Assessment of genetic diversity in Brassica juncea Brassicaceae genotypes using phenotypic differences and SSR markers / Evaluación de la diversidad genética en genotipos de Brassica juncea Brassicaceae utilizando diferencias fenotípicas y marcadores SSR

Brassica mustard species represent one of the most important oilseed crops in India, nevertheless, their genetic diversity is barely known. A better understanding on this topic is essential for the proper utilization of genotypes in breeding programmes. We evaluated the genetic diversity among 44 Indian mustard Brassica juncea genotypes including varieties/purelines from different agro-climatic zones of India and few exotic genotypes Australia, Poland and China. For this, we used A and B genome specific SSR markers and phenotypic data on 12 yield and yield contributing traits. Out of the 143 primers tested, 134 reported polymorphism and a total of 355 alleles were amplified. Dendrograms based on Jaccards similarity coefficients and Manhattan dissimilarity coefficients were generated based on an average linkage algorithm UPGMA using marker data and phenotypic data. Genotypes were grouped into four clusters based on genetic distances. Both the clustering patterns based on Jaccards similarity and Manhattan dissimilarity coefficients, independently, discriminated the genotypes effectively as per their pedigree and origin. PCoA revealed that, the grouping of genotypes based on SSR marker data is more convincing than phenotypic data, however, the correlation between phenotypic and genetic distance matrices was observed to be very low r=0.11. Hence, for diversity studies reliability on molecular markers is worth proving and SSR markers are the stronger tools than quantitative traits in discriminating B. juncea genotypes.

Las especies de mostaza del género Brassica representan uno de los cultivos de semillas oleaginosas más importantes en India, sin embargo, su diversidad genética es poco conocida. Para la utilización de genotipos en programas de cultivos resulta esencial un mayor conocimiento sobre este tema. Debido a ello, se evaluó la diversidad genética entre 44 genotipos de mostaza de la India Brassica juncea incluyendo variedades y líneas puras de diferentes zonas agro-climáticas de la India y algunos genotipos exóticos Australia, Polonia y China. Para ello, se utilizaron marcadores SSR específicos del genoma A y B y datos fenotípicos del rendimiento de 12 cosechas y sus características. De los 143 primers evaluados, 134 reportaron polimorfismo y un total de 355 alelos fueron amplificados. Se generaron dendrogramas a partir de los coeficientes de similitud de Jaccard y de disimilitud Manhattan, basados en un algoritmo de vinculación promedio UPGMA. Se utilizaron datos de marcadores genéticos y datos fenotípicos. Los genotipos se agruparon en cuatro grupos basados en las distancias genéticas. Ambos patrones de agrupamiento, tanto los basados en los coeficientes de similitud de Jaccard como los basados en los de disimilitud Manhattan, separaron independientemente los genotipos por su genealogía y origen, de una manera efectiva. El PCoA reveló que la agrupación de genotipos basada en datos de marcadores SSR, es más convincente que los datos fenotípicos, sin embargo, se observó que la correlación entre las matrices de distancia fenotípica y genética resultó muy baja r=0.11. Por lo tanto, para estudios de diversidad basados en marcadores moleculares es necesario realizar más pruebas. Los marcadores SSR constituyen herramientas más eficientes para discriminar entre genotipos de B. juncea, que las características cuantitativas.

Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgroTM) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34ºC. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel.

Gene ontology based characterization of Expressed Sequence Tags (ESTs) of Brassica rapa cv. Osome.

Chinese cabbage (Brassica rapa) is widely recognized for its economic importance and contribution to human nutrition but abiotic and biotic stresses are main obstacle for its quality, nutritional status and production. In this study, 3,429 Express Sequence Tag (EST) sequences were generated from B. rapa cv. Osome cDNA library and the unique transcripts were classified functionally using a gene ontology (GO) hierarchy, Kyoto encyclopedia of genes and genomes (KEGG). KEGG orthology and the structural domain data were obtained from the biological database for stress related genes (SRG). EST datasets provided a wide outlook of functional characterization of B. rapa cv. Osome. In silico analysis revealed % 83 of ESTs to be well annotated towards reeds one dimensional concept. Clustering of ESTs returned 333 contigs and 2,446 singlets, giving a total of 3,284 putative unigene sequences. This dataset contained 1,017 EST sequences functionally annotated to stress responses and from which expression of randomly selected SRGs were analyzed against cold, salt, drought, ABA, water and PEG stresses. Most of the SRGs showed differentially expression against these stresses. Thus, the EST dataset is very important for discovering the potential genes related to stress resistance in chinese cabbage, and can be of useful resources for genetic engineering of Brassica sp.

Genetic diversity of Brassica oleracea var. capitata gene bank accessions assessed by AFLP

The genetic diversity of 20 cabbage (Brassica oleracea var. capitata, including sub.var. alba and rubra) cultivars and landraces from the Gene bank of Crop Research Institute was estimated using amplified fragment length polymorphism (AFLP) marker technology. Two cultivars of Brassica pekinensis (syn. Brassica rapa var. pekinensis) were used as outliers in the study. Thirty AFLP primer combinations produced a total of 1084 fragments. A total of 806 fragments, 364 (45 percent) of them polymorphic, were found across 20 Brassica oleracea var. capitata accessions. The accessions were clustered into two main groups. Special subgroups, reflecting place of origin, were observed within these groups. Ten selective primer pairs were found to be most informative because each of these uniquely identified all of the accessions used. Furthermore, two accessions of Brassica pekinensis were clearly differentiated from the Brassica oleracea var. capitata accessions. AFLP is an efficient tool for determination of genetic diversity of cabbage gene bank accessions.

Biologia de Plutella xylostella L. (Lepidoptera: Plutellidae) em cultivaresde couve-flor / Biology of Plutella xylostella L. (Lepidoptera: Plutellidae) reared on cauliflower genotypes

This work was conducted to evaluate biological parameters of Plutella xylostella L. reared on leaves of several cauliflower genotypes under laboratory conditions. The experiment was set in a randomized block design and arranged in a 6 x 2 factorial (genotypes x generations). Leaf disks of the cultivars Barcelona, Verona, Piracicaba Precoce, Sharon, Silver Streak, and Teresópolis Gigante were placed in Petri dishes with 12 newly-hatched larvae. Leaf disks were initially changed after the fourth day, but daily afterwards until the larvae reached the pupal stage. The same procedure was adopted for the second generation. Twenty adults of each sex were separated from each genotype to evaluate their longevity, and 10 couples from each treatment were used to assess female fecundity. The lowest larval survival was obtained on the 'Silver Streak' (78.9 percent) and highest on 'Verona' (97.1 percent). The 'Silver Streak' and 'Teresópolis Gigante' showed the lowest pupal weights (4.83 mg and 5.11 mg, respectively), as well as the lowest fecundity, 119.4 and 123.0 eggs/female, respectively, while 'Piracicaba Precoce' the highest (167.7 eggs/female). Males obtained from larvae reared on 'Teresópolis Gigante' and 'Silver Streak' lived shorter (5.1 days), while the short-lived females were obtained from larvae reared on 'Barcelona' and 'Verona' (4.9 and 5.0 days). Insect development was prolonged in the second generation in all tested genotypes.

A duplicated coxI gene is associated with cytoplasmic male sterility in an alloplasmic Brassica juncea line derived from somatic hybridization with Diplotaxis catholica.

A cytoplasmic male sterile (CMS) line of Brassica juncea was derived by repeated backcrossing of the somatic hybrid (Diplotaxis catholica + B. juncea) to B. juncea. The new CMS line is comparable to euplasmic lines for almost all characters, except for flowers which bear slender, needle-like anthers with aborted pollen. Detailed Southern analysis revealed two copies of coxI gene in the CMS line. One copy, coxI-1 is similar to the coxI gene of B. juncea, whereas the second copy, coxI-2 is present in a novel rearranged region. Northern analysis with eight mitochondrial gene probes showed altered transcript pattern only for the coxI gene. Two transcripts of 2.0 and 2.4 kb, respectively, were detected in the CMS line. The novel 2.4 kb transcript was present in floral bud tissue but absent in the leaf tissue. In plants where male sterility broke down under high temperature during the later part of the growing season, the 2.4 kb coxI transcript was absent, which suggested its association with the CMS. The two coxI genes from the CMS line showed two amino acid changes in the coding region. The novel coxI gene showed unique repeats in the 5' region suggesting recombination of mitochondrial genomes of the two species. The possible role of the duplicated coxI gene in causing male sterility is discussed.

Transgenic tobacco plants expressing BoRS1 gene from Brassica oleracea var. acephala show enhanced tolerance to water stress.

Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, the Brassica oleracea var. acephala BoRS1 gene was transferred into tobacco through Agrobacterium-mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows that BoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.

Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower.

A number of factors that are known to influence genetic transformation were evaluated to optimize Agrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized into Agrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the synthetic cryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae

Interspecific hybridization in Brassica juncea and Brassica tournefortii through embryo rescue and their evaluation for biotic and abiotic stress tolerance.

Interspecific hybrids were obtained in an otherwise incompatible cross Brassica juncea x Brassica tournefortii through in vitro culture of hybrid embryos. The best response was observed from culture of embryos excised 20 days after pollination on MS medium supplemented with kinetin, alpha-naphthylacetic acid, gibberellic acid, glutamine and casein hydrolysate. One hybrid plant had many distinct or intermediate characters. It was tolerant to aphid attack, exhibited irregularities in meiotic events and was partially fertile. The F2 open pollinated and BC1 progenies showed a large diversity in their morphological traits and some promising plants with less aphid infection, drought tolerance and high yield were selected.

Cloning and sequencing of an apparently recombinant promoter for napin gene from Brassica campestris genomic library and its evolutionary significance.

From a genomic library of Brassica campestris (brown sarson cv. B54), we have cloned and sequenced about 2 kb of upstream regulatory region from one of the 2S albumin-coding gene family. The sequence has several seed-specific promoter motifs. A sequence alignment of the 5' flanking regions of the available Brassica 2S storage protein genes showed that our sequence is a double crossover recombinant product of the two members of the napin gene family. A possible explanation of this fact is that Brassica species evolved through gene duplication and recombination from a common ancestor with fewer number of chromosomes and genes.

cDNA clones for salt-inducible genes from mustard (Brassica juncea).

Treatment of a salt tolerant variety of mustard (Brassica juncea cv RH30), with 0.2M NaCl for 96 hr was found to induce synthesis of three new polypeptides and accumulation of proline. A cDNA library from mRNAs derived from salt stressed plants was constructed in the expression vector lambda gt11. Screening by differential hybridization and by salt stress specific antibodies gave two clones msc1 and msc2 (mustard salt clone) respectively. msc1 hybridized to transcripts from salt stressed mustard seedlings only after 48 hr of 0.2M NaCl stress as also to transcripts from drought stress induced by 10% PEG for 24 hr. msc 1 cross hybridized with msc2 clone. The results show the common mechanism of stress response as elicited in other plants.