TY - JOUR
AU - Squire, G.R.
AB - Abstract An experiment was conducted to test the hypothesis that phenotypes differing in germination rate and the presence or absence of secondary dormancy at low temperature were not genetically different. Seed of oilseed rape was germinated at 4, 10 and 19 °C, where selections were made in the percentile ranges 1–10 (early), 45–55 (intermediate) and 91–100 (late). Secondary dormancy occurred only in the late selections at the two lower temperatures. Thermal weighting of curves of cumulative germination on time gave circumstantial evidence that early percentiles were similar at all three temperatures and that seeds with secondary dormancy came largely from later percentiles above the 50th. To test for genetic differentiation between phenotypes, 5′‐anchored simple sequence repeat primers were used to generate DNA marker profiles of seedlings raised from seed from each category. Principal coordinate analysis, and more detailed comparisons using the most discriminating markers, confirmed that the early germinators at the three temperatures were not associated with different banding profiles, but seeds entering secondary dormancy, particularly at 10 °C, were genetically distinct from germinators at the same temperature. Secondary dormant seeds at low temperature appear to originate mainly from the late germinating seed at higher temperature. Effects of temperature history and the requirement for alternating temperatures to break secondary dormancy were quantified. The results confirm the existence of genetically discrete sub‐populations differing in ecologically significant traits. Oilseed rape, Brassica napus, germination, dormancy, temperature, DNA markers. Introduction An individual's position in the time‐profile of germination determines the combination of factors it will experience as a seedling. A position in the profile is governed by two types of trait: those that determine whether germination will occur in the present conditions, and those that determine the rate of germination once germination starts. Both types of trait need to be included in any quantitative description of a seed population, but the problem is that they are not independent, in that the estimation of rate, defined as 1/time to germination for a given percentile (Garcia Huidobro et al., 1982), will be influenced by non‐germination in the population. Specifically, if certain conditions cause a seed not to germinate, then the apparent rankings and distribution of rate among the remaining seeds that do germinate will change. The arguments have a particular bearing on the way a germination profile alters in response to an abiotic stress such as low temperature. Defining the set of functions linking rate to temperature for different percentiles has become a practical means of characterizing a population of seeds and predicting germination from environmental temperature (Covell et al., 1986; Ellis et al., 1986; Washitani, 1987). However, a factor such as a low sub‐optimal temperature not only decreases the rate of the germinating seeds but often increases the percentage of non‐germinators. The main question is how these non‐germinators are distributed in the rate‐profile at temperatures at which all or most seeds germinate. For instance, are late germinators at optimal temperature the non‐germinators at low temperature? The present study arose from an interest in quantifying traits in cultivars of oilseed rape, that would increase the likelihood of their going feral following seed drop at harvest. The factor most likely responsible for non‐germination in this context is a secondary dormancy induced by low temperature, which leads to over‐wintering and thereby to extension of the germination profile (Pekrun et al., 1998a, b). Differences in temperature‐ related traits have been identified between B. napus cultivars (Kondra et al., 1983; Wilson et al., 1992) and between seed lots of the same cultivar (Acharya et al., 1983; Nykiforuk and Johnson‐Flanagan, 1994). Earlier work in this laboratory aimed to quantify the variation in traits by standard rate–temperature analysis (Marshall and Squire, 1996; Squire et al., 1997). Subsequent work in the field (Squire, 1999) showed that only the early germinators (low percentiles) displayed consistent rate–temperature responses over a range of temperature. In particular, the early parts of cumulative emergence curves at a range of temperatures were superposed when expressed in thermally weighted time, while the late parts diverged (Squire, 1999). This was circumstantial evidence that the late germinators at optimal temperature were indeed non‐germinators at low temperature, but the very existence of non‐germination at low temperature meant the estimates of the rate–temperature parameters were subject to large extrapolation errors. More direct evidence is needed to show whether the non‐germinating fraction at low temperature arises from any particular part of the population at optimal temperature. In other species, the evidence for the genetic basis of innate dormancy (Garbutt and Witcombe, 1986; Naylor, 1983; Ramsay, 1997) is much stronger than that for rate of germination, but there is still very little information on whether non‐germinators arise from a particular part of the rate profile. The association between non‐germination and rate of germination is therefore explored here in more detail, and in a shorter time scale than required for a full‐scale genetic analysis. No markers specific for germination traits were available. Instead, 5′‐anchored simple sequence repeat DNA primers were used to reveal broad‐scale genetic diversity, which was then compared with germination traits through principal coordinate analysis. Such primers were already known to distinguish numerous cultivars and to reveal within‐cultivar variation in oilseed rape (Charters et al., 1996). Since the range of germination traits and temperatures are potentially very large, the study focused on specific, testable hypotheses cast around the established rate–temperature analysis in oilseed rape. Hypotheses The representation in Fig. 1 shows three percentiles or sub‐sets of a population—early, intermediate and late —as defined by a typical rate–temperature analysis. Percentiles between and outside these are not shown, simply for clarity. Temperature (T) ranges between the lower limit, or base temperature (Tb) where germination rate tends to zero and the optimum temperature (Topt) where rate is fastest. For the purpose of this representation, it does not matter whether the relation between germination rate (reciprocal of time to germination, 1/t) and T is linear or curved. Values above the optimum are not shown, though a similar approach and reasoning can be taken there. For illustration, reference is made to three temperatures: T1, just above the lower limit; T2, midway between the lower and upper limits; and T3, just below the optimum. Solid parts of the curves represent the response for germinating seeds. The dashed parts are extrapolations from the solid parts and indicate combinations of 1/t and T where the non‐germination trait is expressed. The intersections of temperatures T1, T2 and T3 with the three curves represent specific phenotypes characterized by the two‐dimensional space of the graph. So at the near‐optimal temperature T3, E3, I3 and L3 represent early, intermediate and late germinating phenotypes. At T2, E2 and I2 represent early and intermediate germinating phenotypes, while L2, lying on the dashed part of the lower curve, represents a non‐germinating phenotype. At T1, E1 represents an early germinating phenotype, while I1 and L1 represent non‐germinating phenotypes on the middle and lower curves. The assumption in previous analyses is that the phenotype L3 at T3 becomes the phenotypes L2 and L1 at lower temperatures, i.e. it is the same genotype at each of the locations L1…3 whose phenotypic expression is modified by temperature. In reality, the locations of the non‐germinating phenotypes (indicated by dash lines) do not lie in the plane of the graph. A third axis is required which characterizes non‐germination and it is then not clear whether the genotype remains the same in these intermediate and late non‐germinating percentiles as temperature is lowered. The questions regarding the genotypic basis of the rate–temperature response therefore refer to the extent to which an individual's location within the phenotypic‐space bounded by E3, L3, L1, and E1 (shaded region, Fig. 1) has a genetic basis. The null hypothesis is that none of the phenotypes is genetically different. The following statements serve to direct the analysis. (1) Phenotypes along a rate–temperature response curve for a given percentile are not genetically different, so that (a) early germinators, e.g. E1, E2 and E3, are not genetically different at different temperatures; and (b) late germinators at higher temperatures (e.g. L3) are not genetically different from non‐germinators at low temperature (e.g. L1, L2); (2) Phenotypes at the same temperature, along a vertical dashed line, are not genetically different, in particular, non‐germinators at any temperature are not different from germinators at the same temperature. The null hypothesis does not hold if any of the above statements are shown to be false. In order to test the approach and methodology, the association is now investigated between the position of individuals in phenotypic‐space (defined by germination rate, non‐germination and T) and 5′‐anchored simple sequence repeat polymorphisms present in those individuals. The study was primarily a test of concept and carried out using a variety of oilseed rape known to show variation in marker profiles between individual plants. Fig. 1. View largeDownload slide Diagrammatic representation of the variation in the rate of germination with temperature for three sub‐classes of seed (early, intermediate and late germinators) within a seed lot. Dashed segments of the line indicate where viable seeds do not germinate at low temperatures. Tb and Topt are temperatures at which the rate of germination is zero and maximal respectively. E1…3, I1…3 and L1…3 refer to the early, intermediate and late germination selections of seed made at temperatures T1…3, respectively. Fig. 1. View largeDownload slide Diagrammatic representation of the variation in the rate of germination with temperature for three sub‐classes of seed (early, intermediate and late germinators) within a seed lot. Dashed segments of the line indicate where viable seeds do not germinate at low temperatures. Tb and Topt are temperatures at which the rate of germination is zero and maximal respectively. E1…3, I1…3 and L1…3 refer to the early, intermediate and late germination selections of seed made at temperatures T1…3, respectively. Materials and methods Germination and seed selection Seeds were germinated on a temperature gradient plate manufactured by Grant Instruments (Cambridge) Ltd. Measurements of temperature on the plate surface were made using 16 fine wire (0.127 mm diameter) type ‘K’ thermocouples arranged in a regular 4×4 grid (12 at the edge of the plate and the remaining thermocouples in the centre four positions of the grid). The thermocouples were connected to a Campbell 21x datalogger which measured temperatures every minute and recorded their average, maximum and minimum temperatures every hour. A constant temperature gradient (2–25 °C) was applied in one direction across the 0.8 m square plate. Temperatures at locations between thermocouple positions were estimated by linear interpolation. Previous tests had shown that germination was the same whether seeds were kept in the dark or exposed to normal fluorescent room lighting in the laboratory. In this instance, they were exposed to the light. The seed used was from the same lot of the ‘winter’ oilseed rape variety, Martina, that was examined in earlier studies (Charters et al., 1996; Marshall and Squire, 1996; Squire et al., 1997; DETR, 1999). It was bred by Semundo (Germany) and supplied by West Crop, UK. It is of the high erucic acid type, and in terms of DNA polymorphisms, among the more variable of the varieties examined by Charters et al. (Charters et al., 1996). Groups of 100 seeds were placed on the plate at each of three nominal temperatures: 4 °C, 10 °C and 19 °C in 1996. Subsequent processing of the temperature data showed that the average temperatures were 3.6, 9.8 and 18.8 °C, respectively, ±0.1 K. Early, intermediate and late germinators were selected in the following way. Out of each 100 seeds, the following were removed in order when their radicle was 1 mm long: 1–10, 46–55 and 91–100 (where 1 is the first and 100 the last to germinate) referred to hereafter as the early, intermediate and late percentiles. Other seeds were discarded. Previous experience (Marshall and Squire, 1996; Squire et al., 1997) suggested most seeds at 19 °C would germinate at constant temperature, while only a fraction of seeds would at the two cooler temperatures, the non‐germinators requiring increasing or alternating temperature before they would germinate. Since these temperature changes were to be applied to the two cooler seed lots simultaneously, the sowing date of the 10 °C lot (6 May 1996) was 3 weeks later than that of the 4 °C lot (16 April 1996) to ensure that both lots had reached the initial plateau in the germination‐time curve at about the same time. Seeds at 19 °C were sown on 17 April. After the initial flush of germination had ceased, the seeds at the temperatures of 4 °C and 10 °C were moved on 17 June 1996 to higher temperatures of 10 °C and 16 °C, respectively. Further increases of temperature up to 21 °C and 25 °C were made at intervals up to 28 June, then on 3 July seeds that had still not germinated were subjected to alternating temperatures, which induced all of them to germinate. Seeds were always removed as soon as they germinated and the seeds from the selected percentiles were transferred immediately to small pots and grown on in a glasshouse. After about 4 weeks growth, leaf samples of the uppermost youngest leaves were removed, placed on ice, then stored at a temperature of −80 °C. In total, 90 plants, comprising 10 at each of the three temperatures and the three percentile ranges were reared for DNA analysis. DNA extraction The DNA extraction procedure described previously (Hu and Quiros, 1991) with additional steps after the RNase phase (Charters et al., 1996) was used: 25 μl of sodium acetate (3 M) and 500 μl of ethanol were added prior to chilling at −20 °C for 20 min. The solution was centrifuged and resuspended in TE buffer. 500 μl of chloroform: IAA (24:1, v:v) was added and the solution shaken for 15 min. The aqueous layer was transferred to a clean tube, 600 μl of iso‐propanol added, the solution centrifuged and the pellet resuspended in TE buffer. DNA concentrations and purity were determined using an Ultrospec III spectro‐photometer (Pharmacia Biotech) at wavelengths of 260 and 280 nm, respectively. PCR amplification The two primers used for DNA amplification, obtained from the Scottish Crop Research Institute chemistry department, were numbers 1420 (BDB‐CACACACACACACA) and 1425 (BDV‐CAGCAGCAGCAGCAG), previously used in studies (Charters et al., 1996). Both primers consisted of a repeat sequence preceded by a 5′ anchor that has a three variable base position. The anchors were designated the code letters accordingly B=C, G or T; D=A, G or T; V=A, C or G. PCR was conducted (Charters et al., 1996). Electrophoresis The PCR amplification products were separated using a Multiphor II flatbed system, cooled to 10 °C, and precast polyacrylamide gels (all Pharmacia Biotech) (according to the method of Charters et al., 1996) using the following three stage programme (i) 24 min at 200 V max, 20 mA max, 10 W max; (ii) 60 min at 380 V max, 30 mA max, 20 W max; (iii) 60 min at 450 V max, 30 mA max, 20 W max. Silver staining Visual detection of bands was obtained using the following silver staining procedure: (a) 30 min fixing in 250 ml of acetic acid; (b) three 2 min washings in 250 ml distilled water; (c) 20 min silvering in 250 ml of 1% (w/v) AgNO3, with 250 μl of 37% (w/v) formaldehyde; (d) 30 s washing in 250 ml distilled water; (e) 5 min developing in 250 ml of 2% (w/v) NaCO2 with 250 μl of 2% (w/v) Na2SO3 and 250 μl of 37% (w/v) formaldehyde; (f) 10 min stop/desilver in 250 ml of 0.5% (w/v) EDTA‐Na2; (g) 20 min gel impregnation in 250 ml of 9% (w/v) glycerol. Gel scoring and similarity matrix Band positions from individual plant DNA profiles were visually identified and recorded as present or absent. The total number of polymorphic bands identified using primer numbers 1420 and 1425 were 18 and 17, respectively. Data were analysed using the Genstat V software package which generated a similarity matrix, bandmap, and principal coordinate analysis (PCO) (Nei and Li, 1979). Results The progression of germination with time at the three selection temperatures is shown in Fig. 2. At 4 °C (Fig. 2A), the early fraction (1–10) germinated by day 9. Only half the intermediate fraction (46–50) germinated by day 32. One more seed germinated in the following month, after which time the non‐germinated seeds were moved to a warmer temperature of 10 °C (day 60, identified by transition a in Fig. 2A) and within 4 d the remaining part of the intermediate fraction (52–55) had germinated. By day 69, germination had again slowed, reaching a maximum of 63%. The remaining seeds were moved to still warmer temperatures of first 16 °C (b) and then 3 d later to 21 °C (c). Germination resumed, reaching a maximum of 75%. It was only when alternating temperatures (12/12 h at 23/10 °C) were applied at day 77 (transition x) that full germination was eventually achieved and the late fraction (91–100) of seed collected. At 10 °C (Fig. 2B), a greater proportion of seeds germinated more quickly. The early fraction germinated by day 3 and the intermediate fraction within the next 2 d. A maximum of 80% of the seeds germinated by day 15. Moving the seeds first to 16 °C, then 21 °C and finally 25 °C had no effect on the remaining 20% of non‐germinated seed (transitions b, c, d, Fig. 1B). Only when the seeds were subject to alternating temperatures (12/12 h at 25/6 °C) was germination resumed, taking less than 2 d for all the remaining seeds to germinate (transition y). At the warmest selection temperature, initially 19 °C, 95% of the seeds germinated by day 5 (Fig. 2C). The remaining 5% were moved to a constant 25 °C (not shown) at this time, where they germinated after 4 d. They did not require transferring to alternating temperature regimes to germinate. Three categories of seed were therefore identified, reflecting the conditions under which the various seed fractions were selected (Table 1). First are seeds that germinated at the initial temperatures: 1–10% at all temperatures, 46–55% at 10 °C and 19 °C, and 91–95% at 19 °C; these seeds were not induced into secondary dormancy and are classed as germinators. Second are seeds that did not germinate at the initial temperature, but did when temperature was ramped to a warmer, but still constant, value than the initial temperature: the 52–55% selection at 4 °C and the 96–100% selection at 19 °C. Third are the remaining fractions that required alternating temperatures for germination: the 91–100% selections at 4 °C and 10 °C. The second and third classes are both non‐germinators under the initial conditions, but differed in what was required to make them germinate. In the second class, moreover, not all the seeds that would have germinated at a warmer temperature, appeared to germinate when the temperature was first cool, then raised to the warmer temperature, even though adequate time was allowed after a transition. For example, the plateau after transition a (to 10 °C) in Fig. 2A is lower than the first plateau at the same temperature (10 °C) in Fig. 2B. Thermally weighted time Inspection of the relation between 1/time and temperature (T) for selected percentiles (not shown) was consistent with the exponential response found previously (Squire et al., 1997)   1 where a and b are parameters. It was found that the value of b, which defines the degree of non‐linearity, was independent of percentile for a given seed lot and that the value of a, a simple scalar, decreased systematically with increasing percentile (Squire et al., 1997). At constant temperature it is possible to calculate a thermally weighted time for the ith percentile germinating, 1/ai, by rearranging equation 1,   2 where ti is the time to germination of the ith percentile. Thermal weighting of the cumulative germination curves in Fig. 2 should provide indicative evidence of whether lateness at high temperature was linked to non‐germination at low temperature. If 1/a is calculated for each measure of percentage emergence, curves can be drawn showing cumulative percentage germination on cumulative weighted time (Squire, 1999). Provided all percentiles germinate, curves of percentage germination at different temperatures (which are different in chronological time) become superposed when expressed on weighted time. If some percentiles show non‐germination at low temperature, the degree of superposition is a test of whether non‐germinators are evenly distributed throughout the population or not. If rate of germination and the non‐germination trait were not correlated in the population, then the cumulative curves would progressively diverge from the earliest percentiles onwards. The divergence would be increasingly to the right as the germinating temperature was reduced and the expression of the non‐germination trait increased. In contrast, if the non‐germination trait was confined to a sub‐set of the seed, then the cumulative curves would superpose up to that sub‐set of precentiles, then diverge and thereafter continue to cumulate in parallel, again with the coolest curve shifted right‐most. For example, if non‐germination was confined to slow germinators, then the curves would only diverge at the later percentiles, or if confined to the fast would diverge immediately then rise in parallel. Thermal weighting of the curves in Fig. 2 was done using values of −0.7 °C for Tb and 1.08 for b (Squire et al., 1997). As previously, a gompertz function was fitted to the data at 19 °C for percentage germination (y) and 1/a (x),   3 where ymax=maximum germination in the first flush and p and m are parameters. The data at 10 °C and 4 °C displayed only very slight divergence from this curve up to the 30th percentile, but greater divergence thereafter (Fig. 3). Non‐germinators at 10 °C and 4 °C are therefore most likely to arise from the 30th–100th percentiles of the population at 19 °C. Fig. 2. View largeDownload slide Time‐course of germination at initially (A) 4 °C (B) 10 °C and (C) 19 °C, solid circles. The arrows indicate when seeds that did not germinate at their initial temperatures were moved to successively warmer temperatures (a, 10 °C; b, 16 °C; c, 21 °C; d, 25 °C and alternating 12 h periods of x, 23 °C/10 °C or y, 25 °C/6 °C). Note that the scales on the time axes change between graphs. Fig. 2. View largeDownload slide Time‐course of germination at initially (A) 4 °C (B) 10 °C and (C) 19 °C, solid circles. The arrows indicate when seeds that did not germinate at their initial temperatures were moved to successively warmer temperatures (a, 10 °C; b, 16 °C; c, 21 °C; d, 25 °C and alternating 12 h periods of x, 23 °C/10 °C or y, 25 °C/6 °C). Note that the scales on the time axes change between graphs. Fig. 3. View largeDownload slide Replotting of the germination curves in Fig. 2 on a common, thermally weighted time scale (1/a,equation 1) at initial temperatures of 4 °C (○), 10 °C (•) and 19 °C (□). For reference, the Gompertz function fitted through the values at 19°C has parameters (see equation 3) ymax=95%, P=1.05, m=4.45; r2>0.99. Fig. 3. View largeDownload slide Replotting of the germination curves in Fig. 2 on a common, thermally weighted time scale (1/a,equation 1) at initial temperatures of 4 °C (○), 10 °C (•) and 19 °C (□). For reference, the Gompertz function fitted through the values at 19°C has parameters (see equation 3) ymax=95%, P=1.05, m=4.45; r2>0.99. Table 1. Summary of the final temperature requirements for the selection of the germination fractions at the three initial selection temperatures A tick indicates that the seeds selected germinated at the initial temperature, otherwise the warmest constant temperature and/or alternating temperature requirement is given. The cumulative percentage of seeds germinated where less than 100% is given in parentheses. Initial selection temperature   Early   Intermediate   Late     4 °C (51%)  ✓  10 °C (63%)  21 °C (75%)  23/10 °C  10 °C (80%)  ✓  ✓  25 °C (80%)  25/6 °C  19 °C (95%)  ✓  ✓  25 °Ca    Initial selection temperature   Early   Intermediate   Late     4 °C (51%)  ✓  10 °C (63%)  21 °C (75%)  23/10 °C  10 °C (80%)  ✓  ✓  25 °C (80%)  25/6 °C  19 °C (95%)  ✓  ✓  25 °Ca    aNote that only half the seeds in this late selection required a change in temperature to germinate. View Large Principal co‐ordinate analysis Both primers produced a similar number of variable bands and spread of frequencies of occurrence. There were a total of 35 variable bands with the frequency of occurrence ranging from 0.2 to 9.9 in the samples of ten plants. Only two of the 90 plants examined had identical band frequencies. A similarity matrix was constructed with values ranging from a maximum of 100% for the two identical plants, both individuals being from the late percentiles selected at 19 °C, to a minimum of 50%. The PCO plot was then constructed from the similarity matrix. The first three dimensions accounted for 24, 10 and 8% of the variation in principal coordinate or ‘genetic’ space. Any separation into phenotypic sub‐groups (selection temperature or germination fraction) appeared most clearly in the first two principal coordinates. The plots for all plants are compared in Fig. 4A, while B, C and D show only the data relating to each specific question. All the results presented in Fig. 4 are from the one PCO analysis. Given that all but two individuals were genetically different, variation of the individuals in the PCO space is expected. For the null hypothesis to stand, all phenotypes should be distributed randomly throughout the occupied PCO space. For the null hypothesis to be false, at least some phenotype should separate into a distinct cluster: i.e. at least one of statements 1a, 1b or 2 must be shown to be false (see Introduction). Taking each statement in turn: Statement 1a, ‘early germinators selected at different temperatures are not genetically different’, stands since there is no indication in Fig. 4B of any separation of the early germinators that could be related to the temperatures at which they were selected. The majority of these early germinators occupy the right hand side of the graph, a space also occupied by many intermediate germinators (Fig. 4A). The few outliers to the left do not systematically alter the picture. Statement 1b, ‘late germinators at higher temperatures are not genetically different from non‐germinators at low temperature’, is shown to be false. There are clear indications in Fig. 4C that the non‐germinators (associated with the two coolest temperatures) form a distinct group located towards the middle left‐hand side of the genetic space. The tightest clustering of non‐germinators was observed at a selection temperature of 10 °C where all the symbols were to the left of the main group formed principally by early and intermediate germinators (Fig. 4A). The late germinators (late fraction at 19 °C, solid squares), in contrast, are not grouped with the non‐germinators. About half are clustered separately in the top left corner and the other half lie in the main cluster to the right. Statement 2, ‘phenotypes selected at the same temperature (specially non‐germinators and germinators) are not genetically different’, is shown to be false by the separation of non‐germinators from germinators, specially at 10 °C (Fig. 4C). At the highest temperature where all seeds germinated (Fig. 4D), separation is suggested but is not clear cut. Fig. 4. View largeDownload slide Principal coordinate plot of the genetic distances between early (circles), intermediate (triangles) and late (squares) germinating seeds selected at 4 °C (open symbols) 10 °C (gray symbols) and 19 °C (black symbols): data for (A) all seeds; (B) early germinators only; (C) late germinators, (D), seeds selected at 19 °C. Fig. 4. View largeDownload slide Principal coordinate plot of the genetic distances between early (circles), intermediate (triangles) and late (squares) germinating seeds selected at 4 °C (open symbols) 10 °C (gray symbols) and 19 °C (black symbols): data for (A) all seeds; (B) early germinators only; (C) late germinators, (D), seeds selected at 19 °C. Discriminating markers Early percentiles at the three temperatures show a broad consistency between the thermal weighting analysis (Fig. 3) and their forming a common grouping in PCO space (with few exceptions, Fig. 4B). For late percentiles, however, the PCO analysis points to apparent inconsistencies between late germinators at high temperature and the non‐germinators at low temperature. Specifically, a number of germinators at 19 °C are expected to lie within this cluster of non‐germinators at low temperature. None of them do: the late germinators at 19 °C lie either in the small cluster at the top left of the PCO space or within the main group to the right. This suggests the non‐germinator genotypes were not sampled in the 1–10, 45–55 and 91–100th percentiles at 19 °C. The possible origins of this anomaly are now sought in the frequencies of the more discriminating markers. Because there was no genetic separation of the early and intermediate selections in the PCO plot, and phenotypically all seed from these fractions was able to germinate without having to apply alternating temperatures, the analysis concentrates on differences in band frequencies between the extreme selections only. The bands are ranked in order of average frequency of occurrence across early and late selections (Table 2). The maximum frequency of occurrence of a band is 10, there being 10 individuals per seed fraction at each selection temperature. Bands which have a 50:50 presence:absence (5 occurrences out of 10) carry the maximum amount of information and those with low or high frequency of occurrence carry the least. Both primers contributed a similar number of bands with similar ranges of information content. Differences in band frequency between early and late selections where the magnitudes were greater than 5 have been indicated in Table 2. Differences between early and late selections are greatest at 10 °C. Comparison of the occurrence of these bands with the position of individuals in the PCO plot in Fig. 4 showed that six of the bands, three from each primer (primer 1420: bands 7, 10, 14; primer 1425: bands 19, 20, 21; Table 2), largely caused separation of the non‐germinating cluster in Fig. 4C. Four of the bands were generally present and two absent in the non‐germinating type. Moreover, the four late germinators at 19 °C positioned to the top left in Fig. 4C also had this same pattern of bands. They had been drawn away from the non‐germinating cluster along the second axis of the PCO plot mainly by differences in two other bands that occurred sporadically in low frequency throughout the population. It is uncertain whether this was a chance association or whether these bands were associated with additional sorting of the germination rates at 19 °C. Whether genotypes similar to those of the non‐germinators were present in fractions of the germinators that were not sampled (i.e. fractions 11–44% and 56–89%) at 19 °C remains uncertain. Confirmation would be obtained only by genetic analysis of the whole population at each temperature. The distributions of the six bands referred to above among the selected individuals are shown in Fig. 5. The frequency in the population (i.e. the total in early, intermediate and late selections) was generally similar, as shown by the totals at the bottom of each column. Any differences in band frequency between selections would therefore be caused by sorting according to temperature. Of the 30 plants selected at each temperature, 19 had the same banding pattern for these six bands: six at 4 °C, seven at 10 °C and six at 19 °C. The late germinators at 10 °C (all non‐germinators) stand out as having a high frequency of this particular banding pattern: seven of the plants precisely matching the pattern and two others mismatching in only one of the six bands. The early and intermediate germinators at 10 °C had a much lower frequency of this pattern, with only one of the 20 individuals coming close. At 4 °C, the six plants which had this specific banding pattern were all late germinators, but one other late germinator had none of the markers and two others had only one of them. The pattern was completely absent in early and intermediate selections at 4 °C. At 19 °C, however, the specific banding pattern occurred in all three selections. Only four of these six plants were late germinators; a further one was among the intermediate and another among the early. The intermediate selections at 4 °C and the late at 19 °C allow visual comparison of germinators and the first type of non‐germinator (not requiring alternating temperature) which were about the first and second half of each, respectively. No difference is apparent. Fig. 5. View largeDownload slide Distribution of six markers (numbered as in Table 2) among the 30 individuals selected, as early, intermediate and late germinators, at each of the three temperatures. The replicate number indicates the sequence in which the individual germinated (1…30, first to last) at each temperature. A shaded square indicates the presence of a marker except for the two asterisked bands (*; primer 1 band 10 and primer 2 band 20) where it indicates the absence. Values at the bottom are the total numbers of each marker among the 30 individuals selected at each temperature. Fig. 5. View largeDownload slide Distribution of six markers (numbered as in Table 2) among the 30 individuals selected, as early, intermediate and late germinators, at each of the three temperatures. The replicate number indicates the sequence in which the individual germinated (1…30, first to last) at each temperature. A shaded square indicates the presence of a marker except for the two asterisked bands (*; primer 1 band 10 and primer 2 band 20) where it indicates the absence. Values at the bottom are the total numbers of each marker among the 30 individuals selected at each temperature. Table 2. Frequency of band occurrence (maximum 10) of the 35 polymorphic bands produced by primers 1420 (1) and 1425 (2) in the early (E) and late (L) seed selections The phenotype of the seed selections (G, germinating; N, non‐germinating), the magnitude of the difference in band occurrence between early and late selections (Δ) at each temperature and the average frequency of occurrence are indicated. Values in boxes indicate the most discriminating markers (see text).     View Large Discussion The hypothesis that the rate–temperature phenotypes are not genetically different was shown to be false. There was no evidence that early germinating phenotypes differed, but a genetic basis of non‐germination (a form of secondary dormancy) was confirmed. The association between this trait and markers was complex and not consistent between temperatures. No individual band or set of bands was exclusively associated with the trait, but six bands were strongly indicative. About 20% of the sampled plants had the same pattern in these six, another 20% none, and the rest 2, 3, 4 or 5 in common. If a plant had all six matching the pattern, then without exception it was a non‐germinator at the lower temperatures of 4 °C or 10 °C. However, the reverse did not apply in that some non‐germinators at 4 °C had few or none of these bands matching the pattern. Generally, the absence of the pattern was associated with early and intermediate germination, but no difference was found between these categories. The association of the pattern with non‐germination was greatest at 10 °C. All but one of the ten plants formed a tight cluster in the PCO space, this one having only two of the six bands matching the pattern. Selecting at this temperature was most effective in concentrating the occurrence of this banding pattern in the late germinators and removing it from the early and intermediate germinators. That non‐germination at 4 °C was less associated with this banding pattern suggests that the genetic basis of the biochemical processes leading to non‐germination might not be the same across the range of temperature. The connection between the non‐germinating trait at low temperature and the rate of germination at high temperature was less tight. Though most occurrences of this pattern at 19 °C were in the late selection, the overall degree of matching in this selection was only 60% of that in the same selection at 10 °C, while plants with the full pattern also occurred in both early and intermediate selections. The results at 19 °C are prone to the general problem of selecting among rapidly germinating seeds. Selections of early, intermediate and late germinators at 19 °C were made only a few hours apart and any slight difference in rate caused by extrinsic factors might have obscured any genetic control over germination rate. The association between late germination at optimal temperature and non‐germination at low temperature poses certain questions about the biochemical nature of the differences. It is unclear, for instance, whether the two traits are genetically associated, such that the late germinators are intrinsically susceptible to secondary dormancy; or whether one trait is more indirectly a consequence of the other trait, in this instance that the lates remain longer in conditions that are likely to induce secondary dormancy in all or any of the seeds. The second of these statements would be negated if, in some other seed lot, secondary dormancy at low temperature was found to be associated with fast germination at optimal temperature. A general implication of the findings is that the phenotype characterized as the nth percentile in a rate–temperature analysis might not be the same genotype at different temperatures. Likewise, the mean or modal phenotype (or any sub‐set) of a population might not consist of the same genotypes at all temperatures. Comparing physiological responses between temperatures (and by implication, between field populations) will not necessarily be comparing like with like therefore. A more individual‐based, as distinct from population‐based, approach now needs to be explored for modelling or measuring rate–temperature responses in variable seed lots. Ecological implications The results have implications for oilseed rape as a feral plant. There is now substantive evidence that different cultivars, and different seed lots within cultivars of B. napus, might be similar in their rates of germination and growth between 15 °C and 20 °C, but differ at lower temperature (Acharya et al., 1983; Nykiforuk and Johnson‐Flanagan, 1994; Marshall and Squire, 1996). The genetic ‘background’ of a cultivar, whether conventional or transgenic, should therefore influence the performance of the cultivar's feral descendants in ways not predictable from standard germination tests at, say, 20 °C. The previously observed traits of non‐linearity in the rate–temperature response and of non‐germination at low temperature (Marshall and Squire, 1996; Squire et al., 1997) are not the only factors complicating the starting profile of development. The findings here show the history of the temperature environment to which the seed is exposed following wetting is also important. For example, some of the seed that germinated at a constant temperature of 19 °C would not have germinated if they had been first exposed to a constant temperature of 4 °C then ramped gradually to 19 °C. They had to be exposed to alternating temperatures before they would germinate. Even for seed that did not require alternating temperatures to germinate, the maximum number that did germinate at, say, 10 °C (80%) was reduced (to 63%) when the seed was first exposed to an initially cooler temperature. The origins of the within‐population variability at low temperature are uncertain. Many B. napus oilseed rape varieties are produced using inbreeding systems (Jonsson, 1977). However, the presence of differing levels of polymorphism found within breeders’ stocks of several varieties (Charters et al., 1996) indicates that they were produced by partial inbreeding or some other system, such as synthetic or composite breeding, which retains diversity. Synthetics or composites are mixtures of genetically different lines permitted to interbreed over a few generations (Simmonds, 1979). In a partially self‐pollinating species such as oilseed rape, some of the progeny are expected to result from selfing in each line. These selfed progeny would form tight clusters in a PCO space indicating groups of genetically very similar individuals. The distance between clusters would depend on the degree of variation in band occurrence between the lines. This selfing notwithstanding, the seed multiplication process is normally assumed to achieve a well‐mixed population owing to outbreeding. Consequently, the progeny as a whole would occupy a cloud of points in PCO space covering the tight clusters (selfed progeny) and the intermediate space between them. Genes influencing particular physiological traits would also be expected to be mixed throughout the progeny. Ordinarily, therefore, a cluster of individuals having a particular phenotype is not expected to separate out. There are two possible explanations for the finding in this study. The first is that a band or group of bands have been found that by accident fall close to the gene(s) determining the non‐germination trait. Although the primers used had previously been selected to reveal as much genetic heterogeneity as possible among cultivars (Charters et al., 1996), such an association due to genetic linkage is extremely unlikely. Association of markers with traits due to genetic linkage occurs relatively infrequently. Even in the best circumstances for detecting genetic linkage in a trait, a simply‐inherited resistance trait in a doubled haploid population in B. napus (Mayerhofer et al., 1997), only 15 out of 980 markers showed the association. The frequency is considerably less in non‐doubled haploid and randomly inter‐mating populations. A second, more likely explanation for the relatively high numbers of bands associated with the trait, is that one particular component line of a mixture had the non‐germination trait and that the line has, to a large extent, remained separate from the others through self‐pollination. Arguments over the nature of the seed multiplication system should not obscure the main finding here that a trait influencing the germination profile is genetically based. 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TI - Temperature‐dependent germination traits in oilseed rape associated with 5′‐anchored simple sequence repeat PCR polymorphisms
JO - Journal of Experimental Botany
DO - 10.1093/jexbot/51.353.2075
DA - 2000-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/temperature-dependent-germination-traits-in-oilseed-rape-associated-nKC5rykImQ
SP - 2075
EP - 2084
VL - 51
IS - 353
DP - DeepDyve
ER -