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.

Evolutionary aspects of direct or indirect selection for seed size and seed metabolites in Brassica juncea and diploid progenitor species.

Seed size and seed metabolites have been the targets of direct or indirect selection during domestication and subsequent crop breeding. Understanding these traits and associated genetics can prove very useful for plant translational research. Large germplasm assemblage (235) of Brassica juncea and its progenitor species (B. rapa and B. nigra) was evaluated to establish seed trait variations for seed size and seed metabolites. Seeds were smallest in B. nigra and largest in B. juncea. Australian B. juncea and Indian B. rapa var brown sarson types averaged more seed oil content. Seed size and oil content were generally higher in modern cultivars in comparison to the land races. Allelic diversity for known associated genes for seed-size and oil-content (AP2, ARF2, TTG2, GRF2, GL2, CYP78A5, CYP78A6, MINI3, IKU2, IKU1, BRI1, DGAT, GPDH, LPAAT, GPAT and DA1) was studied so as to infer the effect of domestication on seed traits. Three genes (IKU1, IKU2, AP2) in B. rapa, two (TTG2 and GL2) in B. nigra and two (IKU1 and GRF2) in natural B. juncea were identified as targets of selection on the basis of Fst outlier and/or sequence diversity tests. We report parallel divergence for seed traits between B. juncea and B. rapa. Directional selection appeared stronger for seed-size as compared to correlated seed metabolites. Positive selection on seed-size is likely to have played a significant role in structuring regional variation in the germplasm.

Discovery and mapping of Brassica juncea Sdt 1 gene associated with determinate plant growth habit.

KEY MESSAGE: Newly discovered determinate plant growth habit in Brassica juncea is simply inherited and can help in architectural restructuring of Brassica oilseeds. Brassica juncea is naturally indeterminate. This growth habit tends to accentuate intra-plant competition for resources within the plant canopy, leading to unfilled seeds, immature pods and tip sterility. Recent identification of plants with determinate growth habit is expected to open up new avenues for plant architectural modifications in crop Brassicas. Plants with determinate plant growth habit were identified in progenies of resynthesized B. juncea as a de novo variation. F1 plants, developed from crosses of determinate mustard with natural indeterminate genotypes were indeterminate, indicating the dominance of indeterminacy. F2 and F3 segregation revealed monogenic recessive inheritance in the progenies studied. Gene for determinacy (Sdt 1 ) was mapped to the linkage group 15 of B. juncea. Sdt 1 was flanked by SSR markers SJ6842 and Ni4-A10 at distances of 15.9 cM and 14.0 cM, respectively. Determinate progenies showed significant variation for plant height, flowering time and productivity. There appeared to be no adverse association in terms of lower pod density, productivity or oil content. Determinacy was under control of single recessive gene, mapped to the linkage group 15 of B. juncea. Determinate progenies with high agronomic performance were identified.

Population structure and breeding value of a new type of Brassica juncea created by combining A and B genomes from related allotetraploids.

KEY MESSAGE: Derived amphiploidy helped to resynthesize agronomically superior B. juncea germplasm which showed high heterosis in crosses with natural B. juncea . This new procedure facilitates a seamless flow of variation across Brassica digenomics. Brassica digenomics, artificially resynthesized by hybridizing extant genome donor diploids, show poor breeding value due to the linkage drag associated with diploid donors. We recently developed a method that involves resynthesis through hybridization between related allotetraploids. Derived B. juncea was created by combining A and B genomes extant in B. napus and B. carinata, respectively. Large genomic and agronomic modifications resulted. Population structure analysis based on the DNA polymorphism generated using 108 locus-specific SSR primers helped to identify three pools of allelic diversity. Thirteen progenies with determinate plant growth habit were discovered, and these aligned closely with B genome of the donor species like B. nigra and B. carinata. The indeterminate group showed greater genetic affinity with extant B. juncea. Derived genotypes possessed high agronomic potential. Importantly, high heterosis was observed in crosses between derived and natural B. juncea. Some derived juncea progenies figured in heterotic combinations during both the years of F 1 hybrid evaluation. In essence, the hybrids between derived B. juncea and natural B. juncea can be considered as interspecific hybrids between B. juncea and B. napus for A genome and between B. juncea and B. carinata for B genome. This possibly explains their high heterosis-inducing potential. Integrating genetic diversity with the inherent breeding value allowed more efficient prediction of heterosis. Besides generation of new novel variability of huge economic importance and operational simplicity, the method of derived amphiploidy allows a seamless flow of heritable variation across Brassica digenomics.

Heat stress tolerance in relation to oxidative stress and antioxidants in Brassica juncea.

In the present study fifty genotypes of Brassica juncea were evaluated for heat stress tolerance in terms of biochemical components, in four day old seedlings. Heat shock was given at 45 degrees C for 4.5 hr and thereafter survival percentage, electrolyte leakage and chlorophyll content were estimated. Tolerant genotypes (10) registered survival greater than 65%, moderately tolerant (20) between 35-65% and susceptible (20) less than 35%. Electrolyte leakage was significantly (p < 0.001) higher in susceptible genotypes than in tolerant ones with respect to control seedlings. Chlorophyll content showed no significant variation among the tolerant, moderately tolerant and susceptible genotypes, although it registered a decline in response to heat stress. Lipid peroxidation, assessed by malondialdehyde (MDA) in stressed conditions was 4.66 (MDA g(-1) f. wt. of tissue) in tolerant genotypes, 7.44 (MDA g(-1) f. wt. of tissue) in susceptible genotypes and correlated significantly (r = 0.563) with electrolyte leakage. Increase in POD activity under heat stress was maximum in tolerant class with respect to control. CAT activity showed decrease after heat shock treatment in all the three classes but the decrease was 1.3 fold in tolerant genotypes as compared to 1.6 fold in susceptible genotypes. The non-enzymatic antioxidants glutathione and proline registered a significantly (< 0.01) high value in tolerant genotypes on heat shock treatment in comparison to susceptible genotypes corroborating the role of antioxidants in mitigating the effect of heat stress in Bjuncea. The antioxidants and proline seemed to play role in mitigating the effect of heat stress.

Using near-infrared reflectance spectroscopy (NIRS) to predict the nitrogen levels in the stem and root tissues of Brassica juncea (Indian mustard).

Brassica juncea depends heavily on nitrogen (N) fertilizers for growth and accumulation of seed protein. However, it is an inefficient mobilizer of applied N which leads to accumulation of excess N in the soil, posing environmental risks. Hence, it is imperative to systematically examine spatial-temporal pattern of crop N to efficiently manage N application. The Kjeldahl method is commonly used to estimate N status of crops but it is a destructive method that entails the use of perilous and expensive chemicals. Near-infrared reflectance spectroscopy (NIRS) offers a safe, accurate, and non-destructive alternative for large-scale screening of seed metabolites. Currently, no NIRS model exists to quickly estimate N content in shoots and roots from large germplasm sets in any rapeseed-mustard crop. Developing such a model is essential to breed for enhanced nitrogen use efficiency (NUE). We used 738 shoot and 346 root samples from a B. juncea diversity set to construct the NIRS models. A diverse range of genetic variation in N content was recorded in the stem (0.21-6.61%) and root (0.15-3.04%) tissues of the crop raised on two different N levels (N0 and N100). Modified partial least squares (MPLS) method was employed to establish a regression equation linking reference N values with spectral changes. The developed models exhibited strong associations with reference values, with RSQ values of 0.884 for stem and 0.645 for roots. Furthermore, external validation confirms the reliability of the developed models. The developed models have strong predictive capabilities for rapid and reliable N estimation in various tissues of B. juncea plants.

PHF10 is required for cell proliferation in normal and SV40-immortalized human fibroblast cells.

Normal human diploid fibroblasts have limited life span in culture and undergo replicative senescence after 50-60 population doublings. On the contrary, cancer cells typically divide indefinitely and are immortal. Expression of SV40 large T and small t antigens in human fibroblasts transiently extends their life span by 20-30 population doublings and facilitates immortalization. We have identified a rearrangement in chromosome 6 shared by SV40-transformed human fibroblasts. Rearrangements involving chromosome 6 are among the most frequent in human carcinogenesis. In this paper, we extend analysis of the 6q26-q27 region, a putative site for a growth suppressor gene designated SEN6 involved in immortalization of SV40-transformed cells. Detailed molecular characterization of the rearranged chromosomes (6q*, normal appearing; and 6q(t), translocated) in the SV40-immortalized cell line HALneo by isolating each of these 2 chromosomes in mouse/HAL somatic cell hybrids is presented. Analysis of these mouse/HAL somatic cell hybrids with polymorphic and nonpolymorphic markers revealed that the 6q* has undergone a chromosomal break in the MLLT4 gene (alias AF6). This result in conjunction with previous published observations leads us to conclude that SEN6 lies between MLLT4 and TBP at chromosomal region 6q27. Examination of different genes (MLLT4, DLL1, FAM120B, PHF10) located within this interval that are expressed in HS74 normal fibroblast cells reveals that overexpression of epitope-tagged truncated PHF10 cDNAs resulted in reduced cell proliferation in multiple cell lines. Paradoxically, down-regulation of PHF10 by RNAi also resulted in loss of cell proliferation in normal fibroblast cells, indicating PHF10 function is required for cell growth. Taken together, these observations suggest that decreased cell proliferation with epitope-tagged truncated PHF10 proteins may be due to dominant negative effects or due to unregulated expression of these mutant proteins. Hence we conclude that PHF10 is not SEN6 but is required for cell growth.

SEN6, a locus for SV40-mediated immortalization of human cells, maps to 6q26-27.

Normal cells show a limited lifespan in culture and the phenotype of cellular senescence. Tumors and tumor cell lines have typically overcome this form of growth suppression and grow continuously as immortal cell lines in culture. We have exploited the DNA virus SV40 to study the mechanism by which human fibroblasts overcome senescence and become immortal. Multiple steps have now been identified, including inactivation of cellular growth suppressors through direct interaction with SV40 large T antigen and through mutation of a gene on chromosome 6 (designated SEN6). In this study, we sublocalize the site of SEN6 to 6q26-27 based on molecular genetic analysis. Twelve SV40-immortalized fibroblast cell lines share a deletion in this area based on assessment for loss of heterozygostiy (LOH) for seven informative markers on 6q. Two immortal cell lines (AR5 and HALneo) appeared to have retained separate single copies of chromosome 6 despite the fact that they are both derived from the same preimmortal SV40-transformant and should share the same mutated allele of SEN6 (Hubbard-Smith et al., 1992). Detailed analysis by polymerase chain reaction, restriction fragment length polymorphism and fluorescence in situ hybridization shows, however, that although they differ for 17 markers from the centromere to 6q26, they share AR5 derived sequences (eight markers) distal to 6q26 including the minimal deletion region, further supporting the assignment of SEN6 to this region. Since human tumors including non-Hodgkins lymphoma, mammary carcinoma and ovarian carcinoma show LOH in 6q26-27, inactivation of SEN6 may be responsible for immortalization of these tumors as well.

Neurosyphilis in a tertiary care hospital in north India.

BACKGROUND AND OBJECTIVES: The clinical diagnosis of neurosyphilis is very rarely encountered today in the developed world although syphilis remains a significant health problem in few areas of the industrialized countries and in most of the third world nations. This apparent decline may be due to increase in number of asymptomatic neurosyphilis and cases presenting as subtle, illdefined syndromes rather than classic presentation of tabes dorsalis and general paresis in the post penicillin era. This retrospective study was carried out to report the neurosyphilis cases diagnosed at a tertiary care hospital in North India, and to analyse the laboratory and clinical parameters of these cases. METHODS: Suspected cases of neurosyphilis presenting at Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh over a period of 13 yr (January 1990 to December 2002) were identified. Diagnosis of neurosyphilis was based on clinical presentation, prior history of syphilis, routine CSF biochemistry (protein and leukocytes) and serological evidence [serum and CSF venereal disease research laboratory (VDRL) and Treponema pallidum particle agglutination (TPPA) tests]. RESULTS: A total of 25 cases of neurosyphilis were identified, 18 (72%) with reactive CSF-VDRL, 22 (88%) with elevated CSF protein and 24 (96%) with CSF mononuclear leukocytosis. Serum VDRL was reactive in all 25 cases. Three patients were asymptomatic (2 primary syphilis; 1 early latent stage), 8 had secondary and 14 had tertiary syphilis. Two of the neurosyphilis cases were also seropositive for HIV. Radiology was abnormal in 7 (28%) patients. INTERPRETATION AND CONCLUSION: Neurosyphilis still remains a problem in a country like India and a high index of suspicion and clinical expertise are required for appropriate diagnosis and proper management especially in the era of AIDS pandemic.

Screening of patients with acute febrile illness for leptospirosis using clinical criteria and serology.

BACKGROUND: Leptospirosis is one of the common zoonoses but, in most instances, the infection goes unnoticed. Rapid diagnostic modalities are needed to diagnose the disease in the early stages. We assessed the usefulness of clinical criteria and compared these with enzyme-linked immunosorbentassay (ELISA) for the early detection of leptospirosis. METHODS: One hundred patients with a febrile illness for > 7 days were screened by Faine criteria and their sera were subjected to both IgM and IgG ELISA using a commercially available kit (Institut Virion Serion GmbH, Warburg, Germany). RESULTS: Twenty-six patients satisfied the clinical criteria for leptospirosis and 8 of them tested positive for IgM antibodies while 1 patient who was clinically negative tested positive by serology. Thus, Faine criteria had a sensitivity of 88.9%, specificity of 80.2%, positive predictive value of 30.8% and a negative predictive value of 98.6%. Paired serum samples were obtained from 70 patients but the IgG levels of only 2 showed a 4-fold rise. CONCLUSION: Faine criteria has moderate sensitivity and specificity but a high negative predictive value in comparison with IgM ELISA. The high negative predictive value may help to screen patients with acute febrile illness for leptospirosis during the early phase of the disease.

SV40-mediated immortalization of human fibroblasts.

We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

Hypersensitivity of Drosophila mei-41 mutants to hydroxyurea is associated with reduced mitotic chromosome stability.

6 mutant alleles of the mei-41 locus in Drosophila melanogaster are shown to cause hypersensitivity to hydroxyurea in larvae. The strength of that sensitivity is directly correlated with the influence of the mutant alleles on meiosis in that: alleles exhibiting a strong meiotic effect (mei-41D2, mei-41D5, mei-41D7) are highly sensitive; alleles with negligible meiotic effects (mei-41(104)D1, mei-41(104)D2) are moderately sensitive and an allele which expresses meiotic effects only under restricted conditions (mei-41D9) has an intermediate sensitivity. This sensitivity is not a general feature of strong postreplication repair-deficient mutants, because mutants with that phenotype from other loci do not exhibit sensitivity (mus(2)205A1, mus(3)302D1, mus(3)310D1). The observed lethality is not due to hypersensitivity of DNA synthesis in mei-41 larvae to hydroxyurea as assayed by tritiated thymidine incorporation. Lethality is, however, potentially attributable to an abnormal enhancement of chromosomal aberrations by hydroxyurea in mutant mei-41 larvae. Both in vivo and in vitro exposure of neuroblast cells to hydroxyurea results in an increase in 3 types of aberrations which is several fold higher in mei-41 tissue. Since hydroxyurea disrupts DNA synthesis, these results further implicate the mei-41 locus in DNA metabolism and provide an additional tool for an elucidation of its function. The possible existence of additional genes of this nature is suggested by a more modest sensitivity to hydroxyurea which has been detected in two stocks carrying mutagen-sensitive alleles of alternate genes.

P transposition in Drosophila provides a new tool for analyzing postreplication repair and double-strand break repair.

A genetic screen has been developed in Drosophila for identifying host-repair genes responsible for processing DNA lesions formed during mobilization of P transposable elements. Application of that approach to repair deficient mutants has revealed that the mei-41 and mus302 genes are necessary for recovery of P-bearing chromosomes undergoing transposition. Both of these genes are required for normal postreplication repair. Mutants deficient in excision repair, on the other hand, have no detected effect on the repair of transposition-induced lesions. These observations suggest that P element-induced lesions are repaired by a postreplication pathway of DNA repair. The data further support recent studies implicating double-strand DNA breaks as intermediates in P transposition, because the mei-41 gene has been genetically and cytologically associated with the repair of interrupted chromosomes. Analysis of this system has also revealed a striking stimulation of site-specific gene conversion and recombination by P transposition. This result strongly suggests that postreplication repair in this model eukaryote operates through a conversion/recombination mechanism. Our results also support a recently developed model for a conversion-like mechanism of P transposition (Engels et al., 1990). Involvement of the mei-41 and mus302 genes in the repair of P element-induced double-strand breaks and postreplication repair points to a commonality in the mechanisms of these processes.

Recovery and characterization of hybrid dysgenesis-induced mei-9 and mei-41 alleles of Drosophila melanogaster.

X-Linked methyl methanesulfonate (MMS)-sensitive mutations were induced with hybrid dysgenesis using four P strains: pi 2, Harwich, T-007 and OK-1. Mutations were identified after two generations of backcrosses to M strain females to replace the autosomes. Among 51,471 X-chromosomes examined 10 carried stable MMS-sensitive mutations representing 8 independent events. Males of the mutant strains failed to induce gonadal dysgenesis in crosses to Oregon-R females at 28.5 degrees C. Complementation tests showed that 3 of the induced mutations were mei-9 alleles, 2 were mei-41 alleles, 1 was a mus102 allele, and 2 were alleles at a newly identified MMS-sensitive locus, mus112 (map position: 1-32.8). As assayed by in situ hybridization on polytene chromosomes, each X-chromosome had no more than four P element insertions. 4 of the 8 mutations recovered in this study proved to have P element insertions at or very close to sites to which MMS sensitivity has been mapped. Hybrid dysgenesis-induced reversion of 2 mutants, mei-9RT1 and mei-41RT2, is associated with the loss of the P element from regions 4B and 14C respectively.

Complementation of V(D)J recombination defect and X-ray sensitivity of scid mouse cells by human chromosome 8.

Cells derived from mice homozygous for the severe combined immune deficiency (scid) mutation exhibit hypersensitivity to ionizing radiation, and defects in DNA double-strand break repair and V(D)J recombination. Using the technique of microcell-mediated chromosome transfer, we have introduced a number of dominantly marked human chromosomes into scid cells to localize the human homolog of the murine scid gene. Analysis of human-scid hybrid clones revealed that the presence of human chromosome 8 partially restored accurate V(D)J recombination and radioresistance to scid cells. Subsequent loss of the human chromosome 8 from human-scid hybrid clones rendered these cells sensitive to gamma-radiation and impaired their ability to catalyse V(D)J recombination. Introduction of chromosomes 2, 14, 16 and 19 that encode other repair genes did not result in the correction of these two scid defects. These observations demonstrate that the human homolog of the mouse scid gene resides on human chromosome 8.

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.

Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster.

An efficient technique has been developed for performing in vivo site-directed mutagenesis in Drosophila melanogaster. This procedure involves directed repair of P-element-induced DNA lesions after injection of a modified DNA sequence into early embryos. An oligonucleotide of 50 base pairs, whose sequence spans the P-element insertion site, mediates base replacement in the endogenous gene. Restriction mapping, DNA sequencing, and polymerase chain reaction analysis demonstrate that base substitutions present in an injected oligonucleotide are incorporated into genomic sequences flanking a P insertion site in the white gene. This analysis suggests that progeny bearing directed mutations are recovered with a frequency of about 0.5 x 10(-3). Because Drosophila remains a premier organism for the analysis of eukaryotic gene regulation, this system should find strong application in that analysis as well as in the analysis of DNA recombination, conversion, repair, and mutagenesis.

Alternaria incidence in some alloplasmic lines of Indian mustard (Brassica juncea (L.) Coss.).

The cytoplasmic substitution lines of Brassica juncea (L.) Coss were evaluated for their field resistance to Alternaria blight (Alternaria brassicae). The euplasmic B. juncea cv. 'RLM 198' had a mesothetic reaction while alloplasmic B. juncea lines with cytoplasms of B. campestris, B. chinensis, and B. japonica were highly susceptible. B. nigra cytoplasm did not have any effect on the disease reaction of the B. juncea genome. However, the alloplasmic lines with the cytoplasm of B. napus and B. carinata revealed a comparatively higher degree of resistance. The study underlined the utility of cytoplasmic manipulations in modifying the phenotypic expression of nuclear genes.

Male sterility in Indian mustard (Brassica juncea (L.) Coss.) - a biochemical characterization.

Biochemical studies were conducted on some male sterile and their fertile F1 analogues in Indian mustard. The variation in pH activity during microsporogenesis was normal, except in MS-3. Male sterile anthers had deficient sugar metabolism. Cytochemical analysis of sporogenous and tapetal tissue suggested an effect of sterility elements on the anabolic and catabolic fate of DNA and protein during microsporogenesis. Leaves of male sterile lines had a higher chl a/b ratio. Leaf peroxidase activity was low and different isozymes appeared when separated by starch electrophoresis.