PCR analysis of oilseed rape cultivars (Brassica napus L. ssp. oleifera) using 5′ -anchored simple sequence repeat (SSR) primers

Primers complementary to simple sequence repeats (SSRs) and with variable three-base anchors at their 5 end, were used in PCR analyses to compare pooled DNA samples from various Brassica napus and B. rapa cultivars. Amplification products were resolved on polyacrylamide gels and detected by silver-nitrate staining. The resulting banding patterns were highly repeatable between replicate PCRs. Two of the primers produced polymorphisms at 33 and 23 band positions, respectively, and could each discriminate 16 of the 20 cultivars studied. Combined use of both primers allowed all 20 cultivars to be distinguished. The UPGMA dendrogram, based on the cultivar banding profiles, demonstrated clustering on the basis of winter/spring growth habit, high/low glucosinolate content, and cultivar origin (i.e. the breeder involved). Intracultivar polymorphism was investigated using a minimum of ten individuals for each cultivar and was found to vary considerably between cultivars. It is concluded that anchored SSR-PCR analysis is a highly informative and reproducible method for fingerprinting oilseed rape populations, but that intra-cultivar variation should be investigated before using banding profiles from pooled samples for the identification of individuals.     

Functional characterization of β-ketoacyl-CoA synthase genes from Brassica napus L.

Seed-specifically expressed -ketoacyl-CoA synthase genes of Brassica napus (Bn-FAE1.1 genes) were cloned from two cultivars, namely Askari, a high-erucic-acid type, and Drakkar, a low-erucic-acid type. The genes from the two cultivars were found to be nearly identical. They encode proteins of 507 amino acids, the sequences of which differ only at position 282. The Bn-FAE1.1 gene of Askari, unlike that of Drakkar, was functionally expressed in yeast cells suggesting that the single amino acid exchange effects the low erucic acid phenotype at the E1 gene locus. In yeast cells the -ketoacyl-CoA synthase of Askari elongated not only oleoyl but also palmitoleoyl groups as well as saturated acyl groups in such a way that monounsaturated acyl groups of 22 carbons and saturated ones of 26 carbons were formed as main products. A reporter gene fused to the promoter region of the Bn-FAE1.1 gene from Askari showed seed-specific expression in transgenic rapeseed plants. Over-expression of the coding region of the Askari gene in developing seeds of transgenic Drakkar plants resulted in a significant increase in the levels of eicosenoic acid and erucic acid esterified in the seed oil. On the other hand, in transgenic high-erucic-acid rapeseed plants the increase in erucic acid level was at most 60% although the chimeric Bn-FAE1.1 gene was co-expressed with an erucoyl-CoA-specific lysophosphatidate acyltransferase gene enabling trierucoyl glycerol to accumulate in the seed oil.     

Genetic mapping of nuclear fertility restorer genes for the ‘Polima’ cytoplasmic male sterility in canola (Brassica napus L.) using DNA markers

 Co-segregation of male fertility with DNA markers selected by targeted mapping approaches as being potentially linked to the Rfp1 restorer gene for the pol cytoplasmic male sterility (CMS) was analyzed using two canola (Brassica napus L.) backcross populations. Eleven DNA markers (10 RFLP markers and one RAPD marker) directly linked to the Rfp1 locus were identified. The linkage group containing the Rfp1 locus was found to correspond to B. napus linkage group 18 of Landry et al. (1991). A similar pattern of co-segregation between DNA markers and male fertility was observed in a backcross population segregating for the pol restorer gene Rfp2 from line ‘UM2383’; one RFLP marker, cRF1b, showed perfect linkage with both Rfp1 and Rfp2 and detected identical polymorphic fragments in both the Rfp1 and Rfp2 restorer lines. Our findings indicate that restoration of pol CMS is controlled by a single nuclear genetic locus on linkage group 18 and that Rfp1 and Rfp2 are likely allelic. Received: 2 October 1996 / Accepted: 20 December 1996     

Genetic analyses of the host-pathogen system Turnip yellows virus (TuYV)—rapeseed (Brassica napus L.) and development of molecular markers for TuYV-resistance

The aphid transmitted Turnip yellows virus (TuYV) has become a serious pathogen in many rapeseed (Brassica napus L.) growing areas. Three-years’ field trials were carried out to get detailed information on the genetics of TuYV resistance derived from the resynthesised B. napus line ‘R54’ and to develop closely linked markers. F₁ plants and segregating doubled-haploid (DH) populations derived from crosses to susceptible cultivars were analysed using artificial inoculation with virus-bearing aphids, followed by DAS-ELISA. Assuming a threshold of E ₄₀₅ = 0.1 in ELISA carried out in December, the results led to the conclusion that pre-winter inhibition of TuYV is inherited in a monogenic dominant manner. However, the virus titre in most resistant lines increased during the growing period, indicating that the resistance is incomplete and that the level of the virus titre is influenced by environmental factors. Bulked-segregant marker analysis for this resistance locus identified two closely linked SSR markers along with six closely linked and three co-segregating AFLP markers. Two AFLP markers were converted into co-dominant STS markers, facilitating efficient marker-based selection for TuYV resistance. Effective markers are particularly valuable with respect to breeding for TuYV resistance, because artificial inoculation procedures using virus-bearing aphids are extremely difficult to integrate into practical rapeseed breeding programs.     

Assigning Brassica microsatellite markers to the nine C-genome chromosomes using Brassica rapa var. trilocularis–B. oleracea var. alboglabra monosomic alien addition lines

Brassica rapa var. trilocularis-B. oleracea var. alboglabra monosomic alien addition lines (MAALs) were used to assign simple sequence repeat (SSR) markers to the nine C-genome chromosomes. A total of 64 SSR markers specific to single C-chromosomes were identified. The number of specific markers for each chromosome varied from two (C3) to ten (C4, C7 and C9), where the designation of the chromosomes was according to Cheng et al. (Genome 38:313-319, 1995). Seventeen additional SSRs, which were duplicated on 2-5 C-chromosomes, were also identified. Using the SSR markers assigned to the previously developed eight MAALs and recently obtained aneuploid plants, a new Brassica rapa-B. oleracea var. alboglabra MAAL carrying the alien chromosome C7 was identified and developed. The application of reported genetically mapped SSR markers on the nine MAALs contributed to the determination of the correspondence between numerical C-genome cytological (Cheng et al. in Genome 38:313-319, 1995) and linkage group designations. This correspondence facilitates the integration of C-genome genetic information that has been generated based on the two designation systems and accordingly increases our knowledge about each chromosome. The present study is a significant contribution to genetic linkage analysis of SSR markers and important agronomic traits in B. oleracea and to the potential use of the MAALs in plant breeding.     

Analysis of B-genome chromosome introgression in interspecific hybrids of Brassica napus × B. carinata

Brassica carinata, an allotetraploid with B and C genomes, has a number of traits that would be valuable to introgress into B. napus. Interspecific hybrids were created between B. carinata (BBCC) and B. napus (AACC), using an advanced backcross approach to identify and introgress traits of agronomic interest from the B. carinata genome and to study the genetic changes that occur during the introgression process. We mapped the B and C genomes of B. carinata with SSR markers and observed their introgression into B. napus through a number of backcross generations, focusing on a BC(3) and BC(3)S(1) sibling family. There was close colinearity between the C genomes of B. carinata and B. napus and we provide evidence that B. carinata C chromosomes pair and recombine normally with those of B. napus, suggesting that similar to other Brassica allotetraploids no major chromosomal rearrangements have taken place since the formation of B. carinata. There was no evidence of introgression of the B chromosomes into the A or C chromosomes of B. napus; instead they were inherited as whole linkage groups with the occasional loss of terminal segments and several of the B-genome chromosomes were retained across generations. Several BC(3)S(1) families were analyzed using SSR markers, genomic in situ hybridization (GISH) assays, and chromosome counts to study the inheritance of the B-genome chromosome(s) and their association with morphological traits. Our work provides an analysis of the behavior of chromosomes in an interspecific cross and reinforces the challenges of introgressing novel traits into crop plants.     

Accumulation and translocation of heavy metals in the canola (Brassica napus L.)—soil system in Yangtze River Delta, China

Aims and methods

Concentrations of heavy metals such as Cd, As, Hg, Pb, Cr, Cu, Zn and Ni in different tissues (seeds, roots and shoots) of the mature canola (Brassica napus L.) plants and in the associated rhizosphere soils from Yangtze River Delta (YRD) region of China, were determined to evaluate the heavy metals’ pollution in the soils and the canola seeds, and to discuss their accumulation and translocation characteristics in canola plants. At the same time, the phytoextraction potential of the canola plant for the above heavy metals was theoretically calculated and discussed on the basis of above measured data.

Results

The results showed that the concentration ranges of Cd, As, Hg, Pb, Cr, Cu, Zn and Ni in the rhizosphere soils were 0.115–0.481, 3.40–20.5, 0.069–0.682, 9.92–27.4, 46.8–86.6, 17.7–253.3, 65.2–511.7 and 16.0–37.8?mg?kg^?1, respectively. The concentrations of Cu, Zn and Hg at some sampling sites exceeded the 2nd grade threshods of Chinese national environmental quality standard for soils. The potential ecological risk of heavy metals in the canola rhizosphere soils decreased in the order of Zhejiang > Shanghai > Jiangsu provinces. The concentration ranges of above heavy metals in the canola seeds were 0.032–0.067, 0.002–0.005, 0.001–0.005, 0.053–0.165, 0.191–0.855, 3.01–13.20, 34.82–96.95 and 0.343–2.86?mg?kg^?1, respectively, with Cu and Zn at some sampling sites exceeding the permissible concentrations in foods of China. Heavy metals’ concentrations in canola seeds didn’t increase with their increasing concentrations in the rhizosphere soils. The bioconcentration factors (BCFs) of most heavy metals in the canola seeds decreased with their increasing concentrations in the associated rhizosphere soils. The average BCFs of heavy metals decreased in the order of Zn (0.488)>Cd (0.241)>Cu (0.145)>Ni (0.038)>Hg (0.021)>Pb (0.005)=Cr (0.005)>As (0.000) in the canola seeds, Cd (1.550)>Cu (0.595)>Zn (0.422)>Hg (0.138)>Ni (0.085)>Pb (0.080)>As (0.035)>Cr (0.031) in the roots, and Cd (0.846)>Zn (0.242)>Cu (0.205)>Hg (0.159)>Ni (0.031)>Pb (0.025)>As (0.012)>Cr (0.007) in the shoots, respectively. The accumulation capacity for most of the above heavy metals in the mature canola tissues was root > shoot > seed, with the exceptions of seed > root > shoot for Zn and shoot > root > seed for Hg. Except Hg from root to shoot and Zn from root to seed, translocation factors (TFs) of above heavy metals were lower than 1.0.

Conclusions

The concentrations, BCFs and TFs of above heavy metals in the canola tissues indicated that the investigated canola plants did not meet the criteria of hyperaccumulators for the above heavy metals. The phytoextracton potential of the studied canola plants for the above heavy metals from the polluted soils was very limited. It would take 920, 3,170 and 3,762?years (assuming two crops per year) to reduce the initial soil Zn, Cu and Hg concentrations, respectively, from the most polluted soil concentrations to the 2nd grade thresholds of Chinese national environmental quality standard for soils.     

Application of in vitro pollination of opened ovaries to obtain Brassica oleracea L. × B. rapa L. hybrids

This study presents the results of experiments concerning: (1) interspecific hybridization of Brassica oleracea × Brassica rapa via application of in vitro placental pollination and (2) embryological analysis of the process of resynthesis of Brassica napus. In order to overcome certain stigma/style barriers, B. rapa pollen was placed in vitro on an opened B. oleracea ovary (with style removed). Pollinated ovaries were cultured on Murashige and Skoog (MS) medium. After 24-d culture, the developing embryos were isolated from immature seeds and transferred onto MS medium supplemented with 0.47 μM kinetin, 0.49 μM 1-naphthaleneacetic acid, and 10% (v/v) coconut water. When the embryos had turned green, they were immediately placed onto MS medium with 100 μM kinetin. After development of the seedling, plantlets were transferred to soil. Chromosome doubling was achieved after another week. Cytometric analysis of nuclear DNA confirmed the hybrid nature of the plants. Resynthesis of B. napus can be performed through interspecific hybridization of B. oleracea × B. rapa followed by embryo rescue and genome doubling.     

Use of molecular markers in breeding for soluble solids content in tomato — a re-examination

Summbary Through earlier breeding efforts, portions of the genome of the wild species Lycopersicon chmielewskii have been introgressed into the cultivated tomato (Rick 1974). These introgressed chromosomal segments have been reported to increase soluble solids in fruit of certain tomato varieties (Rick 1974). Recently, two of the introgressed segments have been identified with RFLP markers and tested for effects on soluble solids in a single F2 population (Osborn et al. 1987). Based on results from that experiment, it was determined that one of the detected segments contains gene(s) controlling soluble solids and concluded that tomato varieties could be improved for this character by indirect selection for the linked RFLP marker (Osborn et al. 1987). In this report, we have independently tested the association between RFLP and isozyme markers and genes controlling soluble solids and other characters in the above described material. These experiments differ from the previous ones in that a set of 132 molecular markers (isozymes and DNA clones) of known chromosomal position have been used. Three introgressed chromosomal segments from L. chmielewskii have been identified using these markers. They map to the middle and the end of chromosome 7 (> 40 cM apart) and to the end of chromosome 10. The effects of these segments on soluble solids and other horticultural characters were tested in crosses with three different cultivars over a period of two years. Two of the three segments were found to increase soluble solids, however the effect of one of these was dependent on genetic background. Both segments were found to be associated with deleterious characters including increase in fruit pH, lower yield and small fruit. These results confirm the utility of molecular markers for detecting genes underlying quantitative variation but demonstrate the danger in establishing breeding programs around such linkages until the effects of the quantitative genes have been tested in a variety of genetic backgrounds and for associated effects on other characters of agronomic importance.     

Modelling the recovery of hypertrophic L. Glumsø (Denmark)

Diversion of sewage from L. Glumsø reduced phosphorus loading from 6.0 g P.m^–2.yr^–1 to 1.6 g P.m^–2.yr^–1. Chlorophyll levels during summer were reduced from 6–800 mg Chl.m^–3 to about 200 mg Chl.m^–3 mainly by extended periods with phosphorus limitation. Internal phosphorus loading was significant in the first 2 years after nutrient reduction. Predictions of the recovery were made by both simple, empirical models and with complex, dynamic model versions. The actual responses of L. Glumsø were compared with both previously published predictions and predictions made with improved model versions. Objective functions of 0.18 and global correlation coefficients of 0.89 could be achieved.     

Use of an α-d-glucosidase for the specific cytochemical identification of lateral α-d-xylosyl end groups in plant xyloglucans

Summary α-Linked d-xylosyl side chains represent the typical feature common to all xyloglucans not shared by other cell wall polysaccharides. Since no easily available α-d-xyloxidase is known, advantage was taken of the conformational and configurational homologies between α-d-xylopyranose and α-d-glucopyranose to make an α-d-glucosidase-gold complex which was able to recognize α-d-xylosyl terminal residues of xyloglucans. This marker was used together with α-l-fucosidase gold complex for the double labeling on two different structural features of the same macromolecule in plant primary cell wall.     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

The use of microsatellite markers for the detection of genetic similarity among winter bread wheat lines for chromosome 3A

Previous studies with chromosome substitution and recombinant inbred chromosome lines identified that chromosome 3A of wheat cv. Wichita contains alleles that influence grain yield, yield components and agronomic performance traits relative to alleles on chromosome 3A of Cheyenne, a cultivar believed to be the founder parent of many Nebraska developed cultivars. This study was carried out to examine the genetic similarity among wheat cultivars based on the variation in chromosome 3A. Forty-eight cultivars, two promising lines and four substitution lines (in duplicate) were included in the study. Thirty-six chromosome 3A-specific and 12 group-3 barley simple sequence repeat (SSR) primer pairs were used. A total of 106 polymorphic bands were scored. Transferability of barley microsatellite markers to wheat was 73%. The coefficient of genetic distance (D) among the genotypes ranged from 0.40 to 0.91 and averaged D=0.66. Cluster analysis by the unweighted pair-group method with arithmetic averages showed one large and one small cluster with eight minor clusters in the large cluster. Several known pedigree relationships largely corresponded with the results of SSR clusters and principal coordinate analysis. Cluster analysis was also carried out by using 22 alleles that separate Wichita 3A from Cheyenne 3A, and three clusters were identified (a small cluster related to Cheyenne of mainly western Nebraska wheat cultivars; a larger, intermediate cluster with many modern Nebraska wheat cultivars; a large cluster related to Wichita with many modern high-yielding or Kansas wheat cultivars). Using three SSR markers that identify known agronomically important quantitative trait loci (QTL) regions, we again separated the cultivars into three main clusters that were related to Cheyenne or Wichita, or had a different 3A lineage. These results suggest that SSR markers linked to agronomically important QTLs are a valuable asset for estimating both genetic similarity for chromosome 3A and how the chromosome has been used in cultivar improvement.     

Development of microsatellite markers for the endangered grassland perennial herb Vincetoxicum atratum (Apocynaceae–Asclepiadoideae)

Eight microsatellite markers were developed for the endangered grassland perennial herb Vincetoxicum atratum. The number of alleles ranged from 4 to 14, and the expected heterozygosities were from 0.575 to 0.933 in a population of V. atratum. Five of the eight loci did not significantly deviated from the Hardy–Weinberg equilibrium. All eight loci were tested for cross-species amplification in five other species of Vincetoxicum in Japan. These microsatellite loci will be useful for conservation genetics of V. atratum and other species of Vincetoxicum.