Diallel analysis of nitrogen fixation in the rhizosphere of rice

Plant Science Letters, 30 (1983) 129--135 Elsevier Scientific Publ/shers Ireland Ltd.

129

DIALLEL ANALYSIS OF NITROGEN FIXATION IN THE RHIZOSPHERE OF RICE*

S. IYAMA, Y. SANO and T. FUJII** Dept. of Applied Genetics, Genetic Stocks Center, National lnst. of Genetics, Miahima 411 (Japan)

(Received June 9th, 1982) (Revision received November 17th, 1982) (Accepted November 20th, 1982)

SUMMARY

The genetics of nitrogen fLxing ability in the rhizosphere of rice (Oryza sativa L.) was investigated by diaUel analysis. In an 11 × 11 diallel set, WrVr graph analysis revealed the presence of a non-allelic interaction. Excluding the interacting parents, a 7 X 7 diallel set gave more uniform (Wr - Vr) values and regression of nearly unity, indicating the disappearance of nonallelic interaction. The regression line indicated partial dominance. Estimates of genetic c o m p o n e n t s of variation were in close agreement with the results from Wr-Vr graph analysis; the average degree o f dominance was in the range of partial dominance and an excess of dominant alleles over recessive alleles. Correlation between parental performance and (Wr + Vr) values suggested that nitrogen fLxing activity in the rhizosphere of the highest parent was likely governed by recessive alleles. Key words: Acetylene reduction --Nitrogenase activity - - O r y z a sativa L.

INTRODUCTION Since nitrogen fixation was detected in non-leguminous plants by Doebereiner et al. [1,2], much work has been done on nitrogen fixation in the rhizosphere of rice (Oryza sativa L.) and other gramineous plants [1--7]. There have, however, been only a limited n u m b e r o f reports of investigation *Contribution from National Institute of Genetics, Mishima, Japan. No. 1464. This research was supported by Grant-in-Aid for Special Project Research 56112019 from the Ministry of Education, Science and Culture, Japan. **To whom correspondence should be sent.

0804-42111831503.00 © 1983 Elsevier Scientific PubllshmmIreland Ltd. Printed and Published in Ireland

130

of the genetic aspects of nitrogen fixation in the rice rhizosphere. Hirota et al. [8] examined the nitrogen fixing activity in the rhizosphere of 50 different strains of rice and found genetic variation for the activity. Sano et al. [9] investigated nitrogen fixing activity of di~erent Oryza species and found significant variation among and within species. Lee et al. [ 10] w.vestigated 40 rice varieties and found varietal differences in nitrogen fixing activity. To our knowledge, so far there have been no reports of breeding experiments on this character to date. Neal and Larson [11] reported in wheat (Triticum aestivurn L.) that the rhizosphere soil of a substitution line which had the 5D chromosome of Rescue substituted into the Cadet genome showed high activity of nitrogen fixation. Kennie and Larson [12] also studied the effect of a particular chromosome substituted into the recipient genome using the disomic substitution line between Rescue and Cadet and found that plants with certain chromosomes showed an increase of nitrogen fixation in association with specific microorganisms. It should be one of the most challenging subjects in crop breeding to improve the nitrogen fLXingactivity of rhizosphere, assuming that this increased activity is associated with increased crop productivity. It is evident that nitrogen fixation in the rhizosphere of rice is expressed by a combination of host plant characteristics and that of the bacteria in the rhizosphere of the soil [8]. To improve the nitrogen fixing activity in the rice rhizosphere, the following three aspects may be useful: (1) improving rice genotypes, (2) improving bacterial genotypes for efficiency and (3) improving both rice and bacterial genotypes for efficient cooperation. The purpose of this research was to study the inheritance of the nitrogen fixing ability in the rhizosphere of rice by diallel cross analysis [ 13,14]. MATERIALS AND METHODS Diallel crosses were made among 11 strains of rice chosen from the collection kept at the Genetic Stocks Center, National Institute of Genetics (Mishima, Japan). Parents were chosen to cover a wide range of origins as shown in Table I. FI plants were obtained from all possible combinations with some reciprocals. The seeds were germinated in petri dishes in midApril and the seedlings were grown in the greenhouse for 4 weeks and then transplanted individually into a plastic pot (7.2 cm diameter) containing 180 g of soil (pH 6.5). The soil was fumic wet and andosol (Aquic Eutrandept). Pots were kept flooded until harvest. Fertilizer was applied at the rate of 20 mg N, 24 mg P2Os, and 24 mg K20/pot as a basal dressing. Since the rhizosphere of the rice plant exhibits highest activity at the heading stage [ 9], plants were subjected to the measurement of nitrogen fixation 1 day after their heading. Plants reached the heading from August to October. Nitrogen fixation was measured by the acetylene reduction method following the system developed by Hirota et al. [8] and the modi~'ed proceduxe of Sano et al. [9] for handling a large quantity of materials. In order properly

131 t o m e a s u r e t h e n i t r o g e n f i x a t i o n activity in t h e r h i z o s p h e r e o f rice b y the a c e t y l e n e r e d u c t i o n m e t h o d , it is i m p o r t a n t to ensure a c e t y l e n e as a substrate effectively reaches t h e i n n e r p a r t o f t h e sample w h e r e n i t r o g e n f i x a t i o n is active w i t h o u t d i s t u r b i n g t h e r o o t s y s t e m [ 8 , 9 ] . Briefly, the p r o c e d u r e f o r m e a s u r i n g activity was as follows: T h e t o p o f t h e p l a n t was c u t o f f at the

base and removed, and the remainder, with soil,was carefully enclosed in a 900-ml glassjar without disturbing the root system. Rigorous displacement of the gas phase within the jar with anaerobic assay gas was achieved by evacuation and introduction of the gas mixture (C2H~ 10% and Ar 90%) twice and then 30 min later for the third time. Then the jars were incubated at 30°C. After 1 and 2 h of incubation, the amount of C2H4 released in the jar was assayed by using gas~hromatography. The activity was expressed by the amount of C2H4 produced/h/g dry root. The number of F, plants used varied among crosses, ranging from 2 to 6 with a mean of 3.8. Ten plants were grown of each parent strain, being divided into two replications of 5 each. Data were analyzed by the method of diallelanalysis developed by H a y m a n [13]. W h e n the reciprocal crosses were available, means of the reciprocals were used for the analysis of diaUel table. Environmental variance for parental plot means (E) were estimated on the basis of replication in parental strains. Environmental variance for F, plots (E') utilized the above variance E after adjustment by the average number of replicates, i.e.reciprocals. Since there was no replication of the diallelset in this experiment, the standard error of estimates of variance components was calculated from the variance of ( W r -- Vr) as Hayrnan suggested [13]. Wr and V r stand for the covariance between parent and F~, and variance in the rth array of diallel table. TABLE I ORIGIN OF PARENT STRAINS AND NITROGEN FIXING ACTIVITY IN THEIR RHIZOSPHERE Activity expressed as C2H~ produced nmol/h/g dry root. Parent

Acc~sion code

Origin

C2H4 produced (nmol/h/g dry root)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 Pll

T65 108 W339 C5053 C5059 C5080 C5444 C5877 C6063 C8005 C8820

Taiwau Taiwan India India India India India India Thailand India Japan

267 422 229 631 479 401 496 446 576 301 379

± 32 * 43 ± 16 ± 116 * 112 * 74 • 89 * 70 ~ 77 * 47 ~ 52

132 RESULTS Nitrogen fixing activity in the rhizosphere of 11 rice strains used in the diallel cross is presented in Table I. Analysis of variance conducted on the basis of replication means revealed that there was statistically significant variation among the strains (0.01 < P < 0.03). Most F~ plants showed nitrogen fixing activity within the range of those of the parents. It was noticed that F~ between the two highest parents, C5053 and C6063, exceeded the level of the higher parent, i.e., 679 nmol/h/g dry root. Regression coefficient of F, progeny means on their common parents was 0.34, being statistically significant (P < 0.01). A Wr-Vr graph analysis for the 11 X 11 diaUel set, showed that the regression of Wr on Vr differed significantly from unity (b = 0.41, P < 0.05) and (Wr - Vr) was not uniform, suggesting the presence of non-allelic interactions, application of the additive-dominance model being inadequate (Fig. 1). Hence, after eliminating the interacting arrays, P2, P6, P7 and P10, the resulting 7 X 7 diallel set of crosses were reanalysed. In this diallel set, V a r ( W r - Vr) was reduced to 1/3 of the variance in the original 11 X 11 diallel set. The following points were revealed by the Wr- Vr graph (Fig. 2): (1) the regression of Wr on Vr. signLficantly differed from zero (b = 0.91, P <

Wr ×]0 4

1

P4 ~ .~

!

.

-

P3 0

• "

"P2

t

!

1

2

Fig. 1. Wr-Vr graph of 11 X 11 diallel set.

Vr

xlO 4

133

x104 Wr 2

•P3 W•. P8 PI l

i

I

I

2

Vr

xl04

Fig. 2. Wr-Vr graph of 7 x 7 diallel set. 0.01) and did n o t differ from unity (P > 0.6), indicating the absence of nonallelic interactions; (2) the regression line crossed the Wr axis closely above the origin, indicating nearly complete dominance; (3) from the position along the regression line, it was indicated that C5877 and C8820 possessed (Wr+Vr)

Recessive 2 r = 0.62 J

•P4

1

• P9

Pl I

!

-2

-l

Low activity

0 P5

Pll

~1

-

I

I Yr

1

2 High activity

P8

-2

Dominant Fig. 3. Standardized deviations (Yr, Wr + Yr) graph for 7 X 7 diallel set.

134 TA~BLEII ESTIMATES OF GENETIC PARAMETERS FOR NITROGEN FIXING ACTIVITY IN THE RHIZOSPHERE OF RICE Expressed as nmol C2H4 produced/h/g dry root. Genetic parameter

Estimate a

D HI H~

1.7059 0.1464 0.0904 0.6627

F h2 E

a- = HI/D KD/K R = ( ~ K = h3/H~ uv = HJ4H I

± 0.1650 ~ 0.3972 ± 0.3500 -* 0.3958

--0.2662 -* 0.2453 0.5292 -* 0.0583

+ F)/(x/'4-D-H~ --- F)

0.29 4.94 0.15

aEstimates D, H~, H~, F, h ~ and E are to be multiplied by 104.

the greatest excess of d o m i n a n t alleles, C5059 and W339 an excess of dominant, C6063 and C5053 an excess of recessive alleles, and T65 was more or less intermediate. The correlation between parental value (Yr) and (Wr + Vr) calculated was n o t significant (r = 0.62), indicating that the nearly equal proportions of d o m i n a n t and recessive alleles are positive and negative, but high activities of C5053 and C6063 are governed by recessive alleles, while low activity of W339 by d o m i n a n t alleles (Fig. 3). Estimates of the genetic components of variation are given in Table II. Measures of average degree of dominance (a-), the ratio of number of d o m i n a n t alleles to recessive (K D/K R ) and average frequency of negative (u) vs. positive (v) alleles at loci exhibiting dominance in the parents (~'d) were calculated as shown in Table II. Though the estimates indicated the tendency of partial dominance and excess of d o m i n a n t over recessive alleles, and an inequality in the positive and negative alleles, they were n o t reliable since the estimates of F, HI and H2 were small compared with their error. The mean of FI plants (roLl) was nearly equal to the mean of the parent (mL0), resulting in the negative estimate of h 2. Hence, the number of effective factors [15] which are showing some dominance was n o t calculated. DISCUSSION As Jinks [ 14] stated, diallel cross analysis is a powerful m e t h o d for obtaining a rapid, overall picture of genetical structure of a number of parental lines. Inferences can be obtained even from the analysis o f FI data. A WrVr graph analysis of original 11 × 11 diailel set indicated the presence of non~allelic interaction. ~n the study of nitrogen fixation of a substitution line of wheat, Neal and L s n o n [11] s u ~ t h a t t h e i n t e r a c t i o n b e t w e e n the 5D chromosome of Rescue and one or more genes on the recipient Cadet

135

chromosomes might be responsible for the high activity f o u n d in the rhlzosphere soil of the substitution line since neither parent showed such high activity. This indicates possible existence of non-allelic interaction in some parent combinations. After excluding the interacting army, the resulting 7 X 7 diallel set fits well to the assumption of no non-allelic interaction. The results of the present experiment revealed that nitrogen fixation of the rice rhizosphere was heritable and t h a t this activity may be considered as a quantitative character. This implies the possibility of improving the rice genotypes through hybridization and selection among resulting recombinants. Since partial to complete dominance was found, it may be inefficient to apply selection in early generations, but screening may be postponed to later generations. The evidence which showed the presence of recessive genes o f positive effects also indicated the inadequacy 9 f selection in early generations. It is suggested that there may be non-allelic interaction in certain cross combinations. If the non-aUelic interaction is the favorable additive and additive t y p e of interaction, it can be fixed in the progeny genotypes. Considering the fairly large variation among parental strains [8,9] it is likely that there may be at least several genes involved relative to the character. If this is true, the selection limit will n o t be reached easily; however, possibly we can accumulate the favorable genes into a genotype to get much higher activity. The experiment using the later segregating generations is in progress. ACKNOWLEDGEMENT Authors wish to t h a n k Drs. Y. Hirota and H. Morishima for their discussion t h r o u g h o u t this experiment. REFERENCES

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