Utilization of doubled haploid technique in Brassica rapa population improvement

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1 Utilization of doubled haploid technique in Brassica rapa population improvement H. Friesen and R. Scarth Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Received 24 March 1999, accepted 24 August Friesen, H. and Scarth, R Utilization of doubled haploid technique in Brassica rapa population improvement. Can. J. Plant Sci. 80: The utilization of doubled haploid (DH) plants in population improvement of Brassica rapa was studied by randomly intercrossing 4, 8, 12 and 22 DH lines developed from the B. rapa cultivar Reward and the B. rapa breeding line DSC- 3 for two generations to constitute synthetic populations. The synthetic populations and the DH plants used in their formation were evaluated for agronomic performance at two locations in the field in 1996 and for genetic variation using random amplified polymorphic DNA (RAPD) analysis. Intercrossing as few as four DH lines from the breeding line DSC-3 produced a synthetic population with improved performance over that of the contributing DH lines. The synthetic population produced from the interpollination of eight DH lines showed an agronomic performance over that of the contributing DH lines to a level similar to the Reward donor population. RAPD analysis efficiently characterized the genotypic variation present in DH lines and synthetic populations, detecting 22 72% polymorphism between DH lines, 17 53% and 27 47% polymorphism in the first and second synthetic populations, respectively. This characterization may be useful as a tool in the reestablishment of heterogeneity and recovery of agronomic performance in B. rapa synthetic populations derived from DH lines by determining the level of genetic variability among DH lines and therefore optimal population size. Key words: Brassica rapa, synthetic population, doubled haploids, RAPD, agronomic performance Friesen, H. et Scarth, R Utilité de l haplodiploïdisation pour l amélioration génétique des populations de Brassica rapa. Can. J. Plant Sci. 80: Nous avons étudié l utilité de l haplodiploïdisation pour l amélioration génétique des populations de Brassica rapa. Pour ce faire, 4, 8 et 22 lignées dihaploïdes (DH) provenant du cultivar Reward et de la lignée génétique DSC-3 ont subi deux générations d intercroisement aléatoires aboutissant à la constitution de populations synthétiques. Ces populations, et les plantes DH utilisées dans leur formation, ont ensuite été évaluées sur leurs performances agronomiques à deux emplacements au champ en 1996, puis sur la variabilité génétique par l analyse RAPD. En intercroisant aussi peu que 4 lignées DH issues de DSC-3, on a produit une population synthétique démontrant de meilleures performances que les lignées DH parentales. L amélioration, par rapport aux lignées de départ, des performances agronomiques de la population synthétique issue de l interpollinisation de 8 lignées DH était comparable à celle observée dans la population originale du cultivar Reward. L analyse RAPD a permis de caractériser la variation génotypique au sein des lignées DH et des populations synthétiques, détectant 22 à 72 % de polymorphisme parmi les lignées DH et 17 à 53 et 27 à 47 %, respectivement, dans les populations synthétiques de première et de seconde génération. Cette caractérisation peut être utilisée comme moyen de rétablir l hétérogénéité et de relever les performances agronomiques des populations synthétiques de B. rapa issues de lignées DH, en déterminant le niveau de variabilité génétique parmi ces lignées et, par voie de conséquence la taille optimale de la population. Mots clés: Brassica rapa, population synthétique, dihaploïdes, RAPD, performances agronomiques Until 1996, the canola acreage in western Canada was divided approximately equally between the two species Brassica napus and B. rapa, with the latter occupying over 80% of the northern canola growing areas due to the 10 to 15 d earlier maturity of B. rapa cultivars. Since 1996, there has been a significant decline in B. rapa acreage to less than 20% of the total production. This decline has been attributed to the approximately 15% yield deficit of B. rapa compared to B. napus cultivars and the susceptibility of B. rapa cultivars to blackleg disease caused by the fungal pathogen Leptosphaeria maculans, which results in a further reduction in agronomic performance. In addition, the production options offered by the new forms of herbicide resistance are available only in B. napus cultivars. Yield improvement of B. rapa would be beneficial for canola production in western Canada. Traditional breeding 75 methods applied to this sporophytically SI species include mass selection, hybridization and the development of synthetic populations. The production of a synthetic population involves the seed mixture of strains, clones, inbreds or hybrids among them, propagated by interpollination (Poelman and Sleper 1995). Intercrossing promotes the maintenance of heterozygosity and heterogeneity, which has Abbreviations: DH, doubled haploid; DI, disease index; HI, harvest index; NNA, nearest neighbour analysis; PCR, polymerase chain reaction; RAPD, randomly amplified polymorphic DNA; RCB, randomized complete block; RFLP, restriction fragment length polymorphism; SI, selfincompatible; Syn1 first synthetic generation, Syn2 second synthetic generation, UBC, University of British Columbia

2 76 CANADIAN JOURNAL OF PLANT SCIENCE been shown to improve yield and yield stability in B. napus winter annual hybrids (Léon 1991). Doubled haploid line development in B. napus speeds up the breeding process by achieving homozygosity in a single generation and avoiding repeated generations of inbreeding traditionally required to fix desirable traits (Chen and Beversdorf 1990). Doubled haploid lines produced in B. rapa express severe inbreeding depression for agronomic traits such as number of seed per pod, seed and biological yield and time to flowering (Dewan et al. 1998). Exploitation of heterosis or hybrid vigour in synthetic or hybrid cultivars offers the opportunity to restore agronomic performance through recombination. Optimum population size is an important consideration in synthetic production. In B. rapa populations, it is critical to have sufficient variation at the multi-allelic S locus, which controls SI, to ensure an adequate number of compatible mates. The population should be large enough to reduce the chance of mating between close relatives and minimize the occurrence of inbreeding depression (Miranda-Filho and Chaves 1991; Byers and Meagher 1992). Genetic variability can be determined by examining the phenotype of individuals in a population. However, phenotype is influenced by the environment and its interaction with a particular genotype. Molecular DNA techniques such as random amplified polymorphic DNA (RAPD) analysis have been applied to detect genetic variation in various crops (Mailer et al. 1994; Demeke et al. 1996). RAPD analysis has the advantage of identifying polymorphisms independent of both prior DNA sequence knowledge and environmental influence. Genotypes can be selected to maximize genetic variation in a population and this heterogeneity may contribute to improved agronomic performance. The purpose of this study was to determine the most efficient utilization of DH lines in the improvement of B. rapa populations through production of synthetic populations. An optimum population size was determined for the production of synthetics using the DH lines, in order to recover the agronomic performance of the original donor population. This study also reports on the application of RAPD analysis to detect genetic variation in B. rapa DH lines. The maintenance of this variation was investigated through two generations of synthetic populations constituted from different numbers of individual DH lines. MATERIALS AND METHODS DH Line Selection The seed sources for the DH lines were second generation selfed DH lines from the B. rapa cultivar Reward, registered by the University of Manitoba (Scarth et al.1992) and second generation selfed DH lines from DSC-3, a breeding line from Agriculture and Agri-Food Canada. The DH lines were produced using the microspore culture protocol reported by Ferrie and Keller (1995), and the first generation seed was provided by Dr. Allison Ferrie (Plant Biotechnology Institute NRC). Five single plants from the DH seed provided were sown in 15-cm pots and grown in growth chambers with an 18-h photoperiod, 580 µe m 2 s 1 light intensity and a day/night temperature of 15/10 C. These plants were covered with polyethylene isolation bags with 1-mm holes from about 1 d prior to flowering until the end of the flowering period. Bagged plants were selfed by spraying with 3% NaCl spray every 3 d after the first flowers were newly opened until flowering was complete, as described in Friesen and Scarth (1998). Selfed seed harvested from 52 DSC3 and 60 Reward DH lines was used for the 1995 field study. DH lines were evaluated in the field in Winnipeg as randomized complete blocks (RCB), which consisted of two replicates with 3-m rows sown with 100 seeds per row for each DH line. Check rows of cv. Reward were sown between each DH line to control competition between rows. Field conditions in 1995 from the time of seeding on 29 May to the end of June were poor for establishment and growth with the average day temperature of 28.4 C and total precipitation of 25.6 mm. Therefore, DH lines were selected for synthetic development based on the availability of at least 0.6 g of seed, which was required for further field study, a mean fertility index of at least 2 and a mean DI relating to white rust (Albugo candida) of less than 82%. The fertility index was determined by visually rating plants in a row under the 1995 field conditions according to a scale of 1 5 where a 5 indicated the plants had pod and seed development similar to the check cultivar Reward. Disease index was a measure of the percentage of infection of a DH line, calculated based on a rating of about 10 plants inoculated on the cotyledons with spores of white rust Albugo candida race 7A and grown under controlled conditions (Williams 1985). The Williams scale of 1 9 was used to rate plants, where the higher number represents a more severe infection of white rust. Synthetic Population Development Sets of 4, 8, 12 and 22 DH lines from each of the DSC-3 and Reward DH populations were grown inside isolation tents in the greenhouse. The DH lines included in the smaller sets were also included in all larger sets. Cross-pollination was encouraged by brush pollinating the plants within each tent using a feather duster every other day. Two tent replicates were grown for each of DSC3 and Reward DH populations. The position of the plants in the pollination tent was rotated every 4 d after flowering began. Seed produced was harvested from single plants and an equal amount from each plant was bulked to produce the first generation synthetic (Syn1). From each of these Syn1 populations, 60 plants were seeded in individual pots and randomly intercrossed to produce the second generation synthetic (Syn2). The Syn2 was produced from 60 individual Syn1 plants to ensure adequate seed production for the 1996 field season. Synthetic Population Evaluation All Syn1 and Syn2 populations were grown at two field locations in 1996 as separate Syn1 and Syn2 trials with six and four replications in Winnipeg and Carman, respectively. A total of 16 populations were evaluated, made up of four levels of DH line combinations in each of two generations,

3 FRIESEN AND SCARTH UTILIZATION OF DOUBLED HAPLOIDS IN BRASSICA RAPA 77 Table 1. Mean parameter values for DH lines produced from B. rapa Reward and the breeding line DSC-3 and comparison to mean values of the cv. Reward (check) in Winnipeg, 1996 Reward DSC-3 Parameter Check DH lines LSD 0.05 Check DH lines LSD 0.05 Seedling emergence z Days to flowering (d) Days to maturity (d) Height (cm) Lodging (1 5) Seed yield per row (g) Harvest index y Seed oil content (%) x Syn1 (Syn 1-4, Syn1-8, Syn1-12, Syn1-22) and Syn2 (Syn 2-4, Syn2-8, Syn2-12, Syn2-22), from the two DH line sources, cv. Reward and DSC-3 breeding line. The original DH lines with sufficient seed were evaluated in Winnipeg in two replications. In Winnipeg, 3-m rows were planted with 0.3 g of seed and in Carman 5-m rows were planted with 0.5 g of seed with 60-cm spacing between each row, with cv. Reward check rows alternating with each of the DH lines, Syn1 or Syn2 rows. These check rows were used in the data analysis in the calculation of the cv. Reward check mean. The DSC-3 breeding line was not included in the field trial due to lack of seed. Agronomic performance of the DH lines and the Syn1 and Syn2 populations was evaluated by measuring number of plants emerged per metre of row, height at maturity, lodging, days to flowering, days to maturity, seed yield, HI and seed oil content. The height (cm) was measured as the average of all plants in a row once flowering had finished. Lodging was measured according to a scale of 1 5 where a 5 indicated good stalk strength with upright growth. The days to flowering were determined as the number of days from when 50% of the plants had emerged until 50% of the plants in a row had begun flowering. Days to maturity were determined as the number of days from when 50% of the plants had emerged until 50% of the plants in a row were physiologically mature. At maturity, plant rows representing DH lines and replicates of Syn1and Syn2 were hand harvested, tied and stooked in the field. Each plant row was weighed when dry (moisture content was not determined) and threshed in the field when dry using a stationary thresher. Seed yield was determined by weighing seed harvested from each plant row. Harvest index was calculated as the seed yield per total biomass yield measured for each row. Seed oil content was measured for each row from 20 g of seed using a Nuclear Magnetic Resonance spectrometer (Robertson et al. 1979). Data were analyzed through nearest neighbour analysis of variance (NNA) using Agrobase/4 (Mulitze 1992) statistical program. A standard error value was calculated for each parameter using a complete data set. RAPD Analysis RAPD analysis was performed on populations of 22 and 23 DH lines of DSC-3 and cv. Reward, respectively. One of the 23 Reward DH lines had insufficient seed for agronomic evaluation and therefore was not included in the 1996 field trial. At the four- to five-leaf stage, all leaves from plants of each DH line were excised and pooled. The DH lines were grown under controlled conditions in a growth chamber. At the five- to six- leaf stage, four to five leaves from randomly selected single plants were excised from the 1996 field trial and individually prepared for DNA analysis. Each individual plant represented one sample from Syn1 and Syn2. DNA was isolated from the lyophilized tissue based on the method reported by Kidwell and Osborn (1992). DNA extracted from plants grown under controlled and field conditions yielded approximately 114 to 2280 µg and 105 to 2164 µg of DNA, respectively. The field samples were taken at a more advanced stage of plant development than the growth chamber samples and therefore they may have contained more impurities, such as starch, as demonstrated by some 260/280 ratios recorded with the UV spectrophotometer falling outside of the acceptable range of Reduced DNA yields from Brassica were also found by Kidwell and Osborn (1992) when they sampled leaf tissue of different maturity and stress levels. A set of 30 primers (oligonucleotides), obtained from the University of British Columbia, was tested for polymorphism between the donor populations of DCS-3 and Reward. PCR was performed using a PTC-100 (MJ Research, Inc.), as reported by Mailer et al. (1994). RAPD products were separated by electrophoresis on ethidium bromide stained 1.4% agarose gels in 1 TAE buffer. Genomic DNA from B. napus and a negative control were exposed to the PCR conditions as controls with the analysis of each new primer. Lambda DNA digested with HindIII (Pharmacia Biotech) was included as the size marker. Primers that expressed polymorphism between DSC-3 and cv. Reward donor populations were selected from the initial primer set. These primers, shown in Table 1, were tested against each of the DH lines produced from the two donor populations. University of British Columbia primers 329 and 338 were selected for RAPD analysis because they detected the highest expression of polymorphism among all of the primers

4 78 CANADIAN JOURNAL OF PLANT SCIENCE Table 2. Mean values for parameters characterizing the Syn1 populations produced from 4, 8, 12 or 22 DH lines (Syn1-4, Syn1-8, Syn1-12, Syn1-22) of B. rapa Reward, 1996 and the cv. Reward (check). Parameters are seedling emergence (EMER), days to flowering (DTF), days to maturity (DTM), height (HT), lodging (LOD), seed yield (YLD), harvest index (HI) and seed oil content (OIL) EMER z DTF DTM HT LOD YLD HI y OIL x Population (d) (d) (cm) (1-5) (g) (%) L1 w L2 v L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 Check Syn Syn Syn Syn cv. (%) LSD SE w L1 = Carman. v L2 = Winnipeg. Table 3. Mean values for parameters characterizing the Syn2 populations produced from 4, 8, 12 or 22 DH lines (Syn2-4, Syn2-8, Syn2-12, Syn2-22) of B. rapa Reward, 1996 and the cv. Reward (check). Parameters are seedling emergence (EMER), days to flowering (DTF), days to maturity (DTM), height (HT), lodging (LOD), seed yield (YLD), harvest index (HI) and seed oil content (OIL) EMER z DTF DTM HT LOD YLD HI x OIL x Population (d) (d) (cm) (1-5) (g) (%) L1 w L2 v L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 Check Syn Syn Syn Syn cv. (%) LSD SE w L1 = Carman. v L2 = Winnipeg. used to analyze the DH lines (Table 6). The percent of polymorphism was determined by dividing the number of bands that were polymorphic by the total number of bands. The number of individuals analyzed in Syn1 and Syn2 was determined by the frequency of bands expressed in the DH lines. The lowest band frequency (p) with a specific primer was calculated by dividing the number of individuals with the least frequently expressed band by the total number of individual lines in the DH population. Then the number of plants (N) sampled from Syn1 and Syn2 was calculated to give a 95% probability (P) that the least frequent allele (p) would be represented, as shown in the following equation (Jasieniuk et al. 1996): N = ln(1 P)/ ln(1 p). For the Reward source populations, the number of individuals (N) sampled for Syn1 and Syn2 from 4, 8, 12 and 22 DH lines was 12, 12, 12 and 15, respectively, for Primer 329 and 12, 7, 6 and 8 for Primer 338. RAPD fragment sizes were estimated from the gels in relation to the size marker. Data matrix tables were compiled where bands were recorded as either present (1) or absent (0). RESULTS Days to Flowering In 1996, the range in days to flower of the Reward DH lines was d with a mean value of 21.0 d, significantly later than the cv. Reward check (Table 1). The individual DSC-3 DH lines had a similar range of d to flowering, with a mean of 20.0 d. Some of the Reward Syn1and Syn2 populations took significantly longer to flower than the donor population at both Carman and Winnipeg (Tables 2 and 3). Among the Syn1 populations, Syn1-4 at Carman and among the Syn2 populations, Syn2-4 at Carman were the latest to flower. In Carman, Syn2-22 flowered earlier than any other Syn2 population. In Carman, the DSC-3 Syn1-22 and Syn2-22 were earliest to flower within each Syn1and Syn2 population, respectively.

5 FRIESEN AND SCARTH UTILIZATION OF DOUBLED HAPLOIDS IN BRASSICA RAPA 79 Table 4. Mean values for parameters characterizing the Syn1 populations produced from 4, 8, 12 or 22 DH lines (Syn1-4, Syn1-8, Syn1-12, Syn1-22) of B. rapa breeding line DSC-3, 1996 and the cv. Reward (check). Parameters are seedling emergence (EMER), days to flowering (DTF), days to maturity (DTM), height (HT), lodging (LOD), seed yield (YLD), harvest index (HI) and seed oil content (OIL) EMER z DTF DTM HT LOD YLD HI y OIL x Population (d) (d) (cm) (1-5) (g) (%) L1 w L2 v L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 Check Syn Syn Syn Syn cv. (%) LSD SE w L1 = Carman. v L2 = Winnipeg. Table 5. Mean values for parameters characterizing the Syn2 populations produced from 4, 8, 12 or 22 DH lines (Syn2-4, Syn2-8, Syn2-12, Syn2-22) of B. rapa breeding line DSC-3, 1996 and the cv. Reward (check). Parameters are seedling emergence (EMER), days to flowering (DTF), days to maturity (DTM), height (HT), lodging (LOD), seed yield (YLD), harvest index (HI) and seed oil content (OIL) EMER z DTF DTM HT LOD YLD HI y OIL x Population (d) (d) (cm) (1-5) (g) (%) L1 w L2 v L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 Check Syn Syn Syn Syn cv. (%) LSD SE w L1 = Carman. v L2 = Winnipeg. There were no differences in days to flowering between the Syn1and Syn2 populations in Winnipeg (Tables 4 and 5). Days to Maturity In 1996, the range in maturity of the Reward DH lines was d. with a mean value of 57.6 d., significantly earlier than the cv. Reward check (Table 1). The individual DSC-3 DH lines had a narrower range of d to maturity. The Reward Syn1 populations at both locations and the Syn2 populations at Carman matured at the same time as the donor population while all of the Reward Syn2 populations took significantly longer to mature than the cv. Reward check in Winnipeg (Tables 2 and 3). There were no differences in the days to maturity between the DSC-3 Syn1or Syn2 populations g and a range of g. The individual DSC-3 DH lines also expressed low seed yields with a mean yield of 43.1 g. (Table 1) and a range of g. In Carman, Reward Syn1-8 and Syn1-12 yielded significantly more seed than the cv. Reward check and other Syn1 populations (Table 2). This ranking was also observed in the Syn2 generation where the cv. Reward check was lower yielding than all of the Syn2 populations at Carman (Table 3). In Winnipeg, the Syn1-8 population was the highest for seed yield (Table 2). At this location, there were no differences between the Reward Syn2 populations and cv. Reward check (Table 3). There was no difference in seed yield between the DSC-3 Syn1populations (Table 4). The DSC-3 Syn2-4 showed higher seed yield than Syn2-8 at Carman only and Syn2-12 was lower yielding than Syn2-22 the highest yielding Syn2 population at Winnipeg (Table 5). Seed Yield The Reward DH lines had a significantly lower mean seed yield than the cv. Reward check (Table 1) with a mean of Seed Oil Content The Reward and the DSC-3 DH lines demonstrated a significantly lower mean seed oil content than the cv. Reward

6 80 CANADIAN JOURNAL OF PLANT SCIENCE Table 6. Expression of polymorphism in the DH lines from the cv Reward and DSC-3 breeding line, using RAPD analysis Primer Primer Source of Polymorphism (UBC) sequence DH lines (%) z ACG GCG TCA C -3 DSC-3 22 Reward TCC CGA ACC G -3 DSC-3 28 Reward CGG AGA GCG A -3 DSC-3 56 Reward CTG TGG CGG T -3 DSC-3 38 Reward 63 z Number of polymorphic bands divided by total number of bands 100. check (Table 1). However, the Reward and the DSC-3 Syn1 and Syn2 populations had seed oil content equal to the cv. Reward check (Tables 2 5). Other Characteristics The other characteristics showed relatively small differences between the cv. Reward check and the synthetic populations, and among the synthetic populations. There was no difference in seedling emergence among any of the populations of the Reward Syn1or between the Syn1and the cv. Reward check. The Reward Syn2 populations all had higher numbers of seedlings emerging than the cv. Reward check. There were no differences in height, lodging or harvest index among Reward Syn1 and Syn2 populations or between Syn1, Syn2 and the cv. Reward check. There were no differences among any of the DSC-3 Syn1and Syn2 populations in seedling emergence, height and lodging (Tables 4 and 5). RAPD Analysis Polymorphism was detected for 9 of the original 30 primers tested between the DSC-3 and Reward donor populations (data not shown). A total of 98 RAPD products were visualized, including 57 polymorphic and 41 monomorphic bands. The number of bands produced per primer ranged from 4 to 15. When tested on the DH lines in both populations, four primers produced bands that expressed adequate intensity to distinguish between their presence or absence. These primers and their sequences are shown in Table 6. Polymorphism was expected between the DSC-3 and cv. Reward. Primers used in this test differed in their ability to detect genetic variation in each of the collection of DH lines (Table 6). RAPD analysis demonstrated that as the number of individual DH lines included to create each synthetic population increased, there was more genetic variability expressed (Table 7). Data are shown for the cv. Reward populations. The DSC-3 populations showed a similar relationship. After one and two generations of interpollination, all Syn1and Syn2 populations expressed equal or less polymorphism than the corresponding DH lines used in their production. The level of polymorphism that was reached in Syn1and Syn2 was similar for all population sizes. The lowest level of polymorphism was expressed when only four DH lines contributed to Syn1and Syn2 and a similar level of polymorphism was observed over the two generations of interpollination. Table 7. Expression of polymorphism in Syn1 (first synthetic population) and Syn2 (second synthetic population) composed of 4, 8, 12 and 22 DH lines compared to the individual DH lines contributing to each synthetic population in the B. rapa Reward, using RAPD analysis with the two primers 329 and 338 Synthetic population size x Primer DH z 40 y Syn Syn Primer 338 DH Syn Syn z Individual DH lines contributing to each synthetic population. y Polymorphism (%) (number of polymorphic bands divided by the total number of bands 100). x Number of DH lines contributing to each synthetic population. DISCUSSION The DH lines derived from the two sources showed a wide range of agronomic performance, as reported by the Dewan et al. (1998) evaluation of DH lines from five B. rapa breeding populations. Intercrossing DH lines in B. rapa in the production of synthetic populations led to the recovery of population performance equal to or above that of the check for some of the characteristics assessed. The Reward synthetic populations took longer to reach first flower than the cv. Reward donor population but the days to maturity were not different. Therefore, the period of seed filling was reduced. Thurling (1991) demonstrated the importance of the time between anthesis and maturity by introducing genes for early flowering into a B. napus line and a B. rapa population. Thurling (1991) concluded that the earlier flowering resulted in a longer period of dry matter accumulation and greater seed yields. The later flowering did not adversely affect the performance of the synthetic populations in this study with the exception of the Reward Syn2-4, which was later flowering in Carman. There was a positive correlation between days to flowering and seed yield for the Reward Syn2 population in Carman (r = 0.494). A negative correlation between days to flowering and seed yield was found with the Reward and DSC-3 DH lines (r = 0.391) and DSC-3 Syn1and Syn2 populations in Carman (r = and 0.699). Synthetic cultivars have been developed in B. rapa to exploit heterosis for yield, using two or more inbred lines mixed in equal proportions and grown in isolation (Buzza 1995). Development of synthetics utilizes the partial expression of heterosis during a limited number of generations of seed increase (Poelman and Sleper 1995). The heterosis expressed after one or two generations of outcrossing between the DH lines resulted in the improved performance of the Syn1 and Syn2 populations. One theory of heterosis is based on the assumption that the alleles that contribute to vigour and growth are dominant. The DH lines are completely inbred lines and their performance is influenced by the expression of harmful or deleterious recessive alleles.

7 FRIESEN AND SCARTH UTILIZATION OF DOUBLED HAPLOIDS IN BRASSICA RAPA 81 The DH lines involved in the development of the Syn1 populations were selected based on the phenotype of the individual DH lines and not on combining abilities expressed in progeny of controlled crosses. Syn1and Syn2 yields therefore may not represent the highest potential yield attainable if combining ability was tested before interpollination to identify the optimum combination of DH lines. RAPD analysis was used in this study to identify genetic variation in DH lines of B. rapa. Therefore the RAPD pattern expressed in a population is affected by the number of individual plants in the population and the number of generations of inbreeding occurring (Falconer and Mackay 1996). Using RAPD analysis as an indication of genetic variation leads to the conclusion that the expression of heterogeneity would be greatest with interpollination of 22 DH lines, which showed the highest level of genetic variation. This is expected because of the restricted number of lines used to produce Syn1and Syn2. The Syn1-4 and Syn2-4 Reward synthetic populations were significantly lower in seed yield than the Syn1-8 and Syn1-12 and Syn2-8 and Syn2-12 populations, respectively, and showed less heterogeneity than the set of 8, 12 and 22 DH lines. Interpollination results in eventual fixation or loss of alleles. The smaller Syn1and Syn2 populations reached a fixed level of polymorphism faster because they were closer to this fixation of alleles initially. Starting with larger numbers of individual DH lines should therefore optimize the recovery of agronomic performance of B. rapa. However, the agronomic assessment in 1996 of the Reward Syn1and Syn2 demonstrated that sufficient variability existed in eight DH lines to recover population performance equal to and greater than that of the cv. Reward check. Field evaluation and RAPD analysis were in agreement in the assessment of consistent agronomic performance and genetic variation after the two generations of outcrossing. CONCLUSION The production of synthetic populations from DH lines has the potential of enhancing B. rapa cultivar development. As a breeding tool, improvements can be made in B. rapa populations by selecting DH lines with fixed traits followed by random interpollination to recover performance. This method avoids the shortcomings of traditional breeding systems such as the time required for repeated generations of inbreeding and selection for mass selection, and the difficulty in producing a reliable pollination control system for hybrid production. None of the individual DH lines exceeded the cv. Reward performance but, when crossed to establish a synthetic population, equal or better performance was demonstrated. This study shows that only a small number of DH lines are required to recover population performance. To optimize this method further, the selection of DH lines can be made by determining their combining abilities or genetic variation through RAPD analysis. RAPD analysis provides the ability to determine the amount of variation present in a population, reducing the effects of inbreeding and maximizing the recovery of vigour and agronomic performance of B. rapa. ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr. Allison Ferrie, (Plant Biotechnology Institute NRC, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9) for the DH material provided. Dr. Michael Mayne provided a valuable support for the RAPD analysis. Technical assistance provided by Judith Nugent-Rigby, Paula Parks, Audrey Friesen and Allison Brown was also appreciated. The research was funded by NSERC and the University of Manitoba Graduate Fellowship held by H. Friesen. Buzza, G. C Plant breeding. Pages in D. S. Kimber and D. I. McGregor, eds. Brassica oilseeds: Production and utilization. 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