Theoretical and Applied Genetics 39, 206--213 (1969)
Some Genetic Aspects of the Symbiotic Relationship Between White Clover ( Trifolium repens) and Rhizobium Trifolii V. CONNOLLY 1, C. L. MASTERSON 2 a n d D. CONNIFFE 3 P l a n t B r e e d i n g Dept., A g r i c u l t u r a l I n s t i t u t e , Carlow 1, Soil Biology Dept., A g r i c u l t u r a l I n s t i t u t e , W e x f o r d z, Statistics Dept., A g r i c u l t u r a l I n s t i t u t e , D u b l i n s Summary. The results of experiments with white clover (Tri/olium repens) in which time of nodulation and seedling plant weight or vigour were measured are reported. Experiments 1 and 2 were conducted in artificial growth medium in test tubes with controlled inoculation and experiment 3 in soil without controlled inoculation. Experiment I which was preliminary in nature showed the extent of the variation for time of nodulation after inoculation with Rhizobium tri[olii. I t was evident also that plant vigour and the number of days to nodulation were negatively correlated. Experiment 2 forms the major part of the results and is concerned with the analyses and interpretation of the diallel cross progeny of twelve plants selected from experiment 1. The results indicated a rather complex genetic picture for the two characters measured, namely days to nodulation and seedling plant weight at 80 days. Reciprocal (both general and specific) as well as additive (g.c.a.) and non-additive (s.c.a.) effects were present. Experiment 3, in which seed from 22 families of the diallel cross was sown in soil without controlled inoculation, indicated that the results obtained under the laboratory conditions of experiment 2 and those obtained in soil conditions were not correlated. The implications of these results in relation to selection of improved varieties of the host species are discussed.
Introduction T h e s y m b i o t i c r e l a t i o n s h i p b e t w e e n legume a n d n o d u l e b a c t e r i a is one of considerable biological i m p o r t a n c e . The genetic aspect of this r e l a t i o n s h i p is the m a i n i t e m of i n t e r e s t in this c o n t r i b u t i o n . NUTMAN (t946a, b ; 1954a, b ; t959) w o r k i n g with red clover (TriJolium pratense) has d e m o n s t r a t e d b y genetic analysis a n d selection e x p e r i m e n t s t h a t n o d u l e size a n d n u m b e r , earliness a n d lateness of n o d u l a t i o n a n d effectiveness of response all show genetic v a r i a tion. Similarly JONES (t960; t 9 6 2 a , b ; 1963; JONES a n d BURROWS ( t 9 6 8 ) w o r k i n g with white clover (Tri[olium repens) a n d GIBSON'S (1962) studies on lucerne (Medicago sativa) have shown t h a t genetic v a r i a t i o n was p r e s e n t for the characters studied. T h e m a i n objectives of the e x p e r i m e n t s reported here were: (a) to i n v e s t i g a t e in some detail the t y p e s of genetic v a r i a t i o n controlling two characters, n u m b e r of days to p r i m a r y n o d u l a t i o n a n d seedling p l a n t weight at 6 0 - - 8 0 d a y s ; (b) to determ i n e to w h a t e x t e n t the o b s e r v a t i o n s on seedling p l a n t s u n d e r l a b o r a t o r y c o n d i t i o n s m a y be of use in a plant breeding programme.
Materials and Methods The legume species used throughout was white clover
(Tri/olium repens), variety S. 100. In the first two experi-
ments (numbered I and 2) the clover seedlings were grown on nitrogen-free agar medium similar to that described by PURCHASE and VINCENT (1949). Before sowing, the seeds were surface sterilised in mercuric chloride and washed in distilled water. The seeds were germinated on agar plates at 25 ~ The germinated seeds were transferred to 6 in. test-tubes containing nitrogenfree agar medium and after five days each seedling was inoculated with Rhizobium tri[olii strain J. 12. Previous
work had shown that this strain gave an effective response with white clover. All experiments were conducted in a glasshouse during the summer and a u t u m n months of 1962, 1964, 1965.
Experiment I This was a preliminary experiment carried out in the summer of 1962, the object of which was to determine the range of effectiveness of response with strain J. 12 and to provide plants for further work. Five hundred seeds of S. 100 white clover were grown as described above, the n u m b e r of days to primary nodulation was recorded and the effectiveness of the symbiosis was visually assessed after 60 days. This assessment was based on height, n u m b e r of leaves and general vigour of each plant in a manner similar to that described by NUTMAN (1954a). Experiment 2 Experiment 2 was based on the diallel cross progeny of 12 parents selected from the plants of experiment 1. I t was observed (experiment 1) that while there was a correlation between time of nodulation and plant vigour, m a n y plants which were slow to nodulate were among the most vigorous group and vice versa. Plants showing various combinations of the two characters were selected as parents for the diallel cross. The parents were not therefore a random sample from the initial population of plants. Selfed progeny could not be obtained in the diallel set of crosses since the incompatibility mechanism in white clover effectively prevents self-fertilisation; reciprocal crosses were kept separate. The crosses were made by hand without emasculation and all parents were tested for self incompatibility by self pollinating approximately 500 florets on each parent plant. All parents were highly self sterile. For each cross about 250 florets were poliinated. A sample of seed from each family was surface sterilised and germinated in petri-dishes. Fifteen seedlings from each family were transferred to nitrogen-free agar medium in numbered test tubes and inoculated with Rhizobium
Symbiotic Relationship Between W h i t e Clover and Rhizobium Trifolii
Vol. 39, No. 5
tri]olii strain J. 12. T h e e x p e r i m e n t a l design consisted of a single block in w h i c h each p l a n t was located at r a n d o m . 1Randomisation was p e r f o r m e d before inoculation. T h e r o o t s y s t e m of each p l a n t was e x a m i n e d each d a y u n t i l t h e first n o d u l a r swelling was observed. This was t a k e n as t h e t i m e of p r i m a r y nodulation. T h e n u m b e r of d a y s f r o m inoculation to p r i m a r y n o d u l a t i o n and p l a n t fresh w e i g h t at 80 days were recorded for each plant.
207
The Model: Y q ---- I~ + gi + gi + sii + mi + rii + e i i where # = overall mean g~ (gi) = g e n e r a l c o m b i n i n g a b i l i t y effect (g.c.a.) of ith (jth) p a r e n t
Experiment 3 This experiment was conducted to determine w h a t correlation, if any, was present between the results obtained in test-tubes and those observed under soil conditions. A small sample of seed from each family of the maternal arrays of parents 4 and 11 were germinated. Ten germinated seeds of each family were sown in soil in 5112 in. pots. The soil used in this experiment was a light t e x t u r e d s a n d y soil of low nitrogen status (Screen series; GARDINER and RYAN t964). Suitable amounts of potassium, phosphorus and trace elements were a d d e d and the p H was raised with lime to about 6.9. The seed was not inoculated in this experiment, nodulation was effected b y the rhizobium in the soil. The experimental design was similar to t h a t for experiment 2, i.e. single p l a n t randomisation of all the 220 plants in the experiment. The plants were grown in the glasshouse in the summer and a u t u m n of t965. Three harvests were t a k e n from each plant, fresh weight and d r y weight for each harvest was determined.
sii = specific c o m b i n i n g a b i l i t y effect (s.c.a.) of t h e i ~'th cross mi :
g e n e r a l r e c i p r o c a l effect (g.r.e.) of t h e i m parent rij = specific r e c i p r o c a l effect (s.r.e.) of t h e (i j)m v e r s u s t h e (i i)m cross eii= Random error
T h e effects were s u b j e c t to t h e following r e s t r i c t i o n s : z~ gi = 0 i sii : sii;
Z sii = 0 = Z sii i
i
i,i
i,i
i r~i = _ ri~;
Z rii = 0 = Z ri~ i i i.i j~-i
eil were a s s u m e d to be N(0, a*)
Statistical Analysis The Model: The analysis and interpretation apply o n l y to t h e g r o u p of p a r e n t s w h i c h were s e l e c t e d in a n o n - r a n d o m m a n n e r as d e s c r i b e d in t h e p r e v i o u s section. T h e a p p r o p r i a t e a n a l y s i s was t h e r e f o r e t h a t of a m o d e l I or f i x e d effects m o d e l as o u t l i n e d b y ]~ISENHART (1947). T h e s t a t i s t i c a l m o d e l is t h e s a m e as Model A of EISEX et al. (t966) a n d t h e d e f i n i t i o n of t h e p a r a m e t e r s follows t h a t of EISEN et al. (t967).
T h e difference b e t w e e n this m o d e l a n d t h a t of GRIFFING (1956; M e t h o d 3, f i x e d effects) is t h a t t h e recip r o c a l s u m of s q u a r e s are p a r t i t i o n e d i n t o g e n e r a l a n d specific r e c i p r o c a l differences. EISEN et al. (1966) has discussed the relationship between the estimates of t h e p a r a m e t e r s d e r i v e d f r o m b o t h models. T h e l a t t e r does n o t give t h e e x p e c t a t i o n s of m e a n s q u a r e s of t h e p r e s e n t m o d e l a n d t h e s e are t h e r e f o r e p r e s e n t e d in T a b l e 1.
T a b l e 1. Sums o/squares and expectation o/mean squares/or diallel analysis Source of variation
D.F.
S.S.*
Expectation of mean squares
S.S.g.e.a.
a 2 + 2 ( p - - 2) ~
s.s.s.c,a.
~ + 2
p (p
~
Some Genetic Aspects of the Symbiotic Relationship Between White Clover ( Trifolium repens) and Rhizobium Trifolii V. CONNOLLY 1, C. L. MASTERSON 2 a n d D. CONNIFFE 3 P l a n t B r e e d i n g Dept., A g r i c u l t u r a l I n s t i t u t e , Carlow 1, Soil Biology Dept., A g r i c u l t u r a l I n s t i t u t e , W e x f o r d z, Statistics Dept., A g r i c u l t u r a l I n s t i t u t e , D u b l i n s Summary. The results of experiments with white clover (Tri/olium repens) in which time of nodulation and seedling plant weight or vigour were measured are reported. Experiments 1 and 2 were conducted in artificial growth medium in test tubes with controlled inoculation and experiment 3 in soil without controlled inoculation. Experiment I which was preliminary in nature showed the extent of the variation for time of nodulation after inoculation with Rhizobium tri[olii. I t was evident also that plant vigour and the number of days to nodulation were negatively correlated. Experiment 2 forms the major part of the results and is concerned with the analyses and interpretation of the diallel cross progeny of twelve plants selected from experiment 1. The results indicated a rather complex genetic picture for the two characters measured, namely days to nodulation and seedling plant weight at 80 days. Reciprocal (both general and specific) as well as additive (g.c.a.) and non-additive (s.c.a.) effects were present. Experiment 3, in which seed from 22 families of the diallel cross was sown in soil without controlled inoculation, indicated that the results obtained under the laboratory conditions of experiment 2 and those obtained in soil conditions were not correlated. The implications of these results in relation to selection of improved varieties of the host species are discussed.
Introduction T h e s y m b i o t i c r e l a t i o n s h i p b e t w e e n legume a n d n o d u l e b a c t e r i a is one of considerable biological i m p o r t a n c e . The genetic aspect of this r e l a t i o n s h i p is the m a i n i t e m of i n t e r e s t in this c o n t r i b u t i o n . NUTMAN (t946a, b ; 1954a, b ; t959) w o r k i n g with red clover (TriJolium pratense) has d e m o n s t r a t e d b y genetic analysis a n d selection e x p e r i m e n t s t h a t n o d u l e size a n d n u m b e r , earliness a n d lateness of n o d u l a t i o n a n d effectiveness of response all show genetic v a r i a tion. Similarly JONES (t960; t 9 6 2 a , b ; 1963; JONES a n d BURROWS ( t 9 6 8 ) w o r k i n g with white clover (Tri[olium repens) a n d GIBSON'S (1962) studies on lucerne (Medicago sativa) have shown t h a t genetic v a r i a t i o n was p r e s e n t for the characters studied. T h e m a i n objectives of the e x p e r i m e n t s reported here were: (a) to i n v e s t i g a t e in some detail the t y p e s of genetic v a r i a t i o n controlling two characters, n u m b e r of days to p r i m a r y n o d u l a t i o n a n d seedling p l a n t weight at 6 0 - - 8 0 d a y s ; (b) to determ i n e to w h a t e x t e n t the o b s e r v a t i o n s on seedling p l a n t s u n d e r l a b o r a t o r y c o n d i t i o n s m a y be of use in a plant breeding programme.
Materials and Methods The legume species used throughout was white clover
(Tri/olium repens), variety S. 100. In the first two experi-
ments (numbered I and 2) the clover seedlings were grown on nitrogen-free agar medium similar to that described by PURCHASE and VINCENT (1949). Before sowing, the seeds were surface sterilised in mercuric chloride and washed in distilled water. The seeds were germinated on agar plates at 25 ~ The germinated seeds were transferred to 6 in. test-tubes containing nitrogenfree agar medium and after five days each seedling was inoculated with Rhizobium tri[olii strain J. 12. Previous
work had shown that this strain gave an effective response with white clover. All experiments were conducted in a glasshouse during the summer and a u t u m n months of 1962, 1964, 1965.
Experiment I This was a preliminary experiment carried out in the summer of 1962, the object of which was to determine the range of effectiveness of response with strain J. 12 and to provide plants for further work. Five hundred seeds of S. 100 white clover were grown as described above, the n u m b e r of days to primary nodulation was recorded and the effectiveness of the symbiosis was visually assessed after 60 days. This assessment was based on height, n u m b e r of leaves and general vigour of each plant in a manner similar to that described by NUTMAN (1954a). Experiment 2 Experiment 2 was based on the diallel cross progeny of 12 parents selected from the plants of experiment 1. I t was observed (experiment 1) that while there was a correlation between time of nodulation and plant vigour, m a n y plants which were slow to nodulate were among the most vigorous group and vice versa. Plants showing various combinations of the two characters were selected as parents for the diallel cross. The parents were not therefore a random sample from the initial population of plants. Selfed progeny could not be obtained in the diallel set of crosses since the incompatibility mechanism in white clover effectively prevents self-fertilisation; reciprocal crosses were kept separate. The crosses were made by hand without emasculation and all parents were tested for self incompatibility by self pollinating approximately 500 florets on each parent plant. All parents were highly self sterile. For each cross about 250 florets were poliinated. A sample of seed from each family was surface sterilised and germinated in petri-dishes. Fifteen seedlings from each family were transferred to nitrogen-free agar medium in numbered test tubes and inoculated with Rhizobium
Symbiotic Relationship Between W h i t e Clover and Rhizobium Trifolii
Vol. 39, No. 5
tri]olii strain J. 12. T h e e x p e r i m e n t a l design consisted of a single block in w h i c h each p l a n t was located at r a n d o m . 1Randomisation was p e r f o r m e d before inoculation. T h e r o o t s y s t e m of each p l a n t was e x a m i n e d each d a y u n t i l t h e first n o d u l a r swelling was observed. This was t a k e n as t h e t i m e of p r i m a r y nodulation. T h e n u m b e r of d a y s f r o m inoculation to p r i m a r y n o d u l a t i o n and p l a n t fresh w e i g h t at 80 days were recorded for each plant.
207
The Model: Y q ---- I~ + gi + gi + sii + mi + rii + e i i where # = overall mean g~ (gi) = g e n e r a l c o m b i n i n g a b i l i t y effect (g.c.a.) of ith (jth) p a r e n t
Experiment 3 This experiment was conducted to determine w h a t correlation, if any, was present between the results obtained in test-tubes and those observed under soil conditions. A small sample of seed from each family of the maternal arrays of parents 4 and 11 were germinated. Ten germinated seeds of each family were sown in soil in 5112 in. pots. The soil used in this experiment was a light t e x t u r e d s a n d y soil of low nitrogen status (Screen series; GARDINER and RYAN t964). Suitable amounts of potassium, phosphorus and trace elements were a d d e d and the p H was raised with lime to about 6.9. The seed was not inoculated in this experiment, nodulation was effected b y the rhizobium in the soil. The experimental design was similar to t h a t for experiment 2, i.e. single p l a n t randomisation of all the 220 plants in the experiment. The plants were grown in the glasshouse in the summer and a u t u m n of t965. Three harvests were t a k e n from each plant, fresh weight and d r y weight for each harvest was determined.
sii = specific c o m b i n i n g a b i l i t y effect (s.c.a.) of t h e i ~'th cross mi :
g e n e r a l r e c i p r o c a l effect (g.r.e.) of t h e i m parent rij = specific r e c i p r o c a l effect (s.r.e.) of t h e (i j)m v e r s u s t h e (i i)m cross eii= Random error
T h e effects were s u b j e c t to t h e following r e s t r i c t i o n s : z~ gi = 0 i sii : sii;
Z sii = 0 = Z sii i
i
i,i
i,i
i r~i = _ ri~;
Z rii = 0 = Z ri~ i i i.i j~-i
eil were a s s u m e d to be N(0, a*)
Statistical Analysis The Model: The analysis and interpretation apply o n l y to t h e g r o u p of p a r e n t s w h i c h were s e l e c t e d in a n o n - r a n d o m m a n n e r as d e s c r i b e d in t h e p r e v i o u s section. T h e a p p r o p r i a t e a n a l y s i s was t h e r e f o r e t h a t of a m o d e l I or f i x e d effects m o d e l as o u t l i n e d b y ]~ISENHART (1947). T h e s t a t i s t i c a l m o d e l is t h e s a m e as Model A of EISEX et al. (t966) a n d t h e d e f i n i t i o n of t h e p a r a m e t e r s follows t h a t of EISEN et al. (t967).
T h e difference b e t w e e n this m o d e l a n d t h a t of GRIFFING (1956; M e t h o d 3, f i x e d effects) is t h a t t h e recip r o c a l s u m of s q u a r e s are p a r t i t i o n e d i n t o g e n e r a l a n d specific r e c i p r o c a l differences. EISEN et al. (1966) has discussed the relationship between the estimates of t h e p a r a m e t e r s d e r i v e d f r o m b o t h models. T h e l a t t e r does n o t give t h e e x p e c t a t i o n s of m e a n s q u a r e s of t h e p r e s e n t m o d e l a n d t h e s e are t h e r e f o r e p r e s e n t e d in T a b l e 1.
T a b l e 1. Sums o/squares and expectation o/mean squares/or diallel analysis Source of variation
D.F.
S.S.*
Expectation of mean squares
S.S.g.e.a.
a 2 + 2 ( p - - 2) ~
s.s.s.c,a.
~ + 2
p (p
~
