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CELL BIOLOGY & MOLECULAR GENETICS

Improvement of Bacterial Blight Resistance of `Minghui 63', an Elite Restorer Line of Hybrid Rice, by Molecular Marker-Assisted Selection

^a National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, People's Republic of China

qifazh{at}public.wh.hb.cn

	ABSTRACT

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`Minghui 63' is a restorer line widely used in hybrid rice production in China. However, this line has become increasingly susceptible to bacterial blight (BB), resulting in a rapid decline of its use in rice production. The objective of this study was to improve the BB resistance of Minghui 63 by introgressing Xa21, a broad-spectrum BB resistance gene, into Minghui 63 by molecular marker-assisted selection (MAS). A polymerase chain reaction (PCR)-based MAS system was established consisting of a marker that is a part of Xa21, a marker located at 0.8 centimorgans (cM) from the Xa21 locus on one side, and a marker at 3.0 cM from the gene on the other side. A total of 128 restriction fragment length polymorphism (RFLP) markers, evenly distributed on the 12 chromosomes, were used to recover the genetic background of Minghui 63. The resulting improved version of Minghui 63, or `Minghui 63(Xa21)', was exactly the same as the original except for a fragment of less than 3.8 cM in length surrounding the Xa21 locus. Both Minghui 63(Xa21) and its hybrid with `Zhenshan 97A' referred to as `Shanyou 63(Xa21)' showed the same spectrum of BB resistance as the donor parent. Field examination of a number of agronomic traits showed that Minghui 63(Xa21) and Shanyou 63(Xa21) were identical to Minghui 63 and Shanyou 63, when there was no disease stress. Under heavily diseased conditions, Minghui 63(Xa21) showed significantly higher grain weight and spikelet fertility than Minghui 63, and Shanyou 63(Xa21) was significantly higher than Shanyou 63 in grains per panicle, grain weight, and yield.

Abbreviations: BB, bacterial blight • cM, centimorgan • MAS, marker-assisted selection • PCR, polymerase chain reaction • RFLP, restriction fragment length polymorphism • Xoo, Xanthomonas oryzae pv. oryzae

	INTRODUCTION

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MINGHUI 63 is the restorer line for a number of widely used hybrids in rice production in China. The general characteristics of the hybrids produced with this restorer line include high yield and wide adaptability, which enabled these hybrids to occupy a total area of approximately 7 million hectares per year in the late 1980s and early 1990s, accounting for more than 25% of the total rice production area in China during that period. The most prominent example of these hybrids is `Shanyou 63', a cross between the male sterile line `Zhenshan 97A' and Minghui 63, that by itself accounted for a total area of 6.7 million hectares per year in its peak period. Clearly, this line has made a major contribution to the success of hybrid rice in China.

In recent years, however, there has been a rapid decline of the area planted to these hybrids. One of the main reasons for this reduction of production area is the breakdown of resistance to bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), one of the most destructive diseases of rice worldwide (Mew, 1987). It is known that Minghui 63 carries the BB resistance gene Xa4 (Y. F. Tan, unpublished data), and was considered to be BB resistant when it was first released. However, its resistance has largely become ineffective during the period of extensive cultivation as a result of evolution of the pathogen population.

A large number of genes for BB resistance have been identified that are available for cultivar improvement (Ogawa et al., 1989; Khush et al., 1990; Lin et al., 1996). However, it has been difficult to use these genes to improve the resistance of the parents for the purpose of hybrid improvement. Incorporation of a resistance gene is difficult with conventional breeding methods because of linkage with undesirable traits that is very difficult to break even with many generations of backcrosses (Young and Tanksley, 1989).

Marker-assisted selection (MAS) has been advocated as a highly efficient breeding method, because it can offer rapid and precise selection of the targeted gene (Tanksley et al., 1989). Recent developments in genome research have provided a large number of molecular markers in many crop species and also diverse techniques for detection, which have made MAS a reality for application in breeding programs. In rice, for example, there have been studies demonstrating the feasibility of using MAS to pyramid genes for BB resistance (Yoshimura et al., 1995; Huang et al., 1997).

The objectives of the study reported in this paper were to improve the BB resistance of Minghui 63 by introgressing Xa21, a gene that is highly resistant to a broad spectrum of the pathogen races (Khush et al., 1990), by means of MAS in the process of recurrent backcrossing; and to evaluate the effects of such improvement on the agronomic performance of Minghui 63 and the hybrid under both BB stressed and non-stressed conditions.

	Materials and methods

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Cultivars, Combinations, and Populations
The plant materials used in this study included four indica (Oryza sativa ssp. indica) cultivars (or lines): (i) `IRBB21', an isogenic line of `IR24' containing Xa21 (Ikeda et al., 1991) kindly provided by the International Rice Research Institute, was used as the donor parent of Xa21; (ii) Minghui 63, the restorer line for a number of elite hybrids widely grown in China, was the recipient parent to be improved; (iii) Zhenshan 97A, the male sterile line for a number of widely grown hybrids, was used to make hybrids with Minghui 63 as well as the improved version of Minghui 63; and (iv) `Zhenzhuai', a cultivar highly susceptible to all the Xoo races, was used as a susceptible check.

Xoo Strains, Inoculum Preparation, Inoculation, and Disease Scoring
A total of 17 Xoo strains were used in this study (see Table 1 for representative examples), including nine strains from China kindly provided by Q. Zhang and L. Zhu, five strains from the Philippines provided by T.W. Mew, and three strains from Japan provided by T. Ogawa. The methods of inoculum preparation and inoculation were the same as described previously (Lin et al., 1996). The evaluation of BB resistance of the breeding materials was conducted in the disease nursery of Huazhong Agricultural University, Wuhan, China. For disease scoring, the length of the longest lesions of five undamaged leaves of each individual was measured 21 d after inoculation. A plant was classified as resistant if the average lesion length was shorter than 3.0 cm, moderately resistant if the lesion was 3.0 to 6.0 cm, moderately susceptible if the lesion was 6.0 to 9.0 cm, and susceptible if the lesion was longer than 9.0 cm.

View larger version (14K): [in this window] [in a new window]   Fig. 1 The linkage map of the Xa21 genomic region on rice chromosome 11

The experimental procedures for RFLP assays, including DNA isolation, digestion, electrophoresis, and southern blot hybridization, were done essentially as described previously (Liu et al., 1997). DNA for PCR analysis was isolated according to the method of K.L. Zheng et al. (personal communication). The PCR analysis was conducted essentially according to Williams et al. (1996) except that the annealing temperature was lowered to 40°C for C189 that contained a 10-base primer.

The Crossing and Selection Scheme
The Xa21 gene was introgressed into Minghui 63 following a recurrent backcrossing procedure, combined with tandem selection using molecular markers. The entire scheme took three generations of backcrosses and one generation of selfing to complete. In this scheme, the progeny of each backcross was first selected for the presence of the Xa21 gene by means of both PCR and disease inoculation. The Xa21-containing individuals in the BC₁F₁ were selected for recombination between Xa21 and either of the flanking marker loci. In BC₂F₁, the Xa21-containing individuals were selected for recombination between Xa21 and the other marker locus. The Xa21-containing plants in the BC₃F₁ were assayed with a large number of molecular markers covering the entire rice genome to identify individuals that were homozygous for the Minghui 63 genotypes at all marker loci, except the Xa21 locus. The selected individuals were then self-fertilized to produce individuals that were homozygous for the Xa21 gene at this locus, thus completing the breeding procedure.

Collection of Field Data for Agronomic Traits
Agronomic performance of Minghui 63, Shanyou 63, and the Xa21-containing versions of Minghui 63 and Shanyou 63 was compared in Wuhan in the summer of 1998 and in Hainan (South China Sea) Island in the spring of 1999. In the Wuhan test, all the eight lines that were obtained in the BC₃F₂ generation (see the Results section) were planted along with the original Minghui 63 and IRBB21 in a field without artificial inoculation. Shanyou 63 and the Xa21-containing version of Shanyou 63 were planted in the disease nursery in two plots. Plants in one of the two plots were inoculated with ZJ173, a prevalent Xoo strain in rice growing areas of central and southern China, to produce heavily diseased conditions, while the uninoculated plots did not have much disease under natural conditions. In all the cases, each of the plots consisted of three rows with nine plants per row at planting density of 17 cm between plants in a row, and the rows were 27 cm apart. Only the five plants in the middle of the center row were used for measuring the agronomic traits.

In the Hainan planting, all the materials were tested in replicated field trials both under heavily diseased conditions and under natural field conditions that did not show much disease. For testing under heavily diseased conditions, Minghui 63, Shanyou 63, IRBB21, and the Xa21-containing versions of Minghui 63 and Shanyou 63 were each planted in a five-row plot, with ten plants per row. The distance between plants within a row was 10 cm and the rows were 20 cm apart. The three rows in the middle of each plot were inoculated with Xoo strains ZJ173, Pxo99, and LN44, respectively. Each of the plots was replicated twice, and the layout of the plots in the field was completely at random.

For testing under natural field conditions without much disease, the same set of materials was planted in the field without artificial inoculation. The sizes and the layout of the plots were the same as described in the previous paragraph except that the plantings followed a randomized complete block design with two replications.

Measurements were taken for the eight plants in the middle of three central rows (24 plants in total) in each plot for a number of agronomic traits, including heading date, plant height, tillers per plant, number of grains per panicle, weight of 1000 grains, and grain yield per plant.

	Results

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Resistance of the Parents and Their Hybrids
To determine the potential usefulness of Xa21 in rice hybrids, we inoculated Minghui 63, IRBB21, Zhenshan 97A, and their hybrids Zhenshan 97A/Minghui 63 (Shanyou 63) and Zhenshan 97A/IRBB21 in the disease nursery with all the 17 Xoo strains (data for representative examples are given in Table 1). Minghui 63 was moderately susceptible to most of the strains tested, and Shanyou 63 was more susceptible than Minghui 63 to all the strains that could attack Minghui 63. IRBB21 was resistant to all strains except T7147, a strain from Japan belonging to the Japanese racial group 2, which confirmed the previous results that Xa21 is highly resistant to a broad racial spectrum of the pathogen (Khush et al., 1990). The resistance reaction of the hybrid Zhenshan 97A/IRBB21 appeared to be exactly the same as that of IRBB21, indicating a complete dominance of the resistance conferred by the gene(s) of IRBB21. Thus, Xa21 would be very useful for improving BB resistance of the hybrid Shanyou 63.

Mas
The local linkage map of the Xa21 genomic region on chromosome 11, constructed on the basis of 200 individuals of the F₂ population from the Minghui 63/IRBB21 cross, is shown in Fig. 1, from which a MAS system was established. Two markers, 21 and 248, cosegregated with the Xa21 locus. These two markers were used for positive selection, i.e., selecting for the presence of the Xa21 gene. Two additional markers, C189 and AB9, flanked both sides of the Xa21 locus at 0.8 and 3.0 cM, respectively. These two markers were chosen for negative selection, i.e., selecting for recombination between the Xa21 locus and the flanking markers. Such negative selection would ensure that the introgressed segment surrounding the Xa21 locus was shorter than the length between the two flanking markers (3.8 cM).

The molecular marker-assisted backcross breeding was conducted with the above MAS system. Among a total of 49 plants in BC₁F₁ that contained Xa21 as determined by both disease inoculation and PCR selection, one individual was found to be a recombinant between Xa21 and the marker locus AB9 and was subsequently backcrossed to Minghui 63. In the same way, one of the 180 Xa21-containing plants in BC₂F₁ was found to be a recombinant between Xa21 and the marker locus C189. Thus an individual containing an introgressed segment of less than 3.8 cM in length (0.21% of the rice genome, assuming a total length of 1800 cM) was obtained in BC₂F₁, and was further backcrossed to Minghui 63 to obtain the BC₃F₁.

Screening of 354 RFLP probes from the two published high-density maps (Causse et al., 1994; Kurata et al., 1994) identified 158 markers that were polymorphic between Minghui 63 and IRBB21 (Table 2) . These markers were distributed quite evenly in the linkage map except that there were more markers from chromosome 11 than from the other chromosomes. One hundred twenty-eight of the 158 polymorphic markers, representing all the 12 chromosomes with the largest interval less than 30 cM, were used for "cleaning up" the genetic background of the selections. Among a total of 250 plants that carried Xa21 in BC₃F₁, two plants were found to be homozygous for the Minghui 63 genotypes at all marker loci except the RG103 locus residing in the Xa21 gene region. There were six additional plants that were heterozygous at one to four marker loci besides the RG103 locus. The lesion lengths of these eight plants were not significantly different from that of IRBB21 when tested against Pxo99, a strain belonging to Xoo Race 6 of the Philippines (Table 3) .

In the Hainan test without artificial inoculation, Minghui 63 and Minghui 63(Xa21) were identical for all the agronomic traits examined, as were Shanyou 63 and Shanyou 63(Xa21) (Table 5) . Under heavily diseased conditions (Table 5), Minghui 63(Xa21) showed significantly higher grain weight and spikelet fertility than Minghui 63. The differences in yield related traits were even more pronounced between Shanyou 63 and Shanyou 63(Xa21) (Table 5). Yield and all three yield component traits (tillers per plant, grains per panicle, and grain weight) were higher for Shanyou 63(Xa21) than Shanyou 63.

	Discussion

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There are several points that need discussion regarding the efficiency of the MAS procedures in the recurrent backcross scheme. The first point concerns the selection for recombination between the targeted gene locus and the flanking markers. There are two options for obtaining an individual with recombination on both sides of the targeted gene. The first option is selection for simultaneous recombination on both sides, which will result in the desired recombinant in one generation. The alternative is a tandem selection, i.e., selection for recombination on one side in the first generation and selection for recombination on the other side in the next generation. Although the first option would save one generation of backcrossing, it is much more costly than the second option. For example, assuming a distance of 1 cM on both sides, simultaneous recombination on both sides would occur at a frequency of 0.01%. Thus, one would have to screen 10000 positive individuals containing the gene (representing only 50% of the plants in a backcross population) to obtain one double recombinant. This is obviously prohibitive for the molecular marker assay as well as for the hand emasculation involved in making the cross. Additionally, there are possible complications of interference in obtaining a double cross over. In contrast, also assuming a distance of 1 cM on both sides, a recombinant event between the targeted gene and a flanking marker on either of the two sides would occur at a frequency of 2%. Thus, only 50 positive individuals would be needed in the first generation of backcrossing to expect a recombinant event between the targeted gene and one of the flanking markers, and 100 positive individuals would be needed to obtain a recombinant on the other side of the gene in the second generation of backcrossing. Thus, the second option obviously costs much less in labor and resources than the first option.

The second point is related to the time in which background selection should be practiced. At a single locus, the expected frequencies for individuals to be homozygous for the genotype of the recurrent parent would be 0.5, 0.75, and 0.875, in BC₁F₁, BC₂F₁, and BC₃F₁, respectively. These can also be viewed as the expected proportions of loci for individuals to be homozygous for the recurrent parent genotypes. The variances of such proportions in these generations would be 0.5(1 - 0.5)/n, 0.75(1 - 0.75)/n, and 0.875(1 - 0.875)/n, respectively, where n is the number of independent recombinational units in the genome. It is not known how many map units are equivalent to an independent recombinational unit in the rice genome. But it is clear that the variance in BC₁F₁ is much larger than those in subsequent generations, indicating a wider frequency distribution in the BC₁F₁ than the later generations. Also, as discussed in the previous paragraph, the desired individual with recombination between the targeted gene locus and either one of the flanking markers is expected to occur at a much higher frequency in BC₁F₁ than BC₂F₁, which means that it is feasible to practice background selection in the BC₁F₁ generation. Thus, in addition to the background selection in the BC₃F₁ generation, adding one round of background selection in BC₁F₁ to the MAS scheme may greatly increase the efficiency of the program.Ogawa Khush 1989

	ACKNOWLEDGMENTS

 
We thank S.D. Tanksley's group at Cornell University and the Japanese Rice Genome Research Program for kindly providing the probes. We also thank Dr. P.C. Ronald of University of California, Davis, for providing the primer sequences before publication. This study was supported in part by a grant from the National Natural Science Foundation of China and a grant from the Rockefeller Foundation.

Received for publication March 23, 1999.

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TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 

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