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CYTOLOGICAL STABILITY OF DOUBLED HAPLOID LINES DERIVED FROM INTERSPECIFIC CROSSES BETWEEN BRASSICA NAPUS L. AND B. RAPA L.

T.J. Kubik, G.P. Hawkins, and G.R. Stringam

Dept. of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5

ABSTRACT

A study to determine the potential for introgression of traits of agronomic importance between B. napus and B. rapa, including the determination of chromosome number and cytological stability of DH lines, derived from interspecific crosses between these two species. Fifty DH lines were obtained when the interspecific F1 from a cross between B. napus DH line 94-98 and a self-fertile F7 B. rapa line, was backcrossed twice to B. rapa. Meiotic preparations showed that all 50 interspecific DH lines contained 38 chromosomes which formed 15 to 19 bivalents, 1 to 2 quadrivalents, and occasionally 2 univalents, a hexavalent, or chain of 6. Bridge / fragment associations were also observed. Possible explanation as to why a B. napus chromosome number was maintained despite two backcrosses to B. rapa, and the implications of multivalent and bridge / fragment associations in the DH lines, are discussed.

KEYWORDS Chromosome aberations, quadrivalents, bridges, doubled haploid

INTRODUCTION

Agronomic advances in Brassica rapa L. have been limited in part by the difficulties of breeding a self-incompatible species, while in B. napus L., yield performance and disease resistance have been improved dramatically over the past ten years. Interspecific hybridization is an established procedure to introgress traits between Brassica species. As part of a study to determine the potential for introgression of traits of agronomic importance between B. napus and B. rapa, a cytological analysis was conducted on doubled haploid (DH) lines, derived from the F1 and backcrosses of these two species. The DH method was used to reduce the fertility problems associated with chromosomal and genic imbalances which arise in interspecific progeny (Thiagarajah 1994).

Materials and Methods:

Fifty DH lines were obtained when the interspecific F1 from a cross between B. napus DH line 94-98 and a self-fertile F7 B. rapa line, was backcrossed twice to B. rapa. Flower buds were fixed in Newcomers solution to which pure basic ferric acetate was added (3% w/v) as a mordant (Stringam 1970) and chromosomes were stained using 2% acetocarmine. Pollen viability was determined using aceto-carmine staining.

Results

Meiotic preparation showed that in all 50 DH lines tested, 38 chromosomes were present at diakinesis. Within these lines, the frequency of loose homologous pairing ranged from 7 to 44%. Pollen mother cells (PMCs) with 18 bivalents and 2 univalents, as well as 17 bivalents with 1 univalent and 1 trivalent were also observed. The occurrence of univalents located off the metaphase plate and laggards at anaphase confirmed the presence of univalents at diakinesis.

A high degree of chromosome stickiness made chromosome counts difficult in 3 of the DH lines. One to two quadrivalents (rings of 4) were present in 37 of 50 DH lines (Figure 1), a hexavalent (ring of 6) was observed in 3 DH lines and a chain of 6 in 4 DH lines

Non-disjunction and chromosome bridge/fragment associations were observed at anaphase I, as well as telophase I and II (Figure 2). Although the frequency of ring formation was high, the frequency of nondisjunction and improper disjunction was low.

Pollen viability was used as a measure the stability of each DH line. Of the 44 DH lines tested, 22 lines had a pollen viability greater or equal to 90%, 16 had greater than or equal to 80%, 5 greater than or equal to 70% and 1 had a viability of 64%. The presence of previously mentioned aberrations was reflected in decreased pollen viability of each line (Table 1).

Table 1. Pollen Fertility of Parent Lines and Interspecific DH Progeny.

Line

Parent

Fertility (%)

93-1005-3

B. rapa

911.6

94-98

B. napus

960.3

95-376

Interspecific DH1

753.7

95-440-2

B.C. B. rapa

980.3

96-1776-2

95-1062-B

913.8

96-1712-2

95-1061-B

930.8

96-1764-2

95-1061-B

850.6

96-1691-3

95-1061-B

961.1

96-1766-1

95-1061-B

910.5

96-1718-3

95-1061-B

852.2

Conclusions

The mechanism by which a B. napus chromosome number was maintained despite two backcrosses to B. rapa, is unknown. B. napus and the interspecific DH1 were used as the female parents during crossing, prior to microspore culture. These results suggest that there there was a maternal influence on microspore culture resulting in a selection differential in favor of microspores with the B. napus gametic number.

Multivalents could have formed from non-homologous recombination, while bridge / fragments indicate that inversions occurred. These associations are not expected in DH lines unless non-homologous recombination and inversions occurred after the doubling process, suggesting chromosomal instability. These aberrant chromosome configurations influenced pollen fertility levels in some DH lines (Table 1). The persistence of these configurations and their effects on the phenotypic stability of the lines requires further study.

REFERENCES

1. Stringam GR (1970) A simple mordant technique for plants with small chromosomes. Canadian Journal of Botany. 48:1134-1135.

2. Thiagarajah MR, Hawkins GP, Stringam GR, Bansal VK, and Johnson-Flanagan AM. Use of doubled haploids to introgress traits between Brassica juncea and Brassica napus. (abstr.) The methodology of plant manipulation: Criteria for decision making. Eucarpia, section: Genetic maipulation in plant breeding, Cork, Ireland. Pp 11-14. Sept. 1994.

Figure Captions

Figure 1. A univalents in line 96-1776-2 (1000X), B quadrivalent in line 96-1712-2 (1000X), C hexavalent in line 96-1764-2.

Figure 2. A Bridging in line 96-1691-3 at metaphase I (600X), B bridge/fragments in line 96-1766-1 at anaphase I (1000X), C bridging in line 96-1718-3 at anaphase II (1000X).

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