VERN-: BRIDGING THE GAP BETWEEN WINTER AND SPRING CANOLA
Anne M. Johnson-Flanagan1, Zhanao Deng2, Nancy E. Go1 and
Glen P. Hawkins 1*
*1Department of Agricultural, Food and Nutritional Science, Faculty of Agriculture, Forestry and Home Economics, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, Alberta, Canada. T6H 2P5. e-mail aflanaga@gpu.srv.ualberta.ca
2Citrus Research and Education Center. 700 Experiment Station Road. University of Florida. Lake Alfred, Florida. USA. 33850.
Winter Brassicas must be exposed to extended periods of low temperature to flower and set seed. This low temperature exposure also results in the acquisition of freezing tolerance. To investigate the relationship between freezing tolerance and vernalization, homozygous microspore-derived lines were developed from reciprocal crosses between two winter cultivars of Brassica napus, Cascade and Rebel. One line, Vern-, which has very high freezing tolerance was identified. Further physiological and morphological characterization of this line, including determination of photoperiod responses, field studies and SEM of reproductive development, verified that Vern- is a true spring type, having completely lost the vernalization requirement. In order to determine the genetic basis of the phenotype, molecular analysis of the parents and Vern- was undertaken. The results show that Rebel, although traditionally classified as a winter cultivar, carries spring alleles at the major and some of the minor loci that control vernalization requirement and flowering time. Vern- appears to have inherited these alleles from Rebel, while inheriting freezing tolerance alleles from Cascade. Thus, Vern- has lost the vernalization requirement, despite the very high degree of both inherent and acclimation-specific freezing tolerance. The results indicate that the linkage between freezing tolerance and vernalization can be broken in winter B. napus.
KEYWORDS freezing tolerance, double haploid, genotyping, vernalization
INTRODUCTION
The vernalization requirement in winter Brassica napus imposes considerable limitations in both winter and spring canola breeding programmes. On one hand, development of winter cultivars is slowed by up to 6 weeks for every generation because of the vernalization requirement. On the other hand, introgression of winter traits into spring programmes is rarely successful because the resulting progeny are generally slow to mature.
To study the genetics of vernalization and its relationship with freezing tolerance, we produced a doubled haploid population. Vern- is one of a number of lines in the resulting population lacking a vernalization requirement, while retaining a high degree of freezing tolerance. As it is canola quality and has good agronomic performance, this line shows potential as a spring cultivar and breeding parent. Moreover, its winter lineage makes it a desirable resource to bridge winter and spring gene pools. We then utilized a number of markers to examine the genetic basis for the loss of the vernalization requirement and the retention of freezing tolerance in Vern-. The information gained from this study will provide valuable insight into the genetic nature of vernalization and will help define the genetic background required for the development of early flowering lines from winter Brassica.
MATERIALS AND METHODS
Phenotypic characterization
Non-vernalized plants were grown under greenhouse conditions and assessed on the basis of days to first flower, completion of flowering and maturity (measured by the presence of mature seed in the top ¾ of the primary raceme). Vernalization requirement was assessed after 100 and 300 days in the greenhouse. Field testing was conducted using 3 replicates at 3 locations, 2 in Alberta, Canada, and 1 in Saskatchewan, Canada. Seed was sown in 4 row nursery plots and evaluated on days to first flower, completion of flowering and maturity (measured by the presence of mature seed in the top ¾ of the primary raceme). Assessment of freezing tolerance was completed on plants that had been acclimated (vernalized) for 6 weeks at 4°C, using the leaf-disk method of Boothe et al. (1995).
DNA marker and linkage analysis
Freezing tolerance and vernalization requirement were established based on X2 values derived from orthogonal function analysis using SAS (Statistical Analysis Software ver 6.03). Interval mapping of vernalization loci was conducted according to Lander and Bolstein (1989), using MAPMAKER/EXP 3.0 and MAPMAKER/QTL 1.1. (Lincoln et al., 1992). A LOD (log of the likelihood of odds ratio) threshold of 2 was used to identify marker intervals containing vernalization QTLs.
RESULTS AND DISCUSSION
To study the relationship between freezing tolerance and vernalization, we developed a B. napus doubled haploid (DH) population using microspore embryo technology. In this manner, a spring type with no vernalization requirement (Table 1) and a high degree of freezing tolerance (TL50 of >-18°C, as compared to a TL50 of –15.5°C for Cascade and only –7.5°C for Rebel) was derived from a cross between the winter type, Rebel, with a weak vernalization requirement and little freezing tolerance and the winter type, Cascade, with an absolute vernalization requirement and a high degree of freezing tolerance.
|
|
Flowering time (days) |
Maturity (days) |
||
Greenhouse |
Field |
Greenhouse |
Field |
|
|
Vern- |
40.8 ± 1.6a |
46 ± 1.2b |
90 ± 1.75c |
102 ± 1.6e |
|
Quantum |
39.0 ± 1.3a |
49 ± 1.0b |
96 ± 1.0d |
107 ± 2.1f |
|
Westar |
41.8 ± 2.0a |
47 ± 1.0b |
94 ± 1.3d |
103 ± 1.0e |
|
Excel |
42.2 ± 1.8a |
48 ± 1.4b |
95 ± 1.5d |
104 ± 2.0ef |
|
Legend |
38.0 ± 2.3a |
NT |
92 ± 1.1d |
NT |
Values represent the mean ± SE with n=10
a,b,c,d,e,f Significant at 5% level.
The search for freezing tolerance markers has proven to be difficult in B. napus. Teutonico et al. (1995) identified a number of QTLs mapping to freezing tolerance in the diploid species B. rapa (AA). Determination of the genetic basis for freezing tolerance in Vern- was hampered by the lack of suitable markers (Table 2). Nonetheless, of the 2 markers identified, both were contributed to Vern- by Cascade.
Table 2. Association of RFLP Marker Loci for Freezing Tolerance Linkage Groups.
|
Marker Loci |
X2 |
Probability |
Significance |
Cascade |
Rebel |
Vern- |
|
ec3e5 |
12.42 |
0.007 |
Significant |
TT |
SS |
TT |
|
wg1g6 |
8.66 |
< 0.01 |
Significant |
TT |
SS |
TT |
|
wg1g3 |
14.43 |
0.6 |
Non |
na |
na |
na |
|
wg1f6 |
2.22 |
0.2 |
Non |
na |
na |
na |
|
wg4h3 |
0.025 |
0.1 |
Non |
na |
na |
na |
Linkage groups as assigned by Teutonico et al. (1995).
Origin of alleles are represented as T for freezing tolerant and S for freezing sensitive
Genetic studies regarding the vernalization requirement in B. napus were previously reported by Ferreira et al. (1995). They identified 3 linkage groups Lg 9, 12, and 16, which are strongly associated with the requirement for vernalization and show major effects on time-to-flowering in a winter by spring cross in B. napus. We used markers from these to analyze spring and winter type varieties, thereby identifying spring and winter alleles for each marker in question (Table 3). The results show that Vern- arose from a cross between the winter parent, Rebel, which contains predominantly spring alleles, and the winter parent Cascade, which contains predominantly winter alleles. In order for Vern- to be a spring type, it had to inherit the spring allele, wg7b3, in LG12, from Cascade and all other major vernalization and time to flowering alleles from Rebel.
Table 3. Genotypic Comparison of Vern- with Spring and Winter Varieties.
|
|
Linkage group 9 |
Linkage group 12 |
Linkage group 16 |
N10 |
N19 |
|
|||||
|
Line |
Tg6a12a |
Wg8g1b |
WG6B10 |
Ec3g3c |
Wg7b3 |
Wg1g4 |
Wg9c7 |
Wg6b2 |
CoN10 |
CoN19 |
FCA17 |
|
Vern- |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
|
Rebel * |
SS |
SS |
SS,WS |
SS,WS |
WW |
SS |
SS |
SS |
SS |
SS |
SS |
|
Cascade * |
WW |
WW |
WW |
WW |
SS |
WW |
WW |
WW |
WW |
WW |
WW |
|
Jet Neuf * |
WW |
SS |
WW |
WW |
WW |
WW |
WW |
WW |
WW |
WW |
WW |
|
Westar |
SS |
SS |
WW |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
|
Excel |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
|
Quantum |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
|
Legend |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
SS |
* denotes a winter type variety.
Origin of alleles are represented as S for spring and W for winter.
Results from the present study clearly demonstrate that freezing tolerance and vernalization can be inherited separately. Although this would be expected on the basis of the genome mapping work by Osborn and his coworkers (Ferreira et al., 1995; Teutonico et al., 1995), the evidence to support the expectation has not been reported. Our results show that the Vern- line has lost the vernalization requirement while expressing a higher degree of freezing tolerance than is expressed by either parent, both in the absence of acclimation and following acclimation. As such, the linkage between vernalization and freezing tolerance in Vern- has been broken at all loci examined.
The findings from this study have implications that extend beyond furthering our understanding of how plants survive the winter and flower successfully in the spring: first, it indicates that there is a potential to bridge between the winter and spring gene pools; second, it provides homozygous germplasm that has a higher degree of acclimation-specific freezing tolerance than is currently available in winter canola and; finally it provides homozygous early maturing germplasm with inherent freezing tolerance which is superior to that currently available in spring canola.
Boothe JG, deBeus MD, and Johnson-Flanagan AM. (1995) Expression of a low temperature induced protein in Brassica napus. Plant Physiol. 108:795-803.
Ferreira ME, et al. (1995) Mapping loci controlling vernalization requirement and flowering time in Brassica napus. Theor. Appl. Genet. 90:727-732.
Lander ES, and Bolstein D. (1989) Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121:185-199.
Lincoln S, Daly M, and Lander E. (1992) Mapping genes controlling quantitative traits with MAPMAKER/QTL 1.1. Whitehead Institute Technical Report. Cambridge Mass.
Teutonico RA, et al. (1995) Genetic analysis and mapping of genes controlling freezing tolerance in oilseed Brassica. Molecular Breeding. 1:329-339.
This research was supported through grants to AMJ-F by the Alberta Agriculture Research Foundation, the National Sciences and Engineering Research Council of Canada (NSERC # 0036689), and the Western Grain Research Foundation. Financial support for GPH was provided by the Canadian Wheat Board.