total chlorophyll, and total carotenoids in leaves. Conversely, IC-597869, RE-389, and IC-597894 exhibited the highest concentrations of chlorophyll 'b' under an environment characterized by low light intensity, shorter daylights, and low temperatures (E3) during flowering and siliqua formation stages. The combined analysis found NPJ-182, NC-533728, CN-105233, RLC-2, CN-101846, JA-96, PBR-357, JM-3, and DTM-34 as top performers with high stability. Comparative transcriptome analysis with two stable and high-performing genotypes (PBR-357 and DTM-34) and two average performers (name the average performers) revealed upregulation of critical photosynthesis-related genes (ELIP1, CAB3.1, ELIP1.5, and LHCB5) in top performers. This study identified promising trait donors for use in breeding programs aimed at improving the mustard crop's photosynthetic efficiency, productivity, and stability.Understanding the stability of photosynthetic pigments is crucial for developing crop cultivars with high productivity and resilience to the environmental stresses. This study leveraged GGE biplot, WAASB, and MTSI indices to assess the stability of content and composition of photosynthetic pigments in leaves and siliques of 286 Brassica juncea (L.) Czern. genotypes across three environments. The GGE biplot analysis identified NRCQR-9901 as the best genotype in terms of chlorophyll 'a' under conditions of high irradiance and long days (E1). For chlorophyll 'b' and total chlorophyll, NC-533728 performed the best. AJ-2 and NPJ-208 had the maximum total carotenoids levels in leaves. RLC-2 was characterized by maximum values for chlorophyll a, chlorophyll b, and total chlorophyll in the siliques. The low irradiance, short days, and moderate to high temperatures (E2) seemed perfect for the synthesis of photosynthetic pigments. NPJ-182 shows the maximum concentrations of chlorophyll 'a', total chlorophyll, and total carotenoids in leaves. Conversely, IC-597869, RE-389, and IC-597894 exhibited the highest concentrations of chlorophyll 'b' under an environment characterized by low light intensity, shorter daylights, and low temperatures (E3) during flowering and siliqua formation stages. The combined analysis found NPJ-182, NC-533728, CN-105233, RLC-2, CN-101846, JA-96, PBR-357, JM-3, and DTM-34 as top performers with high stability. Comparative transcriptome analysis with two stable and high-performing genotypes (PBR-357 and DTM-34) and two average performers (name the average performers) revealed upregulation of critical photosynthesis-related genes (ELIP1, CAB3.1, ELIP1.5, and LHCB5) in top performers. This study identified promising trait donors for use in breeding programs aimed at improving the mustard crop's photosynthetic efficiency, productivity, and stability.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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