Euphytica 123: 401–409, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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Evaluation of cytoplasmic effects on agronomic and seed quality traits in two doubled haploid populations of Brassica napus L. I. Rajcan1,∗ , K.J. Kasha1 , L.S. Kott1 & W.D. Beversdorf2 1 Department
of Plant Agriculture, Crop Science & Plant Biotechnology Divisions, Crop Science Bldg, University of Guelph, Guelph, Ontario, N1G 2W1, Canada; 2 Novartis Seeds AG, CH-4002 Basel, Switzerland; (∗ author for correspondence; e-mail:
[email protected])
Received 26 October 2000; accepted 2 May 2001
Key words: Brassica napus L., cytoplasmic effect, linolenic acid, inheritance, mitochondrial DNA, RAPD
Summary Cytoplasmic effects have been occasionally implicated in the inheritance of several traits in oilseed rape (Brassica napus L.), including linolenic acid concentration (18:3) in the oil. It is important that these be considered when choosing the direction of cross for producing new breeding populations. To study this phenomenon, a reciprocal cross was made between two genotypes of oilseed rape, Reston and LL09, which differed for their erucic and linolenic acid concentrations in the seed oil. Two DH populations, which were produced by microspore culture from reciprocal F1 plants, were evaluated in the growth room for one generation and in the field at two locations in Southern Ontario in 1993 and 1994. Field notes were taken on days to flower, days to maturity, plant lodging, plant height and, seed quality traits. In the growth room study, the phenotypic distribution of 18:3 differed significantly between the two reciprocal DH populations. In the field, significant reciprocal differences between the population means were detected for 18:3, flowering date and protein content in both years and for days to maturity and oil content in 1993 only. To further study the parental lines, chloroplast (cp) and mitochondrial (mt) DNA from parental lines were isolated and subjected to RFLP and RAPD analysis. Several random primers revealed reproducible DNA polymorphism (RAPD) between the parental mt DNA. It is concluded that the direction of cross should be taken into consideration by oilseed rape breeders relying solely on doubled haploids for developing genotypes with modified seed quality traits in Brassica napus L.
Introduction Cytoplasmic effects causing unknown reciprocal differences in hybridization of crop species could potentially hamper efforts to fully exploit the crosses’ breeding potential. These effects have been reported for a number of species and traits, such as the thousand kernel weight (Ekiz et al., 1998) and anther culture response (Ekiz & Konzak, 1991) in bread wheat, free folic acid in sugar beet (Wang & Goldman, 1997), shriveled seed in peanut (Jakkula et al., 1997), protein, oil and fatty acid content in soybean (Miller et al., 1996) and grain filling traits in maize (Seka & Cross, 1995). In Brassica, only a few reports exist for cytoplasmic effects. Ramsey et al. (1994), who studied the inheritance of quantitative traits in Brassica napus ssp.
rapifera (swedes), found large and unexpected reciprocal differences, particularly for fresh and dry weight yield in all triple test cross generations used and in ten F3 of the first cross. Reciprocal differences were also found for yield traits studied in progeny from a diallel set of crosses involving 12 lines of Brassica juncea (Rawat, 1992). He found significant reciprocal differences for days to flowering, plant height, main shoot length and some other characteristics. Weber et al., (1995) found that the frequency of embryogenesis of Brassica napus isolated microspores depended upon the genotype. Reciprocal effects were noted in the crosses Profit × 91a, Profit × 232a and Tribute × 223a. However, there were no apparent differences in the embryogenic ability of the reciprocal F1 plants (Weber et al., 1995). In the study by Davik & Heneen
402 (1996), a high erucic acid gene was found to function more efficiently in the Yellow Sarson cytoplasm than in two other Scandinavian Brassica rapa lines. Also, more zero erucic F2 -individuals were obtained in crosses where modern low erucic acid lines were used as female parents (Davik & Heneen, 1996). One of the objectives of oilseed rape breeding has been to reduce the level of linolenic acid (18:3), which is easily oxidized to cause off flavour and odour of the oil. Inheritance of linolenic acid in oilseed rape, however, has been a matter of controversy as the results obtained by different groups of researchers varied and often depended upon the materials used. Several groups have reported independently that the cytoplasm played a role in the control of linolenic acid concentration in oilseed rape oil in one of two ways: maternal control or cytoplasmic inheritance (Bartkowiak-Broda & Krzymanski, 1983; Pleines & Friedt, 1989; Diepenbrock & Wilson, 1987). Bartkowiak-Broda & Krzymanski (1983) suggested that linolenic acid concentration in oilseed rape was controlled entirely by maternal plant genotype rather than embryo genotype. Thomas & Kondra (1973) noted that linolenic acid was affected by both the genotype of the maternal sporophyte and the embryo genotype. Diepenbrock & Wilson (1987) found that the concentration of 18:3 acid in triacylglycerol was determined by nuclear and cytoplasmic gene interaction, whereas 18:3 in monogalactosyl diacylglycerol (MGDG) was determined by cytoplasmic factors. Recently, Pleines & Friedt (1989) found significant differences in reciprocal BC1 and BC2 , as well as F1 , indicating maternal control, which was realised by the interaction between maternal genotype and nuclear genes of the embryo. Additionally, they reported that temperature exerted considerable influence on the degree of maternal control. Two cytoplasmic organelles in plants have been known to contain DNA apart from the nuclear genome, chloroplasts (cp) and mitochondria (mt). The differences that exist in these two genomes have been reported to be responsible for some cytoplasmically inherited traits in Brassicas, such as herbicide (triazine) resistance or cytoplasmic male sterility (Vedel et al., 1982). Chloroplast and mt DNA in Brassica have been investigated previously by comparing the RFLP profiles within and among the Brassica species, which was used mostly in phylogenetic studies. The variation in the restriction profiles of the mtDNA makes it possible to differentiate closely related species, subspecies and species with different cytoplasm (reviewed by Lonsdale & Grienenberger, 1992). However, the
variability in the cytoplasmic genomes have a potential to be also used to account for reciprocal differences in crosses involving traits of agronomic interest to the breeder. The existence of conflicting results concerning the inheritance of linolenic acid concentration in oilseed rape oil has prompted us to conduct a study looking at the potential for obtaining cytoplasmic effects on linolenic acid concentration, and possibly other agronomic and seed quality traits, using doubled haploid populations. To achieve this objective, two reciprocal DH populations were produced from a cross involving the oilseed rape cultivar Reston and a low linolenic DH line, LL09. These populations were compared for the means and distribution of linolenic acid levels and for a number of agronomic and other seed quality traits across multiple environments. The segregation of molecular markers associated with 18:3 content and analyses of RFLP and RAPD patterns of the parental cpDNA and mtDNA were used also in an attempt to explain the source of the cytoplasmic effects observed. The ultimate goal of this study, however, was to determine if the direction of the cross should be considered by breeders involved in the development of oilseed rape varieties with modified fatty acid composition.
Material and methods Two oilseed rape lines, a high linolenic-low glucosinolate oilseed rape variety Reston and a low linolenic canola line LL09 were crossed reciprocally to produce F1 plants. These lines were chosen because of their potential to be used for mapping concurrently erucic and linolenic acid genes, which was done in a related study (Rajcan et al., 1999). The F1 gametes were sampled to develop two reciprocal doubled haploid populations using the isolated microspore culture method (Polsoni et al., 1988). Eighty four double haploid (DH) lines were generated from the cross Reston × LL09 (R×L) and 78 DHs from the cross LL09 × Reston (L×R). These DHs were grown in the growth room in 1992, and in the field in two locations in Southern Ontario; Elora in 1993 and, Dundalk & Elora in 1994. Field notes were taken on days to flower, days to maturity, plant height and lodging as described before (Rajcan et al., 1997). Open pollinated seed samples from each field plot were used to measure the oil content using Nuclear Magnetic Resonance (NMR) and the protein content using Near Infra-red Reflectance (NiR). The
403 self-pollinated seeds from single plants obtained from each plot or in the growth room were used for fatty acid analysis by gas chromatography of fatty acid esters as described by Bannon et al. (1982). Another experiment was conducted to compare the inheritance pattern of linolenic acid in reciprocal DH populations with that of reciprocal F2 populations using the same parents. For this purpose, the parental lines Reston and LL09 were crossed again in 1994 to produce two reciprocal F1 plants that were grown to obtain segregating F2 populations. The F2 plants were analysed for fatty acids and the inheritance pattern of 18:3 compared between the reciprocal populations by a chi-square test. The mitochondrial and chloroplast DNA were extracted from 10–12 g of leaf tissue following the method of Kemble (1987). Restriction digests were made using the EcoRI and the cpDNA and mtDNA patterns were resolved by electrophoresis for 16 hours using 0.8% agarose gels. The total genomic DNA of Reston and LL09 for molecular marker analysis was isolated from two leaf discs taken from young plants using the method of Edwards et al., (1991), which was modified by adding 2 additional ethanol precipitations at the end of the process (Rajcan et al., 1999). RAPD-PCR (Williams et al., 1990; Welsh & McClelland, 1990) was performed in 25 µl reaction mixtures containing: 25 ng of genomic DNA, 2.5 mM MgCl2 , 200 mM of each dNTP, 1.25 units of Taq polymerase (Gibco BRL) and 1× PCR Buffer per reaction. For PCR reaction, DNA was amplified using the MJ Research PTC-100 Programmable Thermal Controller. The temperature profile used for PCR was as follows: 94 ◦ C for 1.5 min followed by 45 cycles at 94 ◦ C for 30 sec, 36 ◦ C for 45 sec and 72 ◦ C for 1 min, repeated in sequence, and finally followed by 72 ◦ C for 2 min. Amplified products were separated by electrophoresis on 1.4% agarose gels and visualized under UV light by staining with ethidium bromide. Fifteen random primers were selected on the basis of revealing polymorphism between the total genomic DNA of parental lines (Rajcan, 1996). They were used for the screening of the cpDNA and mtDNA of Reston and LL09. These primers are from the commercial kits available from the University of British Columbia under the following numbers: 28, 35, 44, 55, 210, 242, 310, 322, 350, 389, 440, 574, 586, 589, 597. The mt, cp and total genomic DNA samples of the parental lines Reston and LL09 were run in parallel on a gel for a comparison of RAPD patterns, within and among various DNA species.
To complement the genetic studies, RAPD markers RM350 and RM574 (Rajcan et al., 1999), and RM440 (Rajcan, 1996), which were previously reported to be associated with linolenic acid content, were tested for random segregation against the two DH populations. The RAPD method used in this analysis was the same as described above. The reciprocal populations were compared for all traits considered by using contrasts as calculated by SAS software package. Chi-square tests were calculated and tested against for significance (Gomez & Gomez, 1984). A non-parametric Mann-Whitney U test (Siegel, 1956) was employed to compare the frequency distributions of reciprocal microspore-derived DH populations. In this method, the linolenic acid determinations were combined from the two reciprocal populations and ranked in the order of increasing size. The U statistic was calculated using the following formula: U = n1 n2 + 1 /2 n1 (n1 + 1) – R1 where n1 , n2 are the number of DH lines from the R×L and L×R crosses and R1 is the sum of the ranks assigned to the R×L DH lines. The Z-test was used to test for significance of the U-values (Siegel, 1956).
Results Comparison of the inheritance patterns of 18:3 The frequency distribution of linolenic acid (18:3) in the reciprocal DH populations obtained by analysing the seed fatty acid content in the growth room study is provided in Figure 1. The goodness-of-fit to a three-gene model (Chen & Beversdorf, 1990) was tested using a χ 2 test for each reciprocal population separately. The segregation of linolenic acid content fits a typical tri-genic segregation ratio for a DH population of 1:3:3:1 [low (8.51)] only for the R×L population (χ 2 =1.59, p=0.70) but not for the reciprocal, L×R (χ 2 =17.25, p < 0.01). In contrast, the results of the fatty acid analysis of 120 and 121 F2 plants from the R×L and L×R, respectively, revealed that the segregation of linolenic acid followed the expected tri-genic ratio of 63 (intermediate to high 18:3):1 (low 18:3) for both populations.
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Figure 1. Frequency distribution of linolenic acid concentration in DH lines grown in a growth room from reciprocal DH populations involving parents Reston and LL09. Table 1. The distribution of linolenic acid among phenotypic classes of DH lines produced from reciprocal crosses involving Reston and LL09 Linolenic acid phenotypic class∗
Reston × LL09
LL09 × Reston
Expected tri-genic ratio DH population
Low ( 8.51%)
11 31 35 7
19 32 26 1
1 3 3 1
∗ Phenotypic classification by Chen and Beversdorf (1990) was used because of a similar background
of the parental lines.
Comparison of the means and variances between reciprocal DH and F2 populations i) Growth room study The ranges, minimum, maximum values, standard errors or standard deviation, and means for linolenic acid in DH and F2 populations obtained in the growth room are given in Table 2. The F-test of the linolenic acid
distributions between reciprocal DH populations was statistically significant (p = 0.04). Also, the L×R population was visibly shifted toward lower linolenic acid values when compared to R×L (hatched bars, Figure 1). Linolenic acid distribution in the reciprocal DH populations was also compared by the non-parametric
405 Table 2. Means, standard errors and, minimum and maximum values of linolenic acid (18:3) concentration (%) in reciprocal populations derived from crosses between Reston and LL09 Population DH Reston × LL09 LL09 × Reston F2 Reston × LL09 LL09 × Reston
N
Mean 18:3%
SE or SD∗
Minimum
Maximum
84 78
5.39 4.57
0.20 0.16
2.36 2.20
11.23 9.78
120 121
5.66 5.98
1.38 1.45
3.04 2.87
11.15 9.97
∗ SE, standard error was used for DH populations; SD, standard deviation was
calculated for F2 populations because of no replication of data points. Table 3. Contrast comparisons of agronomic and quality traits between reciprocal DH populations from the crosses of Reston and LL09 using data from Elora 1993 Characteristica
Contrast SSb
F-value
p-value
18:3 content % Oil % Protein Lodging Days to flower Days to maturity Plant height
8.6 2.3 3.8 0.2 35.4 96.5 526
12.20 1.04 4.21 0.58 65.42 8.58 6.97
