1) ORSTOM, LRGAPT, BP 5045, 34032 Montpellier cedex 1, France
2) INRA, Station de genetique vegetale, Ferme du Moulon, 91190 Gif-sur-Yvette, France
Interspecific backcrosses provide a good opportunity to control recombination process between distant species and to look for markers of genes involved in reproductive barrier. The interspecific hybrid between O. sativa and O. longistaminata was extensivel backcrossed with both O. sativa and O. longistaminata to test the rice RFLP linkage map (Tanksley et al. 1991). The progenies were analysed for 6 isozyme markers and controlled by test crosses to identify presumed genotypes for reproductive barrier. Chu and Oka (1970) proposed a genetic model based on the action of two complementary lethal genes, D1 and D2 which are present in O. longistaminata and O. sativa, respectively; complementations between D1 and D2 produce early or late seed deterioration according to their dosage in triploid endospern albumin. Following this model, a marker linked with D2 is supposed to diminish in backcross on O. longistaminata at a proportional rate to the recombination fraction between this marker and D2; reciprocally, a marker linked with D1 is expected to be lowered in backcross on O. sativa in the same condition. Consequently, heterogeneity of segregation between reciprocal backcrosses can evidence a crude relationship with genes involved in reproductive barrier and our results showed such significant deviations for two isozyme loci, Amp-1 and Pgd-1 (Table 1): Amp-11 which was coming from O. sativa was observed in excess in backcross on O. sativa while Pgd-12 was significantly reduced in backcross on O. longistaminata. Thus, a provisional genetic linkage between Amp-1 and D1 and between Pgd-1 and D2 could be deduced from these data.
Table 1. Heterogeneity of segregation at six isozyme loci between reciprocal
interspecific backcrosses involving O. sativa and O. lotigistaminata
===============================================================================
X2 from 1:1 frequency of allele coming
============== from O. sativa in:
Isozyme Chromo- No of plants deviation heterogeneity==========================
locus some ============= total between BCS BCL
BCS BCL back cross ============= ===========
1 2 1 2
===============================================================================
AMP-1 2 223 272 0.16 13.7*** 0.60** 0.62 0.43 0.50
Enp-1 6 132 317 1.87 0.22 0.51 0.58 0.54 0.67*
Sdh-1 12 223 271 1.59 0 0.47 0.46 0.47 0.53
Pgd-1 11 122 285 2.3 6.28 0.55 0.54 0.42** 0.35*
Est-2 6 208 311 11.4*** 0.03 0.57* 0.62 0.57* 0.65*
Est-5 1 180 67 2.14 1.75 0.57 - 0.48 0.37
===============================================================================
*, **, *** significant at 5%, 1% and 0.1% level
BCS and BCL: Backcrosses on O. sativa and O. lotigistaminata, respectively
1: total available data
2: subset of plants kept to evaluate the reproductive barrier through
test-crosses
In order to confirm these results, test-crosses were made to assess presumed
genotypes for reproductive barrier with a subset of 60 individuals coming
from backcross on O. longistaminata and 26 plants from backcross on O.
sativa. Test-crosses with backcross on O. longistaminata were carried out
with a line of O. sativa as female parent and reciprocal tests with
backcross on O. sativa were made with a strain of O. longistaminata as male
parent to display late seed abortion. X2 tests were calculated with
different limits of significance to control the reliability of the two groups
of plants with presumed genotypes for reproductive barrier (Table 2). At any
level of confidence, tests on backcross on O. longistaminata permitted to
cluster easily two groups: the first displayed high crossability and high
level of deteriorated seeds while the second was according to 1 : 1 ratio
between normal and deteriorated seeds. In tests with backcross on O. sativa,
the frequency of deteriorated kernels was found more continuous and revealed
identification of two clear-cut groups even with a highly conservative
1%test; this can be related with probable modifications of D1 gene in F1
hybrid which release more intermediate genotypes in backcross on O. sativa;
these modifications are also supported by the heredity pattern of rhizome
expression in interspecific backcross progenies (Ghesquiere, 1990).
Table 2. Results of test-crosses to identify presumed genotypes for
reproductive barrier (0.1% confidence limit) among interspecific backcross
progenies on O. sativa and O. longistaminata
===============================================================================
Presumed genotype No of No of crossability % X2
Test-cross for reproductive plant pollinated % deteriorated from
barrier (1%) spikelets kernels 1:1
===============================================================================
SxBCL D\1\D\1\ 23 1853 46.4 90.5
D\1\d\1\ 37 3806 9.4 46.2 2.04
BCSxL D\2\D\2\ 8 682 40 84.3
D\2\d\2\ 18 1318 23.6 63.1 29.2***
===============================================================================
***P<0.001
Estimates of recombination values by the maximum likelihood method (Allard,
1956) were computed in taking firstly frequencies of electromorphs in the
subset of plants and secondly complete classification of data in test-crosses
following the different levels of significance (Table 3). Separate data were
not large enough to evidence significant liaisons but gave similar tendencies
particularly for Amp-1 and Pgd-1. Finally, pooled data allowed to keep slight
linkage only for these two isozyme loci with presumed D1 and D2 genes
respectively. This result was quite consistent with the total information on
reciprocal backcrosses.
Interspecific backcross progenies are suitable to construct saturated RFLP maps since polymorphism level is high and effects of pseudolinkage seem reduced in comparison to Indica-Japonica combinations. Thus, our six isozyme loci were found to segregate independently according to their location on isozyme linkage map (Pham et al., 1990). Nevertheless, segregation distortions were also observed in the mapped backcross population and some portions of chromosomes show lowered frequencies of RFLP markers coming from O. longistaminata. These modifications of frequencies can be due to major effects of D1-D2 complementations but can include interactions with other genes involved in sterility and which are also components of reproductive barrier occuring in these
Table 3. Estimates of recombination values between five isozyme markers and
presumed D1 and D2 genes controlling reproductive barrier between O. sativa
and O. longistaminata
===============================================================================
Recombination value with D1 Recombination value with D2
===================================== =================================
Isozyme Source of data Source of data
locus ========================= =========================
Test-cross with BCL BCS Global Test-cross with BCS BCL Global
=================== estimation =================== estima-
5% 1% 0.1% 5% 1% 0.1% tion
===============================================================================
AMP-1 0.42 0.38 0.36 0.38 0.386* 0.54 0.50 0.56 0.50 0.511
Enp-1 0.40 0.37 0.38 0.42 0.395 0.42 0.38 0.50 0.67 0.597
Sdh-1 0.47 0.50 0.58 0.54 0.523 0.54 0.42 0.46 0.53 0.516
Pgd-1 0.51 0.51 0.55 0.46 0.502 0.54 0.35 0.38 0.35 0.373*
Est-2 0.51 0.49 0.47 0.38 0.470 0.54 0.46 0.46 0.65 0.603
===============================================================================
* P<0.05
interspecific progenies. Two RFLP clones, RG2 and CDO348, are flanking Pgd-1
locus and are concerned by such a reduction of frequency, they provide a
reliable confirmation that this segment on chromosome 11 is bearing a QTL
involved in the reproductive barrier between O. longistaminata and O. sativa.
References
Allard, R. W., 1956. Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24: 235-278.
Chu Y. E. and H. I. Oka, 1970. The genetic basis of crossing barriers between Oryza perennis subsp. barthii and its related taxa. Evolution 24: 135-144.
Ghesquiere A. 1990. Reexamination of the genetic control of the reproductive barrier between Oryza longistaminata and O. sativa and relationship with the rhizome expression. In Rice Genetics II. IRRI, P.O. 933, Manila, Philippines. 729-730.
Pham J. L., J. C. Glaszmann, R. Sano, P. Barbier, A. Ghesquiere and G. Second. 1990. Isozyme markers in rice: genetic analysis and linkage relationship. Genome 33: 348-359.
Tanksley S. D., N. Ahn, M. Causse, J. Chungwongse, T. Fulton, P. Ronald, G. Second, K. Wu, Z. Yu, Z. Wang and J. Xiao. 1991. Molecular mapping of the rice genome. Fifth annual meeting of the Rockefeller Foundation International Program on rice biotechnology. Tucson, Arizona, USA. October 2-5, 1991. Abstracts: p. 3-4.