2. Linkage study between molecular markers and genes controlling the reproductive barrier in interspecific backcross between O. sativa and O. longistaminata

A. Ghesquiere1 and M. Causse2

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.