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Gene pyramiding to improve hybrid rice by molecular marker techniques

Yuqing He, Xin Li, Jingfeng Zhang, Gonghao Jiang, Shiping Liu, Sheng Chen, Juming Tu, Caiguo Xu and Qifa Zhang

National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, China. Email: yqhe@mail.hzau.edu.cn

Abstract

Shanyou 63, a cross between Zhenshan 97 and Minghui 63, is an elite hybrid rice cultivar widely grown in China. This study reports on the results of a series of marker-assisted breeding experiments designed to improve the disease and insect resistance of this hybrid. The main results are as follows: (1) two wide-spectrum bacterial blight resistance genes, Xa21 and Xa7, were incorporated into the restorer line Minghui 63 by molecular marker-aided selection (MAS). (2) Minghui 63 was transformed with a Bt δ-endotoxin gene to improve the stem borer resistance. (3) Xa21 and Bt genes were combined into a line with the of Minghui 63 background. (4) Two genes, Pi1 and Pi2, showing broad-spectrum resistance to fungi blast, were introgressed into Zhenshan 97 to improve the blast resistance by MAS. (5) Two genes, for to brown planthopper resistance, Qbph1 and Qbph2, were introgressed into Zhenshan 97. The above versions of improved lines can be combined in various ways to make new hybrids to meet the needs of rice production.

Media summary

Gene pyramiding to improve the disease- and insect-resistance of an elite indica rice hybrid, Shanyou 63, by molecular marker-aided selection.

Key Words

Oryza sativa L; Molecular marker-aided selection; Bt; Bacterial blight; Fungal blast; Brown planthopper

Introduction

Hybrid rice was first cultivated in China in 1976 and is now planted on more than 13 million ha annually. In recent years, several of the most widely cultivated hybrids have shown decreasing resistance to important diseases like bacterial blight and fungal blast, and there is also a urgent need to improve their resistance to insects such as stem borer and brown planthopper, that frequently cause severe damage to the crop.

Marker-aided selection is a very useful approach to maximize utilization of the existent gene resources. Genes controlling different agronomic traits can be quickly brought together in an existing variety. Furthermore, genes responsible for resistance to different races or biotypes of a disease or insect pest can be also pyramided together to make a line have multi-race or multi-biotype resistance. Theoretically, these multi-race or multi-biotype resistances should be more durable than single-race or single-biotype resistance. Gene pyramiding has been successfully applied in several crop breeding programs, and many varieties and lines possessing multiple attributes have been produced (Huang et al. 1997; Wang et al. 2001; Samis et al. 2002).

In this study we used marker-aided selection to improve the insect- and disease-resistance of the hybrid rice Shanyou 63, by improving by introgression of relevant insect and disease resitance genes into the restorer line Minghui 63 and the maintainer line Zhenshan 97.

Methods

Rice cultivars

This study used the following rice lines: (1) Minghui 63, the restorer line of hybrid Shanyou 63. (2) Minghui 63(Xa21), bred with a recurrent backcross using IRBB21 as the donor line and Minghui 63 as recurrent parent by MAS (Chen et al. 2000). (3) Minghui 63(Xa7), with Xa7 transferred from DV85 to Minghui 63 by recurrent backcrossing (also referred to as Kanghui 63) (Wang et al 1996). (4) Minghui 63(Bt), a marker-free transgenic line obtained by particle bombardment transformation (Tu et al. 2000).(5)Zhenshan 97, the maintainer line of hybrid Shanyou 63. (6) BL6 (Pi1/Pi2/Pi3), a line highly resistant to rice blast and kindly provided by IRRI. (7) B5, a variety highly resistant to BPH biotype 1 and 2 in China, with its resistance genes Qbp1 and Qbp2 derived from the wide rice Oryza officinalis.

The crossing and selection scheme

(1) Gene pyramiding of Xa21 and Xa7 was achieved by crossing Minghui 63(Xa21) with Minghui 63(Xa7), followed by selfing to F2 population subjected to MAS to identify individuals homozygous for both genes. The same scheme was used for combining Bt and Xa21 genes from the cross of Minghui 63(Xa21) and Minghui 63(Bt). (2) The Pi1 and Pi2, Qbph1 and Qbph2 genes were introgressed into the Zhenshan 97 background by crossing Zhenshan 97 with BL6 and B5, respectively, followed by three generations of backcrossing and one generation of selfing. In this scheme, the progeny of each backcross was first selected for the presence of the target genes by closely linked markers.

Disease and insect resistance scoring

(1) The bacterial blight and stem borer resistances of the lines followed the procedures of Chen et al (2000) and Tu et al (2001), respectively. (2) Brown planthopper resistance was evaluated as described by Huang et al (2001). (3) Fungal blast resistance was evaluated in a natural blast nursery in a blast hot spot from June to September in 2003 in Yuan’an County, Hubei Province, China. The scoring was according to the method described by Yang et al (1999).

Results

Gene pyramiding of Xa21 and Xa7 to improve the bacterial blight resistance of Minghui 63

The cross between Minghui 63(Xa21) and Minghui 63(Xa7) resulted in 570 F2 individuals. One hundred and seventy two plants were identified to be Xa21 homozygous by marker 248 (Chen et al 2000). All 172 Xa21 homozygous individuals were then assayed by three polymorphism markers, AFLP 1415, STS P3 and M5, tightly linked to Xa7 (Porter et al. 2003). Thirty Xa7 and Xa21 homozygous plants were obtained.

Table 1 Bacterial blight resistance of pyramided lines at booting stage (Wuhan, 2002)

Strains

Minghui 63

Minghui 63(Xa7)

Minghui 63(Xa21)

Minghui 63(Xa21/Xa7)

1

2

3

PXO99

34.204.15

31.904.80

14.654.14

10.771.78

7.861.01

8.881.21

KS-1-21

33.552.02

16.612.47

9.191.84

0.860.25

1.641.04

0.380.18

GX325

21.205.85

6.810.94

8.202.71

0.630.18

0.900.29

0.530.25

PXO145

5.902.72

1.601.08

3.051.70

0.310.21

1.000.67

0.430.26

O249

4.401.53

4.300.97

1.801.86

0.580.12

0.320.27

0.250.19

Zhe173

29.452.58

3.181.36

5.501.78

0.370.13

0.550.14

0.510.19

PXO61

2.041.15

2.901.72

2.601.78

0.580.27

0.790.33

0.330.28

PXO71

17.005.53

1.251.06

2.201.75

0.320.12

0.320.14

0.440.25

T7133

15.172.02

1.800.84

2.752.19

0.290.29

0.490.32

0.210.14

LN44

22.393.26

1.201.10

3.600.65

0.250.25

0.350.15

0.390.29

Ten Xoo races for Minghui 63 were selected to inoculate Minghui 63, Minghui 63(Xa21), Minghui 63(Xa7) and the pyramid lines Minghui 63(Xa21/Xa7) (Table 1). It was shown that Minghui 63 was highly susceptible to the Xoo strains PXO99, KS-1-21, GX325, Zhe173, PXO71, T7133 and LN44. Minghui 63(Xa21) and Minghui 63(Xa7) showed improved resistance to several of the strains compared with Minghui 63. The pyramid line Minghui 63(Xa21/Xa7) showed greatly reduced lesion length with PXO99, and was highly resistant to the other 9 strains. Thus the pyramid line had a much broader spectrum and higher level resistance than their parental lines.

Gene pyramiding of Xa21 and Bt to improve the insect and disease resistance of Minghui 63

A cross was made between Minghui 63(Bt) and Minghui 63(Xa21). In F2, a population of 473 plants was grown and the two genes were traced by PCR analysis. Twenty-one F3 families were positive for both Bt and Xa21 genes without any segregation, thus indicating that the expected homozygous lines were obtained.

The pyramided line Minghui 63(Bt/Xa21) and its derived hybrids were exposed to natural insect infestation and artificial inoculation of Xoo strain mixtures. The symptoms caused by either stem borers or leaf-folders were not observed on the pyramiding line Minghui 63(Bt/Xa21) in both testing years of 2000 and 2001. Similarly, the Bt donor line Minghui 63(Bt) had a percentage of white-head and foliage-folded plants of less than 3.1%. While the negative control Minghui 63 showed white-head symptoms or folded leaves in at least 77.1% of the plants. The bacterial blight resistance was similar to Minghui 63(Xa21) in Table 1.

Gene pyramiding of Pi1 and Pi2 to improve the blast resistance of Zhenshan 97

A cross was made between Zhenshan 97 and BL6 (Pi1/Pi2/Pi3), followed by three generations of backcrosses and one generation of self-fertilization. Pi1 is closely linked to the markers RM144 and RM224 on chromosome 11 and Pi2 is close to AP22 on chromosome 6 (Jiang and Wang 2002; Liu et al. 2003). In BC3F2 population, 14, 12, and 17 plants were identified to contain Pi1, Pi2, and Pi1/Pi2, respectively.

Table 2. Leaf blast and neck blast scores for some BC3F3 lines from a Zhenshan 97/BL6 backcross

Lines

Genotype

Leaf blast score

Neck blast score

Zhenshan 97-2

Pi1+Pi2

1.5

1.0

Zhenshan 97-12

Pi1+Pi2

2.0

1.0

Zhenshan 97-13

Pi1+Pi2

1.5

1.0

Zhenshan 97-19

Pi1

2.0

3.0

Zhenshan 97-20

Pi1

2.5

4.0

Zhenshan 97-22

Pi1

2.0

3.0

Zhenshan 97-30

Pi2

2.0

3.0

Zhenshan 97-35

Pi2

2.0

3.0

BL6

Pi1+Pi2+Pi3

1.0

1.0

C101LAC

Pi1

2.0

3.0

C101A51

Pi2

2.0

3.0

Zhenshan 97

recurrent parent

6.0

a

CO39

control

7.0

a

aThe plant was dead by the time of heading

Resistance to leaf blast and neck blast of the BC3F3 lines with Pi1, Pi2 and Pi1/Pi2 was assessed in a natural nursery at Wangjia Village in Yuan’an County. The controls, e.g. Zhenshan 97 and CO39, were highly susceptible to fungi blast, with a leaf blast score of 6.0 and 7.0, respectively (Table 2). They were killed by blast before heading. BL6, with Pi1, Pi2, and Pi3, was highly resistant to leaf and neck blast. The lines of Zhenshan 97 with Pi1, Pi2, and Pi1/Pi2 were highly resistant to leaf and neck blast. There was no significant difference in resistance among the lines to leaf blast, but Zhenshan 97(Pi1/Pi2) was more resistant to for neck blast than other lines. These findings showed that the genes were successfully introgressed into the background of Zhenshan 97 and gene pyramiding can improve rice resistance to blast disease, especially to neck blast.

Introgressing Qbp1 and Qbp2 to improve the brown planthopper resistance of Zhenshan 97

A cross was made between Zhenshan 97 and B5 (Qbp1/Qbp2). It took three generations of backcrosses and one generation of self-fertilization together with marker-assisted selection. In BC3F1 generation, ten positive individuals from fifty-five individuals were found and eleven of them were selected to self. In the progeny, 35, 38 and 45 individuals were found to be of Zhenshan 97(Qbp1), Zhenshan 97(Qbp2) and Zhenshan 97(Qbp1/Qbp2) , respectively.

The resistance of the BC3F3 lines was evaluated at the day in which TN1, the susceptible control, was completely killed by the insects. The results showed that B5 was strongly resistant to the brown planthopper and Zhenshan 97 was highly susceptible. The severity scores of B5, Zhenshan 97(Qbp1/Qbp2), Zhenshan 97(Qbp1), Zhenshan 97(Qbp2) and Zhenshan 97 (ck) were 1.4, 3.1, 4.4, 4.2 and 7.7, respectively, which showed that the resistance of improved Zhenshan 97 to BPH was improved remarkably.

Conclusion

The main outcome of this study was as follows: (1) Gene pyramiding for insect- and disease-resistance could widen it’s the spectrum and strength of the resistance. (2) Bt, Xa21 and Xa7 were pyramided in the restorer line, Minghui 63, and Pi1, Pi2, Qbp1 and Qbp2 were pyramided in the maintainer line Zhenshan 97. Availability of these lines will greatly improve the resistance of the elite hybrid rice, Shanyou 63, for to bacterial blight, blast, stem borer and brown planthopper.

Acknowledgement

This project was financially supported by National Program of ‘863’ High Technology Development and National Nature Science Foundation in China.

References

Chen S, Lin XH, Xu CG, and Zhang Q (2000). Improvement of bacterial blight resistance of ‘Minghui 63’, an elite restorer line of hybrid rice, by molecular marker-assisted selection. Crop Science, 40, 239-244.

Huang N, Angeles ER, Domingo J, Magpantay G, Singh S, Zhang G, Kumaravadivel N, Bennett J and Khush GS (1997). Pyramiding of bacterial blight resistance genes in rice: marker-assisted selection using RFLP and PCR. Theor Appl Genet 95: 313-320.

Huang Z, He GC, Shu LH, Li XH, Zhang QF (2001) Identification and mapping of two brown planthopper resistance genes in rice. Theor Appl Genet, 102: 929-934

Ikeda, R and Kaneda C (1981). Genetics analysis of resistance to the brown planthopper, Nilaparvata lugens, in rice. Jpn J Breeding, 31: 279-285

Jiang J and Wang S (2002). Identification of a 118-Kb DNA fragment containing the locus of blast resistance gene Pi-2(t) in rice. Mol Genet Genomics 268: 249-252.

Liu SP, Li X, Wang CY, Li XH, He YQ (2003). Improvement of resistance to rice blast in Zhenshan 97 by molecular marker-aided selection. Acta Botanic Sinica 45(11):1346-1350

Porter BW, Chittoor JM, Yano M, Sasaki T and White FF (2003). Development and mapping of markers linked to the rice bacterial blight resistance gene Xa7. Crop Science, 43:1484-1492

Samis K, Bowley S and McKersie B (2002). Pyramiding Mn-superoxide dismutase transgenes to improve persistence and biomass production in alfalfa. J. Exp. Bot., 53(372), 1343-1350.

Tu J, Zhang G, Datta K, Xu C, He Y, Zhang Q, Khush GS, and Datta SK (2000). Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis endotoxin. Nature Biotechnology, 18, 1101-1104.

Wang XY, Chen PD, and Zhang SZ (2001). Pyramiding and marker-assisted selection for powdery mildew resistance genes in common wheat. Acta Genetic Sinica, 28(7), 640-646.

Yang C J, Yang Z H, Hu J F, He G C, Shu L H. Study on the brown planthopper resistance in introgressive lines from wild rice. Acta Phytophylacica Sinica, 1999, 26: 197-202

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