Silicon-mediated genotoxic alterations in Brassica juncea under arsenic stress: A comparative study of biochemical and molecular markers

Arsenic (As), one of the most harmful toxicant at the global level, severely affects plant metabolism when taken up. Interestingly, the presence of silicon (Si) as a fertilizer in As-contaminated soil is an effective strategy to decrease As accumulation in plants. Brassica juncea (var. Varuna) were grown hydroponically to investigate the role of Si at biochemical and molecular levels under arsenite (As³⁺) stress. Seedlings of B. juncea were exposed to As³⁺, Si, and a combination of both elements. Our data demonstrated that seedlings exposed to As³⁺ showed an inhibition in shoot length, chlorophyll, carotenoid, and protein, while co-application of Si improved these growth parameters. Silicon supplementation reduced As accumulation in shoot. Increase/decrease was observed in stress-related parameters (cysteine and proline), antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, and catalase), and oxidative stress markers (malondialdehyde and H₂O₂), which were improved upon co-application of Si as compared to As³⁺ alone treatment. Random amplified polymorphic DNA (RAPD) is a suitable biomarker assay for plants for assessing the genotoxicity. Seven RAPD primers produced a total of 39 and 48 bands in the leaves of the untreated and treated seedlings, respectively. The RAPD band-profiles and genomic template stability were consistent with other growth and physiological parameters. In conclusion, the genotoxic alterations along with the biochemical parameters indicate that the exposure to Si mitigates As³⁺-induced oxidative stress by improving the stress-related parameters and antioxidant system in B. juncea.     

与黄瓜抗枯萎病基因连锁的RAPD标记

以黄瓜抗枯萎病亲本WIS2757和感枯萎病亲本津研2号及其F2分离群体为试材,采用分离群体分组分析法(BSA)进行了与黄瓜抗枯萎病基因连锁的分子标记研究。运用RAPD技术,利用780条RAPD引物对抗、感亲本进行筛选,其中有113条引物在两亲本之间表现多态性,但仅有引物S49在两组间多态性标记与亲本的多态性标记相同。经F2单株分析,引物S49扩增出的特异DNA片段与WIS2757抗黄瓜枯萎病基因连锁,遗传距离为14 cM。DNA标记条带大约为300 bp,定名为S49-300。     

Genetic organization of aromatic rice as revealed by RAPD markers: A case study in conserving crop genetic resources on farm

Summary On-farm conservation of landraces is one strategy to maintain the diversity of crop germplasm in local agro-ecosystems. The genetic structures of landraces are a key biological factor in on-farm conservation strategies. To accumulate a genetic understanding that will help establish a methodology for on-farm conservation, the genetic organization of landraces of aromatic rice in Namdinh province, Vietnam was analyzed using RAPD markers. Eighteen RAPD markers detected 38 genotypes among 320 aromatic rice samples growing at 23 sites of farmers' fields and in the experimental field that derived from 13 sites. Geographical variation was observed in the frequency of genotypes, whereas individual landraces could not be distinguished by RAPD markers. Genetic variation within a site was generally smaller than that among sites. The degree of genetic similarity of the plants in a site varied among sites, as did the number of genotypes. Changes in genetic structure over time were investigated using experimental populations each derived from approximately 30 plants from 13 farmers' fields. The differences detected by DNA markers between the genetic structural in the farmers' fields and those in experimental fields suggested that genetic drift is a major cause of these differences. The present study suggests that DNA markers are an essential means to monitor the genetic structures of heterogeneous landraces of rice, and are useful for selecting study sites for the on-farm conservation of genetic diversity as well as for successive monitoring.     

RAPD markers for discriminating tea germplasms at the inter-specific level in China

For the discrimination of tea germplasms at the inter‐specific level, four tea species and varieties (Camellia sinensis, C. sinensis var. assamica, C. sinensis var. pubilimba, C. sinensis var. kucha) and their 20 wild allied species (C. sp.) preserved in the China National Germplasm Tea Repositories (CNGTR) were investigated using randomly amplified polymorphic DNA (RAPD) markers. Fifteen primers were chosen from the 61 screened for RAPD amplification. The average DNA polymorphic frequency of RAPD primers at the inter‐specific level was 0.30, varying from 0.16 to 0.60, lower than that at the intra‐specific level. Using the presence, sometimes absence of unique RAPD markers, it was possible to discriminate 14 of the germplasms investigated. No single primer could discriminate all the 24 germplasms. However, OPO‐13 provided rich band patterns and it could discriminate 10 genotypes. The combination of two and three primers made it possible to discriminate 15 and 21 germplasms, respectively. Furthermore, the combination of band patterns or the DNA fingerprinting based on specific RAPD markers generated by OPO‐13, OPO‐18, OPG‐12 and OPA‐13 allowed the discrimination of all 24 germplasms investigated. Therefore, RAPD markers also provide a powerful tool to differentiate tea germplasms at the inter‐specific level.     

Use of molecular markers to accelerate the breeding of common bean lines resistant to rust and anthracnose

The main goal of this work was to introduce resistance genes for rust, caused by Uromyces appendiculatus, and anthracnose, caused by Colletotrichum lindemuthianum, in an adapted common bean cultivar through marker-assisted backcrossing. DNA fingerprinting was used to select plants genetically closer to the recurrent parent which were also resistant to rust and to race 89 of C. lindemuthianum. DNA samples extracted from the resistant parent (cv. Ouro Negro), the recurrent parent (cv. Rudá), and from BC₁, BC₂ and BC₃ resistant plants were amplified by the RAPD technique. The relative genetic distances in relation to the recurrent parent varied between 9 and 59% for BC₁, 7 and 33% for BC₂, and 0 and 7% for BC₃ resistant plants. After only three backcrosses, five lines resistant to rust and anthracnose with, approximately, 0% genetic distance in relation to the recurrent parent were obtained. These lines underwent field yield tests in two consecutive growing seasons and three of them presented a good yield performance, surpassing in that sense their parents and most of the reference cultivars tested.     

Homoeological relationships between the f chromosome of Brassica rapa and the e chromosome of Brassica oleracea

Eight plants of the putative double monosomic addition line (DMAL, 2n= 20) were developed by crossing a monosomic chromosome addition line of radish [f(A)‐type monosomic addition line (MAL) (2n= 19)] carrying the f chromosome of Brassica rapa (2n= 20, AA) with another [e(C)‐type MAL (2n= 19)] having the echromosome of Brassica oleracea (2n= 18, CC). The homoeological relationships between the two alien chromosomes were investigated by morphological, cytogenetic and random amplified polymorphic DNA (RAPD) analysis. Seventeen morphological traits that were not present in the radish cv. ‘Shogoin’ were observed in both MALs and these traits were substantially exhibited in DMAL plants. At the first metaphase of pollen mother cells (PMCs), the two parental MALs showed a chromosome configuration of 9II +1I, demonstrating impossibility of recombination between the R and the added chromosomes. The DMALs formed 10II in approximately 73% of PMCs, with one bivalent showing loose pairing between two chromosomes differing in size. In an attempt to identify the two MALs by RAPD‐specific markers using 26 selected random primers, 13 and 20 bands were specific for the f(A)‐type and the e(C)‐type MALs, respectively; 12 bands were common to both MALs (26.7%). In conclusion, the f chromosome of B. rapa is homoeologous to the e chromosome of B. oleracea. The genetic domain (genes) for 17 morphological traits are linked to each homoeologous chromosome bearing 27% of the corresponding RAPD markers.     

栽培罗汉果遗传多样性的RAPD分析

罗汉果(Siraitiagrosvenorii)是单性、雌雄异株的葫芦科球根多年生植物,是特产于我国南部的重要经济植物。其果实是传统中药,果实中还含有一种低热量的天然甜味剂。我们从罗汉果的主产地广西永福县、临桂县和融安县采集了13份雄株和8个品种的62份雌株栽培罗汉果样品,用21个随机引物对这75份样品进行了RAPD分析。共得到130个位点,其中92个(70.77%)是多态性的。用POPGENE软件统计了主要品种及雄株的多态位点百分率和Shannon信息多样性指数;根据样品间的Dice相似性系数,分别对雌株和雄株做了主成分分析(PCA);用UPGMA法进行了单株聚类分析。结果表明主要栽培品种青皮果、红毛果和爆棚果的遗传多样性很低,它们的品种内样品间相似性系数平均值分别为0.961、0.945和0.966,Shannon信息多样性指数分别为0.0997、0.1014和0.0571,这种遗传多样性很低的现状是由于栽培罗汉果主要采用营养繁殖方法进行繁殖的结果。而茶山果和冬瓜果具有相对较高的遗传多样性,样品间相似性系数平均值分别为0.868和0.857,Shannon信息多样性指数分别为0.1876和0.1855。雄株遗传多样性较高,样品间相似性系数平均值为0.873,Shannon信息多样性指数为0.2135。有必要扩大栽培罗汉果的遗传基础。     

Species relationships in the subgenus Ceratotropis (genus Vigna) as revealed by RAPD analysis

Summary The genetic variation among 23 accessions of 5 species in the subgenus Ceratotropis, genus Vigna, were investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 404 fragments amplified with 24 primers were scored and analyzed by cluster analysis. The accessions used were separated into two main groups with an average of 70% differences. Within the main groups, five subgroups were recognized, which are in complete agreement with taxonomic species. Wild forms were always grouped with their most closely related cultivated forms and they showed variation in each species. The largest intraspecific variation was found in V. radiata (mungbean), in which wild forms (V. radiata var. sublobata) were highly different from each other and from cultivated forms. V. angularis (adzuki bean) showed the least variation and thus, was probably differentiated in relatively recent times.     

Genetic variability among elite red clover ( Trifolium pratense L.) parents used in Chile as revealed by RAPD markers

Red clover (Trifolium pratense L.) is one of the main forage species of temperate regions. Cultivars of red clover are heterogeneous which makes their genetic analysis difficult. We applied RAPDs (Random Amplifed Polymorphic DNA) in order to assess the genetic relationship and levels of genetic variability existing among a group of 16 elite red clover parents organised in four subsets of 4 parents each. Out of 55 primers 21 provided reproducible results. A total of 135 reliable and polymorphic RAPD bands were detected which were used to estimate genetic distances among pair-wise combinations of elite parents. Nei and Li's similarity values ranged from 0.60 to 0.77, with a mean of 0.66, which reflects a rather high genetic variability among the genotypes evaluated. Lower levels of genetic variability, as detected by polymorphic loci and mean heterogeneity values, were detected in a subset of parents selected for resistance to the stem nematode. Cluster analyses resolved the different sets of parents in a manner consistent with what is known from their breeding origins. An Analysis of Molecular Variance detected substantial levels of variation within subsets of parents. RAPDs represent a valuable source of genetic information for red clover breeding programmes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Estimating the genetic relationship of hybrid bromegrass to smooth bromegrass and meadow bromegrass using RAPD markers

The two most widely grown bromegrass species in North America are smooth bromegrass, a hay type grass, and meadow bromegrass, a pasture type grass. Hybrid populations between these two species have been developed through hybridization and recurrent selection. The objective of this study was to determine the genetic relatedness of the hybrid bromegrass population S‐9073M to its parental populations using random amplified polymorphic DNA (RAPD) markers and to determine genetic variation within and between populations. Individuals from each of the three populations were genotyped at 43 polymorphic RAPD loci. One of the RAPD fragments was meadow bromegrass‐specific. Cluster analysis showed three groups representing the two parental populations and the hybrid population. An analysis of molecular variance (AMOVA), showed that the hybrid population had the highest within‐population variation, followed by meadow bromegrass and smooth bromegrass. The interpopulation genetic distance (phi‐statistic =Φ_st) was highest between meadow and smooth bromegrass and lowest between smooth and hybrid bromegrass. The hybrid population was genetically intermediate to smooth bromegrass and meadow bromegrass, but closer to smooth bromegrass, possibly reflecting the selection criteria used in the development of this hybrid.