HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (34) Citing Articles via Google Scholar Google Scholar Articles by Sanchez, A.C. Articles by Khush, G.S. Search for Related Content PubMed Articles by Sanchez, A.C. Articles by Khush, G.S. Agricola Articles by Sanchez, A.C. Articles by Khush, G.S.

CELL BIOLOGY & MOLECULAR GENETICS

Sequence Tagged Site Marker-Assisted Selection for Three Bacterial Blight Resistance Genes in Rice

Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines

g.khush{at}cgiar.org

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
IR65598-112 and the two sister lines IR65600-42 and IR65600-96 are promising new plant type (NPT) rice lines with high yield potential. However, these lines are susceptible to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). To improve the resistance of the three NPT lines to Xoo, three bacterial blight (BB) resistance genes, xa5, xa13, and Xa21, were successfully transferred to the NPT lines via a marker-aided backcrossing procedure. Sequence tagged site (STS) markers for the two resistance genes were developed based on DNA sequences of their linked restriction fragment length polymorphism (RFLP) markers (RG556 and RG207 for xa5 and RG136 for xa13). Marker polymorphism for xa5 was detected after digestion of RG556 polymerase chain reaction (PCR) products with MaeII enzyme and digestion of RG136 PCR product with HinfI enzyme for xa13. The STS marker for Xa21 was designed previously from the sequence of a genomic clone RAPD248. Fifty-nine BC₃F₂ near-isogenic lines (NILs) in the three NPT backgrounds containing one to three BB resistance genes in various combinations were developed through marker-assisted selection (MAS) for the resistance genes and phenotypic selection for the NPT. The BC₃F₃ NILs having more than one BB resistance gene showed a wider resistance spectrum and manifested increased levels of resistance to the Xoo races, as compared with those having a single BB resistance gene. Results for two F₂ populations and the progeny testing of their F₃ lines showed that MAS reached an accuracy of 95 and 96% of identifying homozygous resistant plants for xa5 and xa13, respectively. These NPT NILs for BB resistance genes provided valuable materials for breeding and genetic studies of single-gene effects and interaction of several resistance genes. The results demonstrate the usefulness of MAS in gene pyramiding for BB resistance, particularly for recessive genes, such as xa5 and xa13, that are difficult to select through conventional breeding in the presence of a dominant gene such as Xa21.

Abbreviations: BB, bacterial blight • HYV, high-yielding varieties • IRRI, International Rice Research Institute • kb, kilobases • LL, lesion length • NIL, near-isogenic line • NPT, new plant type • MAS, marker-assisted selection • PCR, polymerase chain reaction • QTL, quantitative trait loci • RFLP, restriction fragment length polymorphism • STS, sequence tagged site • Xoo, Xanthomonas oryzae pv. oryzae

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
THE ADVENT of high-density molecular maps coupled with the rapid development of PCR-based techniques has created opportunities for the application of DNA markers both in genetic and breeding research. DNA markers in combination with PCR have enabled MAS to become a practical breeding method. Marker-assisted selection offers a unique opportunity to circumvent many traditional problems associated with phenotypic selection for traits of interest. Marker-assisted selection increases the efficiency and flexibility of a breeding program by selecting for marker genotypes linked to target genes or quantitative trait loci (QTL). Currently, tremendous progress has been made in mapping many agriculturally important genes with DNA markers in many crop plants (Mohan et al., 1997). The identification of DNA markers linked to desirable genes or QTL affecting target traits is a prerequisite for MAS.

Conventional breeding brought major increases in rice production through the modern high-yielding varieties (HYVs). The International Rice Research Institute (IRRI) has designed and bred a new strain of rice, referred to as the NPT, which has the potential to produce higher yield than the modern HYVs. When the final variety is developed, the yield potential is expected to increase by 20% (Khush, 1995). One limitation of the NPT is susceptibility to various pathogens, including BB. Bacterial blight is one of the most destructive rice diseases and can reduce yield by 20 to 30% (Singh et al., 1977). Nineteen resistance genes have been identified so far (Kinoshita, 1995), and pyramiding of multiple resistance genes into rice varieties is one way to develop durable resistance to BB. However, this approach is difficult through conventional breeding due to masking effects of genes such as Xa21, which convey resistance to many BB races. It is impossible to distinguish between plants having Xa21 alone and those having Xa21 and other genes. Marker-assisted selection allows the identification of plants with multiple resistance genes.

Recently, major genes conferring disease resistance in several crop species have been mapped with linked DNA markers, facilitating MAS for disease resistance in these crops (Melchinger, 1990; Kelly, 1995; Penner et al., 1995; Miklas et al., 1996). Marker-assisted selection has been successfully used in selecting for resistance in the absence of pathogens (Melchinger, 1990), pyramiding multiple genes for durable resistance against rice bacterial blight (Huang et al., 1997), and for development of multiple disease–resistant germplasm (Kelly, 1995). In barley (Hordeum vulgare L.), Penner et al. (1995) converted a RFLP marker associated with barley stem rust (Puccinia graminis Pers.:Pers) resistance to an allele-specific PCR-based marker useful for MAS. The same technique was used by Hittalmani et al. (1995) to identify a specific amplicon polymorphism marker for the rice blast (Pyricularia grisea Sacc.) resistance gene Pi-2(t). Marker-assisted selection was often demonstrated solely in populations in which resistance genes were originally mapped with diagnostic DNA markers. However, the effectiveness of MAS must be evaluated in breeding populations of interest to breeders.

In this study, we report the successful use of STS markers to transfer three bacterial leaf blight resistance genes into elite NPT rice breeding lines having high yield potential through marker-assisted backcrossing.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Plant Materials and Marker-Assisted Selection Procedure
We have previously transferred the three bacterial blight resistance genes, xa5, xa13, and Xa21, to IR24 and designated the resulting line as IRBB59 (Huang et al., 1997). This line was used as the donor parent in our backcrosses. The recurrent parents were IR65598-112 and two sister lines, IR65600-42 and IR65600-96, all belonging to NPT lines developed at IRRI. F₁ plants were obtained between the donor and the three recurrent parents and were advanced up to the BC₃ generation by MAS. Starting from the BC₁F₁, and in each of the following BCF₁ generations, approximately 50 plants were genotyped for the selected STS markers (Table 1) . From these, plants carrying resistant alleles of the three target resistance genes (based on their marker genotypes) and phenotypically similar to the recurrent parents were selected as the parents for the next backcross until BC₃F₁. The selected BC₃F₁ plants for each of the recurrent parents were selfed to produce BC₃F₂ seed. Based on phenotypic similarity to their recurrent parents, 21, 20, and 18 BC₃F₂ plants in the genetic backgrounds of IR65598-112, IR65600-42, and IR65600-96, respectively, were selected for homozygosity at the STS marker genotypes and phenotyped for their reactions to the six Xoo races (Kauffman et al., 1973).

Target Genes and Sequence Tagged Site Markers for Marker-Assisted Selection
The target genes selected for the MAS experiment included two recessive genes, xa5 and xa13, and a dominant one, Xa21, conferring resistance to different BB races. Table 1 lists the primers for STS analysis designed based on DNA sequences of both ends of the published RFLP markers linked to the three resistance genes (Huang et al., 1997). The primer pair for Xa21 was previously designed from the sequence of a genomic clone RAPD248 (Chunwongse et al., 1993). This STS marker cosegrates with Xa21 (Williams et al., 1996) and amplified a 900–base pair band from the donor identified as allele 1, and two different alleles from the recurrent parents, IR65598-112 (allele 3) and IR65600-42 or IR65600-96 (allele 2) (Huang et al., 1997).

The STS primers for xa5 were designed based on end sequences of RFLP clones RG556 and RG207 (Table 1). The STS primers designed from RG556 gave monomophic amplification products. The PCR products digested with MaeII detected polymorphism between the parents, producing two bands of 1.3 kilobases (kb) and 0.75 kb for the recurrent parents and of 0.9 and 0.75 kb for the donor. Direct amplification with the RG207 primer pair generated polymorphism between the parents (Fig. 1A) . Multipoint linkage analysis placed xa5 between RG556STS and RG207STS (Yang et al., 1998).

View larger version (88K): [in this window] [in a new window]   Fig. 1 Marker banding pattern of selected BC3F2 rice plants and their corresponding parents for sequence tagged site (STS) markers linked to xa5, xa13, and Xa21. (A) polymerase chain reaction (PCR) using RG207STS as primers for xa5, (B) PCR using RG136STS as primers for xa13, (C) PCR with primers PB7/PB8 for Xa21. Marker = kilobase ladder. The numbers for each lane correspond with the codes for gene combinations given in Table 4. Plant numbers 9, 10, 16, and 24 each carry xa5, xa13, and Xa21 resistant alleles

DNA suitable for PCR analysis was obtained from seedlings of each of the parental lines, the BC progenies, and the F₂ plants by a standard miniscale procedure according to Huang et al. (1997). The PCR procedure for assays of the selected STS marker genotypes of the parental lines, the BC progenies, and the F₂ populations mentioned above followed the standard protocol as described previously (Yang et al., 1998).

Phenotyping Experiments for Reactions to Bacterial Blight
The parental lines, the resulting BC₃F₂ plants and BC₃F₃ lines from MAS and the two sets of F₃ lines were planted in plots of 12 plants, with a spacing of 25 cm between rows and 20 cm between plants. Nine leaves of three different plants (3 leaves per plant) in each of the tested lines were clip-inoculated with six Philippine races of Xoo (Kauffman et al., 1973) at the maximum tillering stage to determine the phenotypic reactions of the tested lines to the Xoo races. The inoculum was prepared by suspending the bacterial mass in sterile water to a concentration of 10⁹ cells mL^-1. Evaluation for resistance was conducted 18 d after inoculation by both visual scoring and lesion length (LL) measurement. The distinction between resistant and susceptible plants was set at LL of 6 cm. Plants with LL of <6 cm were scored as resistant and those with LL of >6 cm were scored as susceptible.

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Reactions of Parents to Xanthomonas oryzae pv. oryzae Races
The donor, IRBB59, showed a high level of resistance to all six races of Xoo. The reactions of the recurrent parent lines ranged from moderately susceptible to highly susceptible (Table 2) . When inoculated with Xoo races 1, 2, and 3, the two sister lines IR65600-42 and IR65600-96 were moderately susceptible with shorter LL than the susceptible check, IR24, and lacked a sharp delineation of advancing lesion expansion. IR65600-42 was moderately susceptible, while IR65600-96 was susceptible to race 4. Both these lines were susceptible to races 5 and 6. IR65598-112 was susceptible to all the six Xoo races.

The disease reactions of the 59 plants to the six Xoo races were verified by inoculation. Based on their marker genotype, the plants were found to carry one to three resistance genes in various combinations (Table 3) . These plants showed resistance to at least one of the Xoo races, as expected from their genotypes at the resistance gene loci (Huang et al., 1997). For example, those plants carrying xa13 were susceptible to five Xoo races but resistant to race 6. Because of intentional selection for the NPT phenotype of the recurrent parents in every BC generation, the resulting BC₃F₃ plants showed a high degree of similarity to their respective recurrent parents. For instance, the BC₃F₃ progenies of IR65598-112 were all semidwarf with dark green leaves, thick stems, and low tillering ability. The BC₃F₃ progenies of both IR65600-42 and IR65600-96 had purple apiculus and stigma, bold grains, thick and sturdy stems, weak tillering ability, and were very similar to their recurrent parents, except for having slightly pale leaves.

Multiple Resistance Genes and the Quantitative Component of Resistance to Xanthomonas oryzae pv. oryzae
The LL of 14 representative BC₃F₃ lines carrying seven different combinations of the resistance genes in the backgrounds of IR65598-112 and IR65600-96 was measured 18 d after inoculation (Table 5) . The two lines with xa-5 alone were resistant to Xoo races 1 to 5 but susceptible to race 6. On the contrary, the two lines with xa13 alone were susceptible to Xoo races 1 to 5 but resistant to race 6. Lines with both xa5 and xa13 were resistant to all six Philippine Xoo races. Hence, a wider spectrum of resistance was obtained upon combining the two resistance genes. Thus, a case of qualitative complementation among lines having two resistance genes was observed, wherein resistance to the sum of all the pertinent races of the pathogen was obtained when two genes each separately conferring resistance to relevant races were combined.

The line IR65598-112 carrying xa-13 had the mean LL of 13.7 cm (caused by Xoo races 1–5), significantly longer than that of IR65600-96 (8.7 cm), which also carried xa-13. A similar difference was observed between their two recurrent parents (Table 2), indicating that the quantitative difference in resistance between the two susceptible parents was heritable.

The high accuracy (>90%) of MAS for either xa5 or xa13, based on single markers, achieved in this study is not surprising since the selected markers are known to be tightly linked to the target genes (Yang et al., 1998; Sanchez et al., 1999). Development of PCR-based markers for the target genes allowed us to genotype a relatively large number of progeny in each of the BC generations with minimum costs so that intensive selection for NPT phenotype was simultaneously practiced. In this way, we were able to develop NILs of the three NPT recurrent lines in only three BC generations even though the recurrent parents and the donor are distantly related. The obtained NPT NILs are phenotypically identical to their recurrent parents, except each has a different combination of the BB resistance genes. In this respect, MAS certainly has a greater advantage over the conventional backcross breeding. As more genes of agronomic importance are being mapped with diagnostic DNA markers in rice, MAS will be increasingly used for genetic improvement of rice.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. M. Yano and T. Sasaki of the Rice Genome Program and Dr. John Bennett of the Plant Molecular Biology Laboratory of IRRI for providing the STS markers used in the study. The technical help of E. Managat and J. Rey is also acknowledged. Financial support from the German Government and the Rockefeller Foundation are greatly appreciated.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Current address of N. Huang is: Applied Phytologics Inc., 4110 N. Freeway Blvd., Sacramento, CA 95834.

Received for publication July 6, 1999.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 

• Blair M.W., McCouch S.R. Microsatellite and sequence-tagged site markers diagnostic for the rice bacterial blight resistance gene xa5. Theor. Appl. Genet. 1997;95:174-184.
• Chunwongse J., Martin G.B., Tanksley S.D. Pre-germination genotypic screening using PCR amplification of half seeds. Theor. Appl. Genet. 1993;86:694-698.
• Hittalmani S., Foolad M.R., Mew T., Rodriguez R.L., Huang N. Development of a PCR-based marker to identify rice blast resistance gene, Pi-2(t), in a segregating population. Theor. Appl. Genet. 1995;91:9-14.
• Huang N., Angeles E.R., Domingo J., Magpantay G., Singh S., Zhang G., Kumaravadivel N., Bennett J., Khush G.S. Pyramiding of bacterial blight resistance genes in rice: Marker-assisted selection using RFLP and PCR. Theor. Appl. Genet. 1997;95:313-320.[ISI]
• Kauffman H.E., Reddy A.P.K., Hsien S.P.Y., Merca S.D. An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Dis. Rep. 1973;57:537-541.
• Kelly J.D. Use of RAPD markers in breeding for major gene resistance to plant pathogens. HortScience 1995;30:461-465.[Free Full Text]
• Khush G.S. Breaking the yield frontier of rice. GeoJournal 1995;35(3):329-332.
• Kinoshita T. Report of committee on gene symbolization, nomenclature and linkage groups. Rice Genet. Newsl. 1995;12:9-153.
• Melchinger A.E. Use of molecular markers in breeding for oligogenic disease resistance. Plant Breed. 1990;104:1-19.
• Miklas P.N., Afanador L., Kelly J.D. Recombination-facilitated RAPD marker-assisted selection for disease resistance in common bean. Crop Sci. 1996;36:86-90.[Abstract/Free Full Text]
• Mohan M., Nair S., Bhagwat A., Krishna T.G., Yano M., Bhatia C.R., Sasaki T. Genome mapping, molecular markers and marker-assisted selection in crop plants. Mol. Breed. 1997;3:87-103.
• Penner G.A., Stebbing J.A., Legge B. Conversion of an RFLP marker for the barley stem rust resistance gene Rpg1 to a specific PCR-amplifiable polymorphism. Mol. Breed. 1995;1:349-354.
• Ronald P.C., Albano B., Tabien R., Abenes L., Wu K., McCouch S., Tanksley S. Genetic and physical analysis of the rice bacterial blight resistance locus, Xa21. Mol. Gen. Genet. 1992;235:113-120.[Medline]
• Sanchez A.C., Ilag L.L., Yang D., Brar D.S., Ausubel F., Khush G.S., Yano M., Sasaki T., Li Z., Huang N. Genetic and physical mapping of xa-13 a recessive bacterial blight resistance gene in rice. Theor. Appl. Genet. 1999;98:1022-1028.
• Singh G.P., Srivastara M.K., Singh R.V., Singh R.M. Variation in quantitative and qualitative losses caused by bacterial blight on rice varieties. Indian Phytopathol. 1977;30:180-185.
• Williams C.E., Wang B., Holsten T.E., Scambray J., de Assis F., da Silva G., Ronald P.C. Markers for selection of the rice Xa-21 disease resistance gene. Theor. Appl. Genet. 1996;93:1119-1122.
• Yang D., Sanchez A.C., Khush G.S., Zhu Y., Huang N. Construction of a BAC contig containing xa5 locus in rice. Theor. Appl. Genet. 1998;97:1120-1124.
• Yoshimura S., Yoshimura A., Iwata N., McCouch S.R., Abenes M.L., Baraoidan M.R., Mew T.W., Nelson R.J. Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers. Mol. Breed. 1995;1:375-387.
• Zhang G., Angeles E.R., Abenes M.L.P., Khush G.S., Huang N. RAPD and RFLP mapping for the bacterial blight resistance gene xa-13 in rice. Theor. Appl. Genet. 1996;93:65-70.

This article has been cited by other articles:

				M. S. Swaminathan An Evergreen Revolution Crop Sci., September 8, 2006; 46(5): 2293 - 2303. [Abstract] [Full Text] [PDF]

				K. Renganayaki, A. K. Fritz, S. Sadasivam, S. Pammi, S. E. Harrington, S. R. McCouch, S. M. Kumar, and A. S. Reddy Mapping and Progress toward Map-Based Cloning of Brown Planthopper Biotype-4 Resistance Gene Introgressed from Oryza officinalis into Cultivated Rice, O. sativa Crop Sci., November 1, 2002; 42(6): 2112 - 2117. [Abstract] [Full Text] [PDF]

				M. C. Willcox, M. M. Khairallah, D. Bergvinson, J. Crossa, J. A. Deutsch, G. O. Edmeades, D. Gonzalez-de-Leon, C. Jiang, D. C. Jewell, J. A. Mihm, et al. Selection for Resistance to Southwestern Corn Borer Using Marker-Assisted and Conventional Backcrossing Crop Sci., September 1, 2002; 42(5): 1516 - 1528. [Abstract] [Full Text] [PDF]

				N. Pal, J. S. Sandhu, L. L. Domier, and F. L. Kolb Development and Characterization of Microsatellite and RFLP-Derived PCR Markers in Oat Crop Sci., May 1, 2002; 42(3): 912 - 918. [Abstract] [Full Text] [PDF]

				Z.-K. Li, A. Sanchez, E. Angeles, S. Singh, J. Domingo, N. Huang, and G. S. Khush Are the Dominant and Recessive Plant Disease Resistance Genes Similar?: A Case Study of Rice R Genes and Xanthomonas oryzae pv. oryzae Races Genetics, October 1, 2001; 159(2): 757 - 765. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (34) Citing Articles via Google Scholar Google Scholar Articles by Sanchez, A.C. Articles by Khush, G.S. Search for Related Content PubMed Articles by Sanchez, A.C. Articles by Khush, G.S. Agricola Articles by Sanchez, A.C. Articles by Khush, G.S.