ABSTRACT

Above the development of different rapeseed genotypes varying in fatty acid composition, improvements of meal quality are still of importance in breeding for quality in rapeseed (Brassica napus). Since reduction of glucosinolates has been achieved already, the protein and crude fibre contents as well as the energy concentration of the meal have gained attention. As compared to usual black seeds yellow seediness is characterized by thinner seed coats causing a lower content of crude fibre and correspondingly higher contents of oil and protein. Regarding the distinction from domestic production of double-low rapeseed (00, Canola) yellow seed colour would have a desirable impact in both, Canola and high-erucic acid rapeseed (HEAR). In the course of a breeding program F₁ material was derived from crosses between dark-seeded HEAR lines and a true-breeding yellow-seeded B. napus (00). Both inbred and doubled-haploid (DH) lines were generated. As compared to the former, DH lines have the advantage that their selfed seed is genetically uniform and the lines are constantly reproducible. Their use for genetic studies is beneficial due to simpler gametophytic genetic ratios and more distinct phenotypic classes, which allow the scoring of rare genotypes in smaller populations. Rather than in F₂ populations differences caused by maternal inheritance are eliminated and analyses of recessive seed traits, such as seed coat colour, can be carried out easier. Since selection for yellow seediness is hindered due to pronounced environmental effects (e.g., temperature during seed ripening), molecular markers linked to gene loci controlling seed colour in B. napus have to be identified by bulked segregant analyses. In addition to visual assessment of seed colour major seed traits are analytically examined and screened by using near-infrared reflectance spectroscopy (NIRS).

INTRODUCTION

Regarding quality improvement of industrial rapeseed (Brassica napus) yellow seed colour has a respectable influence on seed quality and subsequently on the energy concentration of the meal obtained after oil milling. This is explained through the characteristically thinner seed coat of yellow as compared to black seeds, which causes a lower crude fibre content and correspondingly higher proportions of oil and protein. Thus, yellow seed colour enhances the economics of HEAR and the products processed from the seed. A further side-effect would be a clear distinction from domestic production of double-low rapeseed (Canola), which is, so far, entirely black-seeded.

Experimental

Doubled haploid lines were derived from the F₁ generation of crosses between different dark seeded B. napus lines and a yellow seeded B. napus genotype ‘T-25629’ (Table 1). The latter was originally developed at the University of Göttingen (Shirzadegan 1986). The F₁ plants, being selected due to the expression of the character “yellow seediness” in the following generation (F₂S₁), were used as donor plants for microspore culture using a method described earlier (Lühs 1996). Additionally the selection of the corresponding inbred lines was continued regarding seed colour and different agronomic properties, such as vigourness, ripening and disease resistance. Crude seed composition was determined by near-infrared reflectance spectroscopy (NIRS) as described by Daun (1995). Chi-square goodness-of-fit tests were used to compare the observed distribution in the DH populations to those predicted by various genetic models for seed colour inheritance, as follows - black : brown : yellow = 4 : 3 : 1 (Henderson and Pauls 1992), 2 : 5 : 1 or 1 : 6 : 1 (Van Deynze and Pauls 1994a). The data for seed colour were pooled, tested for heterogeneity and fit to appropriate genetic models (cf. Mudra 1958).

Figure 1: Variation of seed colour in different B. napus inbred lines (F₄S₃)

Table 1: Cross combinations of genotypes used for microspore culture

Cross	Pedigree	Haploids (in vitro)	DH lines*
Gi 3	‘DH K26-19’ x ‘T-25629’	143	114
Gi 4	‘DH K26-96’ x ‘T-25629’	231	123
Gi 5	‘DH K26-313’ x ‘T-25629’	106	65
Gi 6	‘Express’ x ‘T-25629’	48	13
Gi 7	‘Sollux’ x ‘T-25629’	5	2
Gi 8	‘Askari’ x ‘T-25629’	0	0
Gi 9	‘Erox’ x ‘T-25629’	116	58
	Total number	649	375

* seed harvested in 1998

RESULTS and DISCUSSION

Development of doubled-haploid lines

Generating the DH populations strong effects due to the different tissue culture ability of the genotypes became obvious as described in the literature (Chuong et al. 1988, Weber et al. 1995). A varying number of haploid plants in vitro, which were obtained from distinct crosses led to different sizes of fertile progenies. The crosses Gi 3 to Gi 5 resulted in a remarkable number of DH lines (Table 1). This might be caused by the dark-seeded DH parents: The latter were already a product of microspore culture, where genotypes were involved demonstrating a high embryogenesis rate or shoot regeneration response in vitro. Following a phase of seed multipli-cation in the field a large number of both DH lines and inbred lines (F₄S₃) were developed, respectively. Regarding seed colour we were able to induce considerable variation for the inbred lines (F₄S₃): bright yellow to brown or black as shown in Figure 1. The corresponding DH lines showing a similar variation of seed colour were used as basis for phenotypic classification for the inheritance study (Table 2).

Seed quality assessment

Besides the seed size the colour of the seed testa has a well-known effect on the composition of storage products, i.e. reduced crude fibre content leading to raised oil and protein proportions (Morgan et al. 1998). Classified by seed colour the scatter plots in Figure 2 display the well-known inverse relationship (r=-0,6 to -0,7) of oil and protein content (cf. Uppström 1995), which was found in the inbred progenies in comparison to high-yielding varieties used as a check.

The box plots in Figure 3 demonstrate the variation in the DH populations and corresponding inbred lines (F₄S₃) of two crosses (Gi 3, Gi 4) in comparison to high-yielding varieties. Due to an increase of protein content, the newly bred rapeseed material - segregating for seed colour - possesses a higher sum of protein and oil with an average ranging from 71.5 to 72.8% (on DM base, measured by NIRS). Regarding the checks only the high-erucic acid cultivar ‘Maplus’ (oil+protein=71.3%) is comparable due to its high oil content (50.6%). Therefore, searching for lines that deviate from the general negative correlation between protein and oil content of the seed (Uppström 1995) by having a high level of both components should help to improve the yield of useful storage products in rapeseed still further. In particular, some of the brown-seeded inbred lines meet this goal. These lines are of primary interest for the following breeding process.

Figure 2: Variation of oil content (NIRS) in relation to protein content (NIRS), found in inbred lines of different seed colour as compared to high-yielding varieties as check

Figure 3. Oil and protein (% of DM) of DH lines, corresponding in-bred populations and the cultivars 'Express', 'Joker' and 'Maplus' as checks; n=number of samples analyzed, number of different DH lines in brackets.

Inheritance of seed colour

In general, inheritance of seed colour in B. napus can be complex due to allotetraploidy (2n=4x=38), multiple gene inheritance, maternal determination and environmental impact (Van Deynze et al. 1993, Rashid et al. 1994, Tang et al. 1997, Meng et al. 1998). Depending on the source of yellow seediness used in the genetic studies in most cases a trigenic inheritance has been proposed. There for, the three genes controlling seed colour must be present in homozygously recessive condition resulting in yellow seeds. Due to Shirzadegan (1986) and Henderson and Pauls (1992) black or brown seeds are genetically determined by epistatic interactions of dominant alleles according to a black : brown : yellow = 4 : 3 : 1 segregation. A similar pattern of genetic control (2 : 5 : 1) has been proposed elsewhere (Van Deynze and Pauls 1994a, Van Deynze et al. 1995). In the present study, the segregation for the DH populations fits a 1 : 6 : 1 ratio (Table 2). Seed colour is inherited in an additive manner: Black seeds occur when all three loci are homozygously dominant, yellow seediness is manifested in the case of three homozygously recessive alleles, whereas all other genetic constitutions result in a more or less brown seed colour. The only discrepancy between predicted and observed segregation ratio was found in the DH population Gi 5. This was proved by a statistical heterogeneity test indicating that pooled data for all four crosses fit very well the predicted segregation (P%=90-95), but in total the material is heterogeneous due to the single cross Gi 5 (P%=1-5). After elimination of Gi 5 the other three populations are homogeneous and the pooled data fit a 1 : 6 : 1 segregation (data not shown). This is an important evidence that the DH populations developed in this study do not show inheritance with epistatic gene action as predicted by Shirzadegan (1986); the latter author used inbred populations rather than DH lines in his study.

Table 2: Observed and expected segregation for yellow seed trait in selected DH populations #

Seed colour	Genotype	Predicted ratio	Gi 3	Gi 4	Gi 5	Gi 9	Pooled	Total	Hetero- geneity
Black	Bl1 Bl1 Bl2 Bl2 Bl3 Bl3	1	6 (12.5)	13 (12.1)	14 (7.9)	5 (6.6)	38 (39)
Brown	bl1 bl1 Bl2 Bl2 Bl3 Bl3 bl1 bl1 Bl2 Bl2 bl3 bl3 bl1 bl1 bl2 bl2 Bl3 Bl3 Bl1 Bl1 Bl2 Bl2 bl3 bl3 Bl1 Bl1 bl2 bl2 Bl3 Bl3 Bl1 Bl1 bl2 bl2 bl3 bl3	6	80 (75.0)	77 (72.8)	39 (47.2)	42 (39.8)	238 (234)
Yellow	bl1 bl1 bl2 bl2 bl3 bl3	1	14 (12.5)	7 (12.1)	10 (7.9)	6 (6.6)	37 (39)
Total			100	97	63	53	313
χ2			3.89	2.47	6.77	0.58	0.19	13.71	13.52
DF			2	2	2	2	2	8	6
P %			10-20	20-50	1-5	50-90	90-95	5-10	1-5

# expected frequencies in brackets

SUMMARY AND PERSPECTIVES

Due to the introgression of the yellow seed trait large variation in seed characters has been generated. Using this breeding material seed colour assessment as well as the impact of seed testa colour on oil, protein and crude fibre content will be analytically examined by NIRS as described earlier (Daun 1995, Van Deynze and Pauls 1994b, Velasco et al. 1996). Since selection for yellow seediness is difficult due to strong environmental effects (e.g., temperature during seed ripening), molecular markers linked to gene loci controlling seed colour in B. napus are being identified by bulked segregant analysis (Van Deynze et al. 1993, 1995). Therefore, the construction of a sceleton map to localize genes for seed colour has been started recently. The DH lines produced in the course of this HEAR breeding program display a suitable basic material for the establishment and validation of an NIRS rapid screening method as well as for marker-assisted selection.

ACKNOWLEDGEMENTS

This work was supported by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF) and the Gemeinschaft zur Förderung der privaten deutschen Pflanzen-züchtung e.V. (GFP), Bonn, Germany. Basic plant material (‘T-25629’) was kindly provided by Dr. J. Koch, Semundo Saatzucht GmbH, Teendorf, Germany.

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