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Field Crops Research 55 (1998) 275-281

Field Crops Research

Quantitative genetics of rice IV. A breeding strategy

M.J. L a w r e n c e a,*, D. Senadhira b
a Wolfson Laboratory for Plant Molecular Biology, School of Biological Sciences, University of Birmingham, Birmingham B15 2TT, UK b Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, PO Box 933, Manila 1099, Philippines

Received 28 March 1997; revised 22 July 1997; accepted 23 July 1997

Abstract A systematic and objective breeding strategy is proposed, involving three stages of selection carried out at the F 1, F 3 and F6 generations of pedigrees founded on single crosses between inbred parents. The first two of these stages involve selection between pedigrees and the third, selection within the pedigrees that survive the first two stages of scrutiny. The key features of this procedure are: that selection within pedigrees is delayed until individuals are effectively homozygous; that all characters are measured directly; and that decisions are based on the performance of plants that are raised in replicated and randomised trials. The replacement of the pedigree method by the new method should allow breeders to recognise and exploit the genetical variation between and within their crosses for characters of interest more efficiently than hitherto at an appreciably lower cost. While this new method has been proposed in response to a specific problem with rice, it is applicable to all crop plant species where the end products of breeding programmes are inbred lines. 1998 Elsevier Science B.V.
Keywords: Biometrical breeding method; Self-pollinating crops

I. I n t r o d u c t i o n W e have shown that there is no evidence of a shortage o f additive genetical variation for yield and other characters o f agronomic importance among m o d e m i n d i c a cultivars o f rice that have been bred for use in transplanted crops in the lowland tropics (Perera et al., 1998). One of the chief causes of the inability o f breeders to increase the yield potential of cultivars of IR8 ideotype over the last 30 years (Flinn et al., 1982; Cassman et al., 1994) is, therefore, the use of breeding methods that fail to exploit

* Corresponding author. Tel.: +44-121-414-5915; fax: +44121-414-5925.

the genetical variation that is present in their crosses. The results obtained by Fahim et al. (1998) support this conclusion. Thus, none of the best F6 lines produced by any of the three methods in which selection was practised for yield during the course of inbreeding, the pedigree, modified pedigree and bulk methods, were significantly better than the best lines produced by single seed descent in either o f the crosses investigated. There is little doubt, therefore, that selection for yield in the early generations of these pedigrees has not been very effective. The inability o f breeders to breach an apparent yield potential barrier of 10 t / h a (Peng et al., 1994) with i n d i c a cultivars is, thus, almost certainly due, in part at least, to their use of inefficient breeding procedures.

0378-4290/98/$19.00 1998 Elsevier Science B.V. All rights reserved. PII S0378-4290(97)00088-9

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In previous papers, we have shown how information from the F 3 generation of a pedigree can be used to assess the potential of a cross (Perera et al., 1998; Fahim et al., 1998; Bentota et al., 1998) with respect to one or more characters. It is obviously impractical, however, to expect breeders to advance all of, say, 50 crosses to the F 3 generation to assess their potential. In the present paper, we introduce a new breeding procedure, derived from the results and conclusions obtained from the analyses reported in the previous papers of this series, which allows the potential of crosses to be assessed and ranked in a completely systematic and objective way.

2. The new procedure

The new procedure involves three stages of selection carried out at the F~, F 3 and F6 generations of pedigrees that are founded from a single cross between two homozygous parents. The first two stages involve discrimination between pedigrees and the third, selection within the pedigrees that have survived the first two stages of scrutiny.
2.1. The F 1 stage

The most promising crosses are those in which the parents are identically homozygous for most of the increasing genes determining the character (e.g., yield), but differ for a minority that are partially dispersed between them, because in these circumstances it should be possible to extract recombinant inbred lines from them that outperform the better parent. Parents that are homozygous for many increasing genes can, of course, be identified from their mean performance; those that differ for a minority of increasing genes that are partially dispersed between them, can be recognised by their heterotic F 1 progeny. Thus, in the absence of overdominance, two conditions have to be met for better parent heterosis to occur: first, that the genes controlling the character display directional dominance, and second, that those of similar directional effect are dispersed between the parents. If, therefore, the F 1 of a cross displays heterosis, at least some of the genes for which the parents differ must be dispersed between them; that is, heterosis indicates this dispersion.

Dominance as such is of little interest to the breeder of small-grain cereals, because the products of their breeding programmes are homozygous lines. Here, it serves as no more than a means of detecting dispersion. It is, of course, possible that the genes for which the parents differ, although dispersed between them, do not display directional dominance; since, in these circumstances, their F~ is not expected to display heterosis, such crosses might be overlooked. The empirical evidence, however, suggests that this is unlikely. Thus, in the two investigations in which triple test cross progenies were raised, (the only design yielding independent estimates of the dominance component of variation, H), the majority of the characters recorded, including yield and its components, displayed dominance in the eight crosses analysed, which for most, had a directional element (Perera et al., 1998). A trial of parents and their F 1 progeny should, therefore, allow the breeder to identify the most promising crosses of a cohort. It is essential, however, that this trial should be appropriately randomised, and that plants should be measured for characters of interest, yield, for example, being recorded as the amount of grain produced by the plants of each family in the trial. Although plants were raised in completely randomised designs in our experiments, breeders will probably find it more convenient to raise theirs in small plots, which have the additional advantage of avoiding competition between plants of different genotype. Suppose a breeder starts with 50 crosses involving 100 different parents. Then, a design in which each of the 150 families is represented by a pair of plots containing 10 plants, whose position in the trial is assigned at random, would give unbiased estimates of the means of these families, and would allow statistical tests to be carried out between the parents and their F l progenies. It is possible, however, that plots of this size are known to be poor predictors of performance of plants in larger plots in which individuals are raised at agricultural densities. If this is the case, it would be necessary to increase the size of the plots in this yield trial. The disadvantage of increasing plot size is that, while the amount of parental seed available is unlikely to be a limiting factor, that used to raise the F l progenies could be, because this seed has to be produced by emasculation and hand-crossing.

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As at this first stage of selection, five of the crosses, perhaps, will be retained and the rest discarded.
2.2. The F 3 stage

An analysis of the F 3 families of a pedigree provides information about four points concerning the potential of the cross. First, an analysis of variance of the data from these families allows the breeder to find out whether all characters of interest are heritable; if it turns out that one or more characters are not heritable, the cross is obviously incapable of yielding transgressive segregants for these characters. Second, examination of the F 3 family means may reveal some that achieve the desired target for one or more characters. A cross that exhibits transgressive variation as early as the F 3 generation has obvious potential, since it will certainly do so in later generations of the pedigree. Third, given that a character is heritable and normally distributed, estimates of the mean, m, and additive genetical variance, D, of the distribution of recombinant inbred lines that can be extracted by single seed descent from the cross obtained from these families, can be used to predict the proportion of such lines whose means equal or exceed the desired target for each character in turn (Jinks and Pooni, 1976, 1980). If the probability of obtaining a line of the desired level of performance for an important character appears to be small ( < 5%), the breeder can either increase the number of single seed descent lines extracted from the cross ( > 100), to compensate for this low probability, or discard the cross. Fourth, the components of covariance analyses carried out on all pairs of characters can be used to estimate the genetic correlations between these characters. It has been argued by some that the chief cause of genetic correlation between characters in crop plant species is pleiotropy (e.g., Simmonds, 1979). However, apart from a small number of obvious pleiotropic relationships, such as that between, for example, tiller and panicle number, we have found that the pattern of genetic correlations varies considerably over crosses (Perera, 1985; Fahim, 1995; Sriyoheswaran, 1995; Bentota et al., 1998). This suggests that in rice, at least, the chief cause of these correlations is the linkage disequilibrium of genes that are linked in

their inheritance. This being the case, it is possible to identify crosses where the genetic correlations are favourable to the breeder's objectives; for example, crosses in which the genetic correlations between characters for which greater expression is required are large and positive, are more likely to yield recombinant inbred lines that meet the targets for all of these characters, than those in which these correlations are zero or negative. A knowledge of the genetic correlations between characters, therefore, can assist the breeder in achieving the targets for all characters of interest, and crosses can be ranked on this information, as well as on their potential in respect of single characters. As at the F 1 stage, it is essential that the individuals of the F 3 families should be raised in a randomised trial, and that characters should be measured and not assessed by eye. Each of the crosses which survive the F 1 stage should be represented by 30-40 families that are raised in the same trial. Assuming, again, that breeders would find it convenient to raise these families in plots, each family could be represented by a pair of independently randomised plots containing, say, 10 plants. Duplicate plots of each of the parents should also be included in the trial. Alternatively, where the performance of recommended varieties of the appropriate age class is to be used to provide the targets for improvement, duplicate plots of these should be raised instead. It is difficult to forecast how many of these crosses will survive scrutiny at the F 3 stage of the programme, but, perhaps, the most promising pair might be retained and the rest discarded.
2.3. The F 6 stage

The design of the F6 trial can be similar to that of the F 3 families, each family being represented by a pair of independently randomised 10 plant plots; the trial should also include duplicate plots of the parents of each cross or of recommended varieties, where these are used to provide targets for improvement. The number of families to be included in this trial requires, however, consideration. Thus, while the single character predictions made on the information obtained from F 3 families can be used to suggest the number of F6 families that need to be raised in the trial to give a reasonable chance of obtaining at

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is why a knowledge of the genetic correlations between characters is useful in a qualitative sense, at least, in assessing the odds of obtaining a line that meets all of the desired targets. This problem of achieving the targets for all components of the desired aggregate phenotype in a single recombinant inbred line is, of course, common to all breeding programmes, and is almost certainly as important a cause of the failure to breach the apparent 10 t / h a yield barrier with indica cultivars as the inefficient exploitation of the genetical variation for these components. The conclusion here is obvious, namely, that if breeders want to maximise the chance of obtaining one or more lines that achieve all of the desired targets with one cycle of inbreeding, they should raise as many families in the F6 trial as their resources allow.

least one that meets the desired target for each character, breeders will nearly always want to improve at least several characters simultaneously. In principle, it is possible to calculate the proportion of recombinant inbred lines that meet the desired targets for any number of characters; Pooni and Jinks (1978) have shown how this can be done for two and three characters simultaneously, which take into account the genetic correlations between characters. The calculations involved, however, are rather tedious and are, for this reason, unlikely to be willingly undertaken by breeders. In any case, the chief problem that faces the breeder is that, unless the number of component characters in the aggregate phenotype are few, the probability o f obtaining a line that meets the targets for all is very small. Suppose, for example, that there are three components, x, y and z, of the aggregate phenotype, for each of which increasing expression is desired. Suppose, further, that these characters are completely uncorrelated. Then the proportion of recombinant inbred lines, P , that are expected to achieve the targets for all three components simultaneously is simply the product of the three single character predictions; that is, P = Px Py Pz. Suppose Px = Py = e z = 0.2 or 20%; then P = 0.008 or 0.8% and it would be necessary to raise not less than 570 lines to be 99% certain of obtaining at least one that achieved all three targets simultaneously. If, on the other hand, the genetic correlations between these characters were positive, the proportion of lines that achieved all three targets is expected to be larger than 0.8% by an amount that depends on the magnitude of these correlations; this

2.4. Logistical considerations

As with all breeding programmes, the implementation of this procedure will involve some careful planning. It is assumed that while breeders will want to carry out the F l, F 3 and F6 trials in the dry season, the production of seed, including that of the single seed descent lines, can be carried out at any time of the year in the nursery. It should be possible, therefore, to carry out a complete cycle of the procedure within a period of three years as shown in Table 1. The number of plants shown in the last column of this table assumes that the programme is initiated with 50 crosses, that the best five of these are advanced to the F 3 generation and that two of these

Table 1 A summary of the new breeding method Year Season Generation 1 Wet
Dry

Task Nursery Production of F1 seed

-

Field Fl trial and production of F2 seed F3

No. of plots in trials 300 400 -

Total no. of plants 100 3000 2500 2500 + 4000 1000 1000 20,000

Parents
F1

2 3
-

Wet Dry -

F2 F3 F4 F5 F6

Dry

Production of F3 seed SSD ~ F 4 SSD ~ F5 SSD ~ F6 -

trial

F6 trial

2000

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are advanced to the F6 generation. It is further assumed that each family is represented by 10 plants in each of a pair of independently randomised plots in each of the three trials; that 40 families are raised in each cross in the F 3 trial; and that 500 single seed descent lines are raised in each cross in the F6 trial. The number of plots raised in each of the trials, shown in the penultimate column of Table 1, indicates the sizes of the random permutations required to randomise these trials. To ensure that the single seed descent lines are advanced to the F6 generation by the dry season of the third year of the programme, it will be necessary to initiate the single seed descent phase of the programme as soon as F 2 seed is available, that is, before the results of the F 3 trial are available. Hence, it will be necessary to initiate single seed descent for each of the five crosses in this trial. Once, however, the best pair of crosses have been identified in this trial, attention can be concentrated on advancing them as quickly as possible to the F6 generation and the lines of the remaining three crosses discarded. Again, while the plants of the early generations of the single seed descent phase of the programme can be raised at high densities in trays in the nursery, it will be necessary to raise those of the F5 generation at a lower density to ensure that they produce enough good seed for the F6 trial. 3. Discussion The key features of this new procedure have emerged from the investigations reported in previous papers of this series (Perera et al., 1998; Fahim et al., 1998; Bentota et al., 1998); it is, therefore, well founded on both quantitative genetics theory and the empirical evidence. Because, however, it involves a radical departure from current practice, breeders may initially be reluctant to use this procedure. It is worthwhile, therefore, reiterating the importance of the three chief ways in which this procedure differs from conventional methods. The first of these is that selection within pedigrees is deferred until the F6 generation, the F~ and F 3 stages of the procedure being used solely to discriminate between crosses. With the pedigree method, on the other hand, selection is practised from the F 2 generation onwards, initially on single plant perfor-

mance and later on family means. On theoretical grounds, this selection cannot be very efficient because the heritabilities of characters of interest are, at best usually only moderate. The estimates of the heritability of grain yield in the crosses investigated by Perera et al. (1998), for example, were 16% and 11%, for those in experiment 2, and 31%, 44% and 43%, for those in experiment 3, the latter pair of estimates being unusually high. Estimates of these magnitudes indicate that most of the phenotypic variation seen by the breeder in segregating populations in the early generations of the pedigree is environmental in origin, rather than genetic, so that in attempting to practice selection for yield, the breeder must be doing little more than choosing plants or families that have enjoyed a favourable environment. The empirical evidence is consistent with the theory since, as mentioned earlier, Fahim et al. (1998) found that none of the F6 lines produced by the pedigree method had a significantly higher yield than the best lines produced by single seed descent in either cross. The conclusion here is obvious, namely, that it is better to defer selection within pedigrees until the F6 generation when plants are effectively homozygous for genes controlling characters of interest, so that their family means provide a reliable guide to their genetic merit. Where, however, breeders need to carry out selection for a qualitative character, such as resistance to a particular disease or pest, that is known to be controlled by a single major gene, selection could be carried out on F 2 individuals in the nursery, provided that both the expressivity and penetrance of the gene is known to be complete, and that the screening of these individuals is sufficiently thorough to eliminate escapes. In these special circumstances, single seed descent could be initiated from F 2 individuals known to have the desired properties. Second, with the new procedure, all quantitative characters are measured directly, yield, for example, being scored as the total weight of grain produced by the plants of each plot in the trial. With the pedigree method, on the other hand, the usual practice is to assess characters indirectly by eye in the early generations of pedigrees at least. Apart from the fact that scoring by eye introduces a further source of error, in addition to that caused by uncontrollable environmental variation, causing the heritability of a charac-

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ter to be lower than if the character had been measured, it has long been known on theoretical grounds that indirect selection is expected to give a better response than direct selection on a character only in rather special circumstances, which are unlikely to be encountered in practice (Falconer and Mackay, 1996; Fahim et al., 1998). The conclusion here is again obvious, namely, that characters of interest should be assessed directly, rather than indirectly, and that they should be measured, rather than scored by eye. Third, with the new procedure, all decisions are based on the performance of plants raised in appropriately replicated and randomised trials, so that it is possible to determine whether differences between families are of a magnitude that can be ascribed to sampling variation, or whether, alternatively, these are sufficiently large as to indicate that they differ genetically. With the pedigree method, however, family plots are neither replicated, nor randomised over the area occupied by the trial. In these circumstances, differences between plots caused by the environment are completely confounded with those caused by genetical differences between them, making the recognition of their genetic merit difficult, to say the least. Fisher (1925, 1935) first drew attention to the crucial importance of replication and randomisation in experiments, as a result of his analysis of data from long-term field trials at the Rothamsted Agricultural Research Station; it is surprising, therefore, that it should, apparently, still be necessary to emphasise their importance some 70 years later. Because characters are measured, rather than assessed by eye, the new procedure differs from conventional methods in one further respect: it entails the processing and statistical analysis of a considerable amount of numerical data. In the past, this requirement could have limited the number of characters that breeders could handle. The present widespread availability of personal computers and statistical software, however, now makes this task straightforward. There are three further points worth raising about this procedure, the first of which concerns the task of evaluating F6 families with respect to all characters of interest. It was pointed out earlier that the probability of extracting a line from a cross that achieves the targets for all characters simultaneously is likely

to be very low unless the number of components of the desired aggregate phenotype is small, or the genetic correlations between these components are unusually favourable. The simple calculation on which this conclusion was based, however, tacitly assumed that all components of the aggregate phenotype were of equal importance. This is unlikely to be the case in practice; yield, for example, will almost always be regarded as more important than other components. It is, of course, possible to consider that some components are more important than others when making judgements about the value of lines, as was done subjectively by Fahim et al. (1998) when evaluating their F6 families. This task could be carried out more efficiently, however, by computing an index that recognised not only that the components of the desired aggregate phenotype are genetically correlated, but also that some are more important than others. The construction of selection indices is summarised by Falconer and Mackay (1996), reviewed by Lawrence (1981) and their application to plant breeding by Baker (1986). The use of indices in evaluating and ranking F6 families would put this task on the same objective and systematic basis as the assessment of crosses at the F 1 and F 3 stages of the procedure. Second, although the F 3 and F6 stages of this procedure have been tested on a small scale against three conventional methods with two crosses, which showed it to be at least as effective as the best of the latter at a considerably lower cost (Fahim et al., 1998) in a single season in Sri Lanka, it would, of course, be desirable to carry out larger scale tests involving more crosses, raised in different seasons at different sites in the lowland tropics, in order to demonstrate that its theoretical superiority over the pedigree method was consistently realised in practice. Lastly, although this new breeding method has been developed in the context of solving a particular problem with rice, it is, of course applicable to any crop plant species where the end products of a breeding programme are inbred lines. References
Baker, R., 1986. Selection Indices in Plant Breeding. CRC Press, Boca Raton, FL.

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Bentota, A.P., Senadhira, D., Lawrence, M.J., 1998. Quantitative genetics of rice: III. The potential of a pair of new plant type crosses. Field Crops Res. 55, 267-273. Cassman, K.G., De Datta, S.K., Olk, D.C., Alcantara, J.A., Samson, M., Descalsota, J., Dizon, M., 1994. Yield decline and the nitrogen economy of long-term experiments on continuous, irrigated rice systems in the tropics. In: Lal, R., Stewart, B.A. (Eds.), Experimental Basis for Sustainability and Environmental Quality. Lewis/CRC Publishers, Boca Raton, FL, USA, pp. 181-222. Fahim, M., 1995. Quantitative inheritance of agronomic characters and comparison of different breeding methods in rice (Oryza sativa L.). PhD thesis, Univ. of Birmingham. Fahim, M., Dhanapala, M.P., Senadhira, D., Lawrence, M.J., 1998. Quantitative genetics of rice: II. A comparison of the efficiency of four breeding methods. Field Crops Res. 55, 257-266. Falconer, D.S., Mackay, T.F.C., 1996. Introduction to Quantitative Genetics, 4th edn. Longman, Harlow, Essex. Fisher, R.A., 1925. Statistical Methods for Research Workers, 1st edn. Oliver and Boyd, Edinburgh. Fisher, R.A., 1935. The Design of Experiments, 1st edn. Oliver and Boyd, Edinburgh. Flinn, J.C., De Datta, S.K., Labadan, E., 1982. An analysis of long-term rice yields in a wetland soil. Field Crops Res. 5, 206-210. Jinks, J.L., Pooni, H.S., 1976. Predicting the properties of recombinant inbred lines derived by single seed descent. Heredity 36, 253-266.

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Jinks, J.L., Pooni, H.S., 1980. Comparing predictions of mean performance and environmental sensitivity of recombinant inbred lines based upon F 3 and triple test cross families. Heredity 45, 305-312. Lawrence, M.J., 1981. Multiple trait selection--a review. In: Gallais, A. (Ed.), Quantitative Genetics and Breeding Methods: Proc. of the 4th Meeting of the Biometrics in Plant Breeding Section of EUCARPIA, Poitiers, France, pp. 263284. Peng, S., Khush, G.S., Cassman, K.G., 1994. Evolution of the new plant type ideotype for increased yield potential. In: Kassman, K.G. (Ed.), Breaking the Yield Barrier; Proc. of a Workshop on Rice Yield Potential in Favorable Environments, Int. Rice Res. Inst., Los Bafios, Philippines, pp. 5-20. Perera, A.L.T., 1985. Biometrical studies in rice (Oryza sativa). PhD thesis, Univ. of Birmingham. Perera, A.L.T., Fahim, M., Sriyoheswarn, S., Dhanapala, M.P., Senadhira, D., Lawrence, M.J., 1998. Quantitative genetics of rice: I. No evidence of a shortage of exploitable genetical variation in modem indica cultivars. Field Crops Res. 55, 245-256. Pooni, H.S., Jinks, J.L., 1978. Predicting the properties of recombinant inbred lines derived by single seed descent for two or more characters simultaneously. Heredity 40, 349-361. Simmonds, N.W., 1979. Principles of Crop Improvement. Longman, New York. Sriyoheswaran, S., 1995. Quantitative inheritance of some agronomic characters of three wide crosses of rice. PhD thesis, Univ. of Birmingham.