1 Brassica Genetics Research Group, John Innes Centre, U.K.
2 Department of Agriculture, University of Newcastle Upon Tyne, U.K.
3 CPB Twyford Limited, Church Street, Thriplow, Herts. U.K.
4 Molecular Genetics Section, Agriculture Canada, Saskatoon, Canada.
During anthesis, the dense floral canopy produced by oilseed Brassicas compromises the yield potential of the crop, as it reduces the transmission of solar radiation to the photosynthetic tissues. This study describes the development of apetalous near-isogenic lines by introgressing three loci controlling the apetalous character from an inferior genotype into the commercial breeding line, Tapidor. The apetalous near-isogenic lines were selected from a B3S1 generation segregating for the apetalous character. This generation was derived from an individual that possessed the most enriched genome for recurrent parental alleles. This B3S1 generation had on average 96% of the alleles present in its genome returned to the recurrent parental genotype. A further backcross generation was completed that will allow for the development of apetalous near-isogenic lines further enriched for the recurrent parental genotype.
The generation of these genetic lines will enable for the first time the accurate assessment of the physiological effects of the apetalous character upon yield.
KEYWORDS: Molecular Markers, RFLP mapping, Marker Assisted Selection (MAS), Light penetration, Yield improvement.
Oilseed rape (Brassica napus) is a major oilseed crop grown throughout Europe, North America and China for its edible oil. B. napus is an amphidiploid crop formed from an interspecific hybridization between the diploid Brassica rapa and Brassica oleracea as proposed by U (1935). The Brassica species possess a high level of natural polymorphism and so lend themselves to analysis using molecular markers. Recently, genetic linkage maps have been constructed in B. napus using RFLP technology (Ferreira et al. 1994; Uzunova et al. 1995; Sharpe et al. 1995; Parkin et al. 1995). Once established the genetic marker technologies and genetic maps can be utilized to further marker-assisted breeding (Paterson et al. 1991; Lydiate et al. 1995) and will enable marker accelerated backcrossing programmes. This involves the simultaneous selection for desirable segments of a donor genotype and for the genotype of the recurrent genetic background over several generations.
The potential for seed yield in oilseed rape is primarily determined by the dry matter accumulation of the crop prior to flowering (Mendham, 1981). However, the realization of this yield potential is determined by the availability of photosynthetically active radiation (PAR) and water availability post-anthesis (Evans, 1984). The dense, highly absorptive and reflective floral canopy impairs the transmission of the photosynthetically active radiation to the leaf canopy. Chapman et al. (1984) determined that 24% and 7% of the available PAR reached the leaf canopy at early and late flowering respectively. Fray et al. (1996) determined that 22% of the available PAR reached the photosynthetically active tissues, with the floral canopy being responsible for absorbing 64% and reflecting 14% of the available PAR. Daniels et al. (1986) proposed that the reduced transmission of the PAR to the leaf canopy is responsible for an accelerated rate of leaf senescence.
Variants with reduced petal number have been identified within the Brassica species. An apetalous variant controlled by two loci with additive effects was reported by Buzza et al. (1983) in a spring oilseed rape. Fray et al. (1997) described another apetalous variant in a spring oilseed rape where the petals are converted to sterile stamen. This phenotype is also under the control of two loci. However, the linkage between the apetalous phenotype and a shriveled leaf phenotype could not be broken, resulting in a reduction in the agronomic value of that trait. A third variant was reported by Fray et al. (1996) with a reduced petal number. This character is under the control of three loci and possesses no obvious pleiotropic effects. Data obtained from physiological analysis has indicated that it is likely that the introgression of an apetalous phenotype into oilseed rape will increase the yield potential of the crop without increasing agricultural inputs (Rao et al. 1991; Fray et al. 1996). Physiological studies have determined the benefit of introgressing the apetalous character into oilseed rape (Rao et al. 1991; Fray et al. 1996). These physiological studies have determined that there is a substantial increase in the amount of PAR transmitted to the leaf canopy in the apetalous B. napus lines compared to the fully petalled elite B. napus lines. Although these studies have demonstrated the potential effect upon yield of the apetalous character, no comparisons of yield differentials can be made as the genetic backgrounds of the apetalous variant and the elite breeding line differ. To answer the question as to the effect the apetalous character has upon yield, the alleles controlling the apetalous phenotype should be introgressed into the elite breeding line. This would generate lines that possess the same genetic background but only differ at the loci controlling the apetalous character (apetalous near-isogenic lines). This would remove the interaction that the apetalous genetic background has on yield and make a direct comparison of yield a fair assessment.
Two major loci and a third locus with a modifying effect control the recessive character of the B. napus apetalous variant N-o-112 described by Fray (1995). This apetalous variant should be of considerable agricultural use despite the fact that the apetalous phenotype is not fully penetrant. The residual petals present on the plant (<1PPF) should not effect the overall benefit of the improved light penetration provided by this character. The identification of RFLP markers flanking the three APET loci have made possible the marker assisted selection of backcross individuals that are heterozygous at the three APET loci. These can be selected for at each generation of a backcrossing programme using a fully petalled elite cultivar as the recurrent parent. This has the advantage of reducing the time and cost required for the breeding programme as self or test cross generations are not required to identify individuals carrying recessive alleles. This backcrossing programme has resulted in the development of apetalous near isogenic lines at a backcross three generation.
MATERIALS AND METHODS.
G. Robbelen (University of Gottingen, Germany), originally donated the B. napus apetalous variant (N-o-112) used in this study. Other than a substantial reduction in the number of petals per flower N-o-112 shows normal floral morphology. TapidorDH1 is a doubled haploid line derived from the commercial oilseed rape variety Tapidor via microspore culture (Howell et al. 1996). A single N-o-112 individual plant was used to pollinate TapDH1 to produce F1 seed. A single F1 plant was used to pollinate both parental plants to generate a mapping backcross population (N-o112 x F1) and an introgression population (TapDH1 x F1). Assessment of the floral phenotype was made by calculating the mean average number of petals per floret (PPF). This statistic was calculated from the total number of petals present on the first ten florets present on the terminal, first, second and third racemes. All plants sown in a peat and sand mixture and were grown in a controlled environment glasshouse (16°C, 16hr photoperiod). All plants were vernalized for six weeks (4°C, 8hr photoperiod) to induce flowering.
DNA extraction, restriction digestion, alkaline transfer and Southern hybridization were carried out as described previously by Sharpe et al. (1995). The RFLP probes used were also described in Sharpe et al. (1995).
The identified 120 polymorphic loci were arranged by linkage analysis at a LOD score of >3 into 18 linkage groups, with six unlinked loci. The recombination frequencies were converted to map distances using Kosambi (1944) mapping function. Seventy-six of these polymorphic loci were equivalent to polymorphic loci positioned on 18 of the 19 linkage groups of pre-existing RFLP maps of oilseed rape (Sharpe et al. 1995; Parkin et al. 1995).
The crossing strategy used throughout this study to develop the apetalous near-isogenic lines is displayed in figure 1. This strategy has been used to generate apetalous near isogenic lines (NILs) by successive generations of marker accelerated backcrossing. To enable this selection, a genetic map has been constructed from a backcross population (figure 1). The observed phenotypic segregation data indicated that the apetalous character was controlled by alleles present at three loci. These APET loci and their flanking RFLP marker loci are displayed in figure 2.
Figure 1. The crossing strategy used to map the loci controlling the apetalous character and to develop apetalous near isogenic lines in oilseed rape.
Figure 2. Displays the three linkage groups to which the loci controlling the apetalous character map in oilseed rape. The length of the genetic intervals (cM) between the APET loci and their flanking markers are displayed.
Selecting individuals that possess the donor (N-o-112) RFLP alleles at the flanking RFLP loci identifies the apetalous heterozygotes throughout the backcrossing programme. The genetic intervals between the flanking RFLP marker loci are short enough to ensure that genetic interference prevents double recombination events from occurring between them. Selection has been carried out simultaneously selecting for the desired donor alleles from N-o-112 and for recurrent parental (Tapidor) alleles throughout the remainder of the genome. This has the effect of accelerating the reduction of genetic drag. The results of this selection are displayed in figure3. PT13-12-65 is the backcross three individual that was most enriched for recurrent parental genotype. The individual plants derived from line PT13-12-65 have returned their genetic background to recurrent parental genotype to 96% on average. The selfed seed derived from this individual segregated for the apetalous phenotype. An apetalous individual and a fully petalled individual were selected from this population for seed multiplication to generate the apetalous near-isogenic lines. The estimation of enrichment for recurrent parental genotype was achieved using RFLP markers that cover the polymorphic regions of the genome in this cross. It is estimated that 55% of the B. napus genome is covered by the polymorphic RFLP loci identified in this cross. A backcross three generation has a mean average of approximately 93% of its genome returned to the recurrent parental genotype. This mean average figure is used for the 45% of the genome that can not be classified as having donor or recurrent alleles using RFLP marker loci. The remaining 55% of the genome that can be classified as originating from the donor or recurrent parental genotype allows for the identification of the individual genome with the most enriched recurrent parental genotype.
Figure 3. Graphical representation detailing the extent of the return to recurrent genotype of the B3S2 apetalous near isogenic lines. 96% of the genome of line PT13-12-65 has been returned to recurrent genotype, this line has been selected for use in field trial trials to assess the physiological effects of the apetalous character. Plots of the apetalous near isogenic line will be compared to the commercial cultivar Tapidor and fully petalled near isogenic lines.
Marker assisted backcrossing offers an effective way of accelerating the development of near-isogenic lines. It is possible to transfer the alleles controlling an agronomically important trait by carrying out selection, not directly on the trait of interest but on molecular markers linked to that trait. Due to the time constraints placed upon this study, the development of the apetalous near-isogenic lines was achieved in the B3S2 generation. However, the backcrossing programme was extended to the B4 generation. This allows the possibility of developing apetalous near-isogenic lines in further generations with an increase in the enrichment for recurrent parental genotype. Identification of the apetalous near-isogenic line and different fully petalled near-isogenic lines was achieved from a segregating population derived from the B3 individual which possessed the genome with most enriched recurrent parental genotype. This individual was PT13-12-65 (see figure 3). The selfed seed from this B3 plant segregated for the apetlous character. The segregants were analyzed using RFLP markers to identify true apetalous individuals along with number of individuals that are fully petalled and possess different alleles at the APET loci. The use of the apetalous near-isogenic lines, the fully petalled near-isogenic lines and the commercial breeding line Tapidor in field trials will allow for the first time, an accurate assessment of the physiological effects of the apetalous charatcer upon yield. The apetalous near-isogenic lines have the potential to return a greater seed yield compared to fully petalled near-isogenic lines due to improved light penetration through the floral canopy and a lower incidence of Sclerotina sclerotiorum infection. However, the value of the apetalous character would be reduced if the gain of extra assimilates of an apetalous crop are used to increase the number of branches and pods as opposed to filling existing pods.
I would like to thank the Ministry of Agriculture Fisheries and Food and Cambridge Plant Breeders, Twyfords for their support and funding of this project.
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