Intraspecific Variation in Floral Organs and Structure in Brassica Rapa L. Analyzed by Principal Component Analysis

Breeding Science 56 : 189–194 (2006)

Note

Intraspecific Variation in Floral Organs and Structure in Brassica rapa L. analyzed by Principal Component Analysis

Syafaruddin1), Yosuke Yoshioka1), Atsushi Horisaki2), Satoshi Niikura2) and Ryo Ohsawa*1)

1) Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan 2) Tohoku Seed Company, 1625 Nishihara, Himuro, Utsunomiya, Tochigi 321-3232, Japan

Key Words: anther-stigma separation, floral characteristics, F1 seed production.

Many biologists have been interested in the evolution incompatible parental lines are planted and pollination is of floral organs as adaptations for effective animal pollina- performed by wild insects, such as honeybees, bumblebees tion. Much of their interest has focused on the effect of floral and hoverflies. The seed yields of F1 hybrids depend on the morphology on the efficiency of pollen removal and deposi- pollinators’ flights among individuals of different parental tion during pollinator visits (Campbell 1989, Murcia 1990, lines, and on the pollination efficiency of individual flowers Young and Stanton 1990, Nishihiro et al. 2000, Yang et al. during pollinator visits. Therefore, to improve F1 seed pro- 2002, Kudo 2003), and the attractiveness to a pollinator for duction, a detailed investigation of floral organs and struc- which there is a high probability that pollen will be placed ture is required. The objective of the present study was to where fertilizing a conspecific ovule is likely to occur investigate the variability of floral characteristics in a wide (Schemske and Bradshaw 1999). Many investigators have range of B. rapa cultivars. indicated significant effects of the characteristics of floral We used four inbred parental lines of some F1 hybrids, organs and structures, especially the spatial relationship be- which become genetically homogeneous through five to tween stigma and anthers, on the flowers’ pollination effi- seven generations of self-crossing, and 30 cultivars that rep- ciencies, in both self-fertilizing and outcrossing species resented high genetic variability across eight varietal groups (Holtsford 1992, Conner et al. 1995, Karron et al. 1997, of B. rapa, in order to investigate intraspecific variation in Motten and Stone 2000, Elle and Hare 2002). In other stud- floral characteristics (Table 1). The plants were grown in a ies, it was reported that the floral size and number play an glasshouse at the Agriculture and Forestry Research Center, important role in attracting pollinating insects (Andersson University of Tsukuba (Tsukuba, Japan), from November 1996, Conner and Rush 1996), such as bees that will use the 2002 until May 2003. We chose three plants per line or cul- flower shape as a visual cue in foraging (Gegear and Laverty tivar, and sampled three flowers from the main stem per 2001, Giurfa and Lehrer 2001). plant (306 flowers in total). The flowers that had newly From a plant breeder’s point of view, floral morpholo- opened on each sampling day were used. gy should be considered to have the potential to increase or After removal of the four petals, each flower was decrease seed production in both self-fertilizing and out- placed sideways on a stage, and its image was projected onto crossing crops. In fact, in many investigations of floral or- a computer display via a Keyence digital microscope (VH- gans, it was showed that the floral morphology influenced 5000, Keyence Co., Osaka, Japan). We measured eight char- the pollination efficiency (Levin et al. 1994, Uga et al. acteristics of the floral organs — long stamen length (LSL), 2003a, 2003b, Yoshioka et al. 2005), and that a lower degree short stamen length (SSL), long anther length (LAL), short of stigma exsertion increased the rate of self-fertilization anther length (SAL), ovary length (OL), stigma height (SH), (Namai et al. 1992, Yashiro et al. 2001, Syafaruddin et al. stigma width (SW) and style length (SL) (Fig. 1) — using 2002, Kobayashi et al. 2004). In crops that outcross by in- image analysis software (VH-Analyzer, Keyence Co.). sect pollination, it remains to be determined what kind of To summarize the information about the eight charac- floral characteristics attract pollinating insects and, in partic- teristics of the floral organs, we performed a principal com- ular, what kind of floral morphology is advantageous to pol- ponent analysis (PCA) based on a variance–covariance ma- lination efficiency. trix. The scores of the principal components (PC) were used In seed-production fields of Brassica rapa L., self- in the analysis to identify the characteristics responsible for the majority of the variation in the floral structure. To deter- Communicated by Y. Takahata mine the effect of each PC, we reconstructed the floral struc- Received June 17, 2005. Accepted January 11, 2006. ture from the eight variables, reverse-calculated by letting *Corresponding author (e-mail: [email protected]) the score for a certain PC be equal to the mean ± 2 standard 190 Syafaruddin, Yoshioka, Horisaki, Niikura and Ohsawa

Table 1. List of lines and cultivars across eight varietal groups used in the present study and mean values of eight floral characteristics. SE and CV denote the standard error of the mean and coefficient of variation, respectively Line no. Cultivar name Varietal group LSL SSL LAL SAL OL SH SW SL LVC-01 Chirimen-hakusai pekinensis 0.72 0.51 0.20 0.21 0.46 0.06 0.11 0.16 LVC-02 Nagasaki-hakusai pekinensis 0.78 0.58 0.20 0.20 0.58 0.07 0.10 0.15 LVC-03 Hankekyuu-santosai pekinensis 0.89 0.66 0.21 0.20 0.44 0.06 0.10 0.15 LVC-04 Maruba-santosai pekinensis 0.78 0.60 0.21 0.19 0.53 0.08 0.11 0.26 LVC-05 Bansei-osakashirona pekinensis 0.79 0.58 0.20 0.18 0.44 0.08 0.11 0.19 LVC-06 Banseimana pekinensis 0.78 0.59 0.19 0.17 0.46 0.08 0.11 0.26 LVC-07 Hiroshimana pekinensis 0.82 0.61 0.21 0.20 0.47 0.06 0.11 0.20 LVC-08 Nabana campestris 0.85 0.61 0.21 0.19 0.53 0.08 0.11 0.21 LVC-09 Seppakutaina chinensis 0.82 0.62 0.19 0.18 0.51 0.07 0.12 0.21 LVC-10 Nagaokana chinensis 0.77 0.57 0.21 0.24 0.47 0.08 0.12 0.25 LVC-11 Shigatsu-shirona chinensis 0.75 0.55 0.21 0.18 0.42 0.07 0.09 0.16 LVC-12 Shinobu-fuyuna rapifera 0.82 0.61 0.21 0.19 0.57 0.08 0.09 0.26 LVC-13 Niigata-tona campestris 0.90 0.61 0.22 0.22 0.59 0.09 0.13 0.27 LVC-14 Sendai-yukina chinensis 0.79 0.60 0.21 0.20 0.51 0.07 0.10 0.21 LVC-15 Kukitachina campestris 0.99 0.76 0.28 0.26 0.60 0.07 0.10 0.26 LVC-16 Katsuyama-mizuna campestris 1.25 0.96 0.32 0.32 0.78 0.07 0.11 0.26 LVC-17 Kumamoto-kyona rapifera 0.90 0.65 0.23 0.22 0.52 0.08 0.11 0.27 LVC-18 Osakina rapifera 0.80 0.61 0.25 0.24 0.43 0.07 0.12 0.30 LVC-19 Wase-aburana campestris 0.88 0.66 0.24 0.23 0.53 0.09 0.13 0.29 LVC-20 Shinshu-yukina campestris 0.93 0.67 0.28 0.27 0.58 0.07 0.11 0.29 LVC-21 Shizuoka-kyona campestris 0.84 0.63 0.25 0.22 0.52 0.07 0.09 0.18 LVC-22 Tatsai narinosa 0.79 0.56 0.17 0.17 0.42 0.08 0.11 0.12 LVC-23 Chugoku-saishin parachinensis 0.90 0.66 0.19 0.19 0.62 0.08 0.13 0.19 LVC-24 Kotsaitai chinensis 0.90 0.68 0.23 0.22 0.58 0.08 0.12 0.21 LVC-25 Pakchoi chinensis 0.90 0.76 0.23 0.23 0.58 0.09 0.13 0.22 LVC-26 Sensuji-kyomizuna japonica 0.89 0.61 0.23 0.22 0.67 0.07 0.09 0.22 LVC-27 Mibuna japonica 0.84 0.63 0.28 0.29 0.57 0.07 0.10 0.23 LVC-28 Nozawana rapa 0.87 0.63 0.27 0.27 0.53 0.07 0.09 0.21 LVC-29 Gensuke-kabuna rapa 0.85 0.62 0.26 0.25 0.45 0.06 0.10 0.23 LVC-35 Kamokabu rapa 0.78 0.57 0.22 0.21 0.46 0.06 0.11 0.17 HaO-017 pekinensis 0.67 0.41 0.20 0.20 0.45 0.07 0.12 0.18 HaO-251 pekinensis 0.72 0.48 0.21 0.21 0.46 0.08 0.13 0.21 HaO-263 pekinensis 0.84 0.60 0.24 0.23 0.42 0.07 0.11 0.21 HaO-314 pekinensis 0.85 0.59 0.23 0.22 0.44 0.08 0.10 0.18 Total Mean 0.84 0.62 0.23 0.22 0.52 0.07 0.11 0.22 SE (10−1) 0.17 0.15 0.06 0.06 0.14 0.01 0.02 0.08 CV 0.12 0.14 0.14 0.16 0.16 0.12 0.11 0.21 LSL: long stamen length (cm), SSL: short stamen length (cm), LAL: long anther length (cm), SAL: short anther length (cm), OL: ovary length (cm), SH: stigma height (cm), SW: stigma width (cm), SL: style length (cm). deviations (SD) and the scores for the remaining PCs be yses, we used PA and ASP as petal characteristics. zero. We also calculated the ratio (referred to as SP hereafter) To examine the differences in the floral characteristics of long stamen length (LSL) to pistil height (sum of OL + SL and PCs, we performed nested ANOVAs among the 34 lines + SH) for each flower. Stigma area (SA) was calculated as and cultivars, since the samples displayed a hierarchical the product of SH by SW for each flower. In addition, we structure with three sources (i.e. variety, plant and flower). photographed each petal with a standard marker (100 mm2) Pearson’s product-moment correlation analyses were also per- using a digital camera (Coolpix 950, Nikon, Tokyo), and formed in order to investigate the relationships between PA measured the width, length and area (PA) of each petal by and each PC. In the correlation analyses, we used the mean image analysis. In a pilot study, we investigated in detail pet- values for each line or cultivar. All the statistical analyses al shape variations in B. rapa using elliptic Fourier descrip- were performed with the JMP 5.0 software (SAS Institute tors and PCA (Iwata and Ukai 2002), and concluded that Inc. 2002). most of the variation could be explained by the aspect ratio There were large differences in the petal characteristics of petals. Therefore, we directly calculated the ratio (re- among the lines and cultivars (Table 2). For example, the ferred to as ASP hereafter) of petal length to width as an cultivars LVC-15 and LVC-16 showed the largest PA, equal index of petal shape in the present study. In subsequent anal- to about 1.8 times that of the smallest, LVC-24, while LVC- Variation in floral organs and structure in Brassica rapa L. 191

Table 2. Mean values of two petal characteristics and stigma area. SE and CV denote the standard error of the mean and coefficient of variation, respectively Line no. PA ASP SP SA LVC-01 6.13 1.32 0.94 0.63 LVC-02 7.06 1.64 1.04 0.77 LVC-03 6.42 1.46 0.73 0.62 LVC-04 7.25 1.77 1.13 0.86 LVC-05 5.91 1.37 0.89 0.91 LVC-06 6.68 1.43 1.03 0.86 LVC-07 6.61 1.51 0.89 0.67 LVC-08 7.15 1.58 0.97 0.85 Fig. 1. Schematic illustration of the eight floral characteristics mea- LVC-09 7.27 1.79 0.96 0.83 sured in the present study. 1, long stamen length (LSL); 2, LVC-10 7.25 1.56 1.04 0.96 short stamen length; 3, long anther length (LAL); 4, short an- LVC-11 5.90 1.75 0.86 0.69 ther length (SAL); 5, ovary length (OL); 6, stigma height (SH); LVC-12 7.47 1.89 1.13 0.73 7, stigma width (SW); 8, style length (SL). Leftmost curved LVC-13 7.18 1.65 1.06 1.13 line represents the corolla tube (part of a petal). LVC-14 6.16 1.44 1.00 0.68 LVC-15 8.16 1.91 0.94 0.72 LVC-16 8.16 1.81 0.90 0.83 26 exhibited the highest ASP scores, indicating that in this LVC-17 7.37 1.67 0.97 0.79 cultivar petals were extremely elongated compared with LVC-18 6.21 1.48 1.02 0.86 those of the other cultivars. The differences in both petal LVC-19 6.91 1.52 1.03 1.12 characteristics among lines and cultivars were significant at LVC-20 7.23 1.74 1.01 0.86 the 1% level (Table 3). The proportions of the variance LVC-21 6.91 1.80 0.91 0.61 LVC-22 5.68 1.39 0.79 0.83 component associated with the differences among lines and LVC-23 4.72 1.95 0.98 1.02 cultivars were 57.03% for PA and 82.50% for ASP. The LVC-24 4.54 1.73 0.97 0.97 proportion of the variance component for ASP at the LVC-25 5.40 1.38 0.99 1.19 whole-plant level was higher than that at the flower level. By LVC-26 4.59 2.90 1.08 0.63 contrast, the proportion of PA at the flower level was higher LVC-27 5.39 2.01 1.03 0.66 than that at the plant level (Table 3). LVC-28 6.50 1.57 0.93 0.62 The eight floral characteristics could be divided into LVC-29 6.43 1.49 0.88 0.59 two groups (stamen vs. pistil). One group consisted of the LVC-35 6.09 1.42 0.88 0.66 stamen (LSL, SSL) and anther (LAL, SAL), while the other HaO-017 5.70 1.15 1.05 0.90 consisted of the ovary (OL), stigma (SH, SW) and style HaO-251 5.87 1.17 1.05 1.00 (SL). The most remarkable variations were apparent in the HaO-263 5.72 1.41 0.84 0.86 HaO-314 5.58 1.38 0.83 0.79 female organs, especially SL. That is, the scores of the co- Total Mean 6.40 1.62 0.96 0.81 efficient of variation (CV) for the female organs were rela- −1 tively higher than those for the male organs (Table 1 and SE (10 ) 1.59 0.54 0.16 0.27 CV 0.14 0.19 0.10 0.20 Table 2). Nested ANOVAs indicated that all the floral char- 2 acteristics differed significantly at the 1% level among the PA: petal area (mm ), ASP: ratio of petal length to width, SP: ratio of long stamen length to pistil height, SA: stigma area (mm2). lines and cultivars (Table 3). However, compared with the variance components at the line and cultivar levels, the proportions of the characteristics of the pistil group, especially associated with the relative position of the stamen against SL, and of the two stigma characteristics (SH and SW) were the pistil. The relative locations of the stamen and pistil were typically smaller than those of the male organs. In addition, mainly due to the ovary and anther length in the second PC, there were significant differences among the eight varietal and to the style length and stigma size in the third PC. Al- groups in two petal characteristics (PA and ASP), anther though the eigenvalue of the fourth PC was relatively low, length (LAL and SAL) and two female organs (OL and SL). this PC seemed to express the size of anthers rather than oth- The Eigenvalues and contributions to the total variance er floral characteristics. of the first four PCs of the eight variables are shown in The nested ANOVAs indicated the presence of signifi- Figure 2. The contribution of the first PC was very high and cantly large differences in the PCs among the lines and cul- accounted for as much as 68% of the total variation. The cu- tivars (Table 3). The scatterplots of these four PC scores mulative contribution from the first to fourth PCs was 94%. clearly indicated the presence of wide variations among the Figure 2 also shows the effect of each PC on the floral struc- lines and cultivars in B. rapa (Fig. 3). In this figure, we di- ture. The first PC provided a good measure of the overall vided the four PCs into two groups: size factors (PC1 and size of the floral organs. The second and third PCs were both PC4) and relative spatial positioning between anther and 192 Syafaruddin, Yoshioka, Horisaki, Niikura and Ohsawa

Table 3. Proportions (%) of variance components in the floral organs and principal components of eight floral characteristics from each of the four hierarchical sources, as estimated by nested ANOVA Petal Source df SP SA PA ASP Line/Cultivar 33 57.03 ** 82.50 ** 32.78 ** 20.95 ** Plant 68 11.08 ** 13.01 ** 32.56 ** 14.73 ** Flower 204 31.88 4.41 34.66 34.66 Characteristics related to male organ Source df LSL SSL LAL SAL Line/Cultivar 33 65.21 ** 61.36 ** 54.77 ** 46.07 ** Plant 68 19.87 ** 23.41 ** 20.46 ** 5.58 ns Flower 204 14.91 15.23 24.77 48.35 Characteristics related to female organ Source df OL SL SH SW Line/Cultivar 33 52.57 ** 35.69 ** 14.86 ** 19.58 ** Plant 68 20.28 ** 28.51 ** 12.90 * 18.16 ** Flower 204 27.15 35.80 72.24 62.26 Source df PC1 PC2 PC3 PC4 Line/Cultivar 33 68.35 ** 30.38 ** 35.89 ** 34.51 ** Plant 68 19.69 ** 27.44 ** 24.07 ** 22.62 ** Flower 204 11.96 42.18 40.03 42.87 ** P < 0.01; * P < 0.05; ns, non-significant. stigma (PC2 and PC3). For example, LVC-8 showed the However, environmental factors were also important, as ob- smallest mean value in the first PC, indicating that in this served at both plant and flower levels. In several studies a cultivar the floral organs were typically large compared with significant environmental effect on the flower morphology those of the other cultivars (Fig. 3a). Both LVC-35 and (Williams and Conner 2001, Yoshioka et al. 2004), and thus LVC-20 showed average values in the first PC. However, on the pollination success (Holtsford 1992, Elle and Hare the fourth PC score of LVC-35 was the highest among the 2002), was revealed. The wide variation in the morphology lines and cultivars, and that of LVC-20 was the lowest, indi- of the B. rapa flowers which is due to both a high genetic cating that the sizes of the anthers were considerably differ- variability across the eight varietal groups and the responses ent from one another. LVC-10 and LVC-12 commonly had to environmental factors, may influence pollination success longer pistils than stamens. However, in the former, the style in this species. was relatively large and the ovary was small. By contrast, in Correlation analyses did not indicate any relationships the latter, the style was relatively small and the ovary was between the size of the floral display and the structure asso- large (Fig. 2 and Fig. 3b). There was a significant difference ciated with PCs. That is, a large flower size did not necessar- among the eight varietal groups in PC1, but not in the re- ily correspond to a large floral structure. In fact, LVC-9 had maining PCs. In addition, scatterplots could not clearly re- a large petal (PA), not a large overall floral structure (PC1). veal the differences among the eight varietal groups (Fig. 3). In LVC-27, the anther was long but the petal was small. Correlation analyses indicated that PA was not correlated Presently, it remains to be determined whether a large floral with PC1 (r = −0.33, n = 34, P = 0.06), PC2 (r = 0.14, n = 34, structure is advantageous to pollination success, although a P = 0.42), PC3 (r = 0.28, n = 34, P = 0.10) or PC4 (r = 0.06, large flower size is considered to be an advantage in attract- n = 34, P = 0.75). ing pollinating insects (Conner and Rush 1996). Among the It was interesting to note that the B. rapa flowers exhib- anemophilous crops, cultivated rice, as a self-pollinated crop, ited such a wide variation in their floral organs and structure. generally displays a lower stigma exsertion and smaller Although the nested ANOVA test was not designed to esti- pistils and stamens than its wild relatives, which show partial mate heritability, the significant effects of line or cultivar outcrossing (Virmani and Athwal 1973). Although different level indicated that a genetic component was involved in the factors affect the pollination success of anemophilous and floral characteristics examined in the present study, as in entomophilous plants, the large variation in the size of the other plant species such as other Cruciferae species (Conner floral structure in B. rapa offers the potential to increase or et al. 1996, Kobayashi et al. 2004), the genus Lycopersicon decrease pollination success. (Levin et al. 1994, Georgiady et al. 2002, Bernacchi and A common floral difference associated with the mating Tanksley 1997, Chen and Tanksley 2004) and the genus system is the degree to which the stigma is either exserted Oryza (Virmani and Athwal 1973, 1974, Uga et al. 2003b). above the anthers (promoting outcrossing) or recessed below Variation in floral organs and structure in Brassica rapa L. 193

Fig. 3. Scatterplot diagrams for (a) PC1 vs. PC4 and (b) PC2 vs. PC3 for the 34 lines and cultivars. Capital H plus numbers corresponds to the line number of the HaO series, and numbers to Fig. 2. Effect of each principal component on the floral structure. PC1 the line numbers of the LVC series listed in Table 1. Symbols to PC4 corresponds to the first to fourth principal components. denote the mean values. The letters or numbers in (b) represent From the right, each column shows the case where the score the lines or cultivars in which SP > 1.0. takes +2 SD, mean, and −2 SD. 1), Eigenvalue of each principal component. 2), proportion relative to the total variance of ternal factors (Horisaki et al. 2003). To improve the efficien- the eight floral characteristics. cy of F1 seed production, including high yield and purity, it is necessary to increase the pollinators’ flights between differ- the anthers (promoting self-pollination). Self-pollinating ent parental lines, and to improve the pollination efficiency forms of B. juncea (Yashiro et al. 2001) and Raphanus of individual flowers so as to prevent self-fertilization and sativus (Namai et al. 1992, Kobayashi et al. 2004) typically enforce outcrossing (Namai et al. 1992). In this context, de- deposit more pollen on their stigma than the outcrossing termining the sources of variation in floral morphology is forms. Our results demonstrated the presence of a wide and crucial to gain a full understanding of the mechanisms under- continuous variation in anther-stigma separation, from out- lying the improvement of F1 seed production. Our approach crossing to intermediate to self-pollinating forms. Therefore, and the results reported here would be a basis for further this variation must be taken into account when parental lines studies and breeding programs. of F1 hybrids are established. In addition, outcrossing forms of B. rapa were generated by expansion of the ovary or style. Acknowledgments For example, the flowers of both LVC-12 and LVC-10 were outcrossing forms. However, the former was on outcrossing We thank Dr. H. Namai, University of Tsukuba, for his form mainly due to the elongation of the ovary, and the latter guidance in the planning of this study and for the critical dis- to the elongation of the style (Fig. 2 and Fig. 3). Further stud- cussions. We also thank Dr. Y. Fujita and Dr. S. Matsuura, ies should be carried out to determine the impact of this phe- Tohoku Seed Company, for providing the seeds of the nomenon on the pollination efficiency and seed-setting abil- Brassica lines and cultivars used in this study. This work ity. was supported by a grant for a Research Project for Utilizing Although self-incompatible parental lines are used to pro- Advanced Technology in Agriculture, Forestry and Fisheries duce F1 seeds in most B. rapa cultivars, self-incompatibility from the Ministry of Agriculture, Forestry and Fisheries of is not complete. The self-incompatibility is due to the con- Japan, and by a Grant-in-Aid for Exploratory Research from trol by a series of multiple alleles acting sporophytically the Japan Society for the Promotion of Science. (reviewed by Watanabe et al. 2003). It is assumed that the incomplete self-incompatibility is affected by internal and ex- 194 Syafaruddin, Yoshioka, Horisaki, Niikura and Ohsawa

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