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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (11): 1612-1620.doi: 10.3724/SP.J.1006.2018.01612

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Development and Application of Functional Markers for Rice Blast Resistance Gene Bsr-d1 in Rice

Jun WANG1,2,Jie-Yu ZHAO3,Yang XU1,2,Fang-Jun FAN1,2,Jin-Yan ZHU1,2,Wen-Qi LI1,2,Fang-Quan WANG1,2,Yun-Yan FEI1,Wei-Gong ZHONG1,Jie YANG1,2,*()   

  1. 1 Institute of Food Crops, Jiangsu Academy of Agricultural Sciences / Nanjing Branch of Chinese National Center for Rice Improvement / Jiangsu High Quality Rice R&D Center, Nanjing 210014, Jiangsu, China;
    2 Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China
    3 College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, Jiangsu, China
  • Received:2018-03-28 Accepted:2018-07-20 Online:2018-11-12 Published:2018-07-25
  • Contact: Jie YANG E-mail:yangjie168@aliyun.com
  • Supported by:
    This study was supported by the National Key Research and Development Program(2017YFD0100400-3);the Exploratory Project of the Jiangsu Academy of Agricultural Sciences(ZX(17)2014);Jiangsu Province Key Research and Development Program Project(BE2017368)

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Abstract:

Rice blast is one of the most serious rice diseases, breeding resistant cultivars is the most effective way to prevent this disease. Bsr-d1 is one vital non race-specific resistance gene for rice blast disease. To improve the selection efficiency of rice blast resistance gene Bsr-d1 in breeding, we selected different types of gene marker, CAPs5-1 and 3Bsr-d1/3bsr-d1, based on the single nucleotide polymorphism in the functional region of alleles Bsr-d1/bsr-d1. Furtherly confirmed by sequencing, we found both CAPs5-1 and 3Bsr-d1/3bsr-d1 could obviously distinguish different genetypes of Bsr-d1 locus. 3Bsr-d1/3bsr-d1 was employed to genotype 34 indica rice varieties, 110 japonica varieties certificated in Jiangsu Province, 13 japonica varieties certificated in different provinces, 148 local japonica cultivars and 19 local indica cultivars from Taihu Basin. There were only 11 indica varieties carrying Bsr-d1 gene, while Bsr-d1 gene was not detected in 271 japonica rice samples, indicating that Bsr-d1 mainly exists in indica germplasm, and is rarely found in japonica rice. This study facilitates the use of Bsr-d1 gene in rice blast resistance breeding and marker assisted selection.

Key words: rice (Oryza sativa L.), rice blast, Bsr-d1, functional marker, CAPs, allele specific PCR

Table 1

Functional markers and sequencing primers designed based on mutation site"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 片段长度
Expected size (bp)
CAPs1F AGTCTAGCATCCACCGTTCCAC 313
CAPs1R GTAGGCAGGCAGTGGGATGA
CAPs2F TTTTATAGGACAGAGGGAATATGTA 368
CAPs2R GCAGTGGGATGAACCTGTAC
Bsr-d1-F AGTCTAGCATCCACCGTTCCAC
1Bsr-d1-R CTTTTCGCTTATACTTATATTTATCAGC 241
1bsr-d1-R CTTTTCGCTTATACTTATATTTATCAGT 241
2Bsr-d1-R CTTTTCGCTTATACTTATATTTATCgGC 241
2bsr-d1-R CTTTTCGCTTATACTTATATTTATCgGT 241
3Bsr-d1-R CTTTTCGCTTATACTTATATTTATCAaC 241
3Bsr-d1-R CTTTTCGCTTATACTTATATTTATCAaT 241
C1F AGTCTAGCATCCACCGTTCCAC 755
C1R ATGATTTGATGGGATTGATTGC
C2F TTTTATAGGACAGAGGGAATATGTA 983
C2R GCGAGGTACTCCTCCTTGTTGAT

Fig. 1

Gradient PCR amplification products of Digu using two new designed primersM: DL2000; Annealing temperature of 1-12: 45.1°C, 45.3°C, 45.9°C, 46.8°C, 47.9°C, 49.1°C, 50.4°C, 51.7°C, 52.8°C, 53.8°C, 54.5°C, 54.8°C."

Fig. 2

Enzyme digestion results of PCR amplification products using CAPs5-1Left of M is PCR amplification products; Right of M is enzyme-digested products; M: DL2000; 1-6: Digu, Nipponbare, Nanjing 9108, Guanglu,ai 4, Wuyujing 3, Nanjing 45."

Fig. 3

Sequence alignment of Bsr-d1 functional domain for partial rice varieties"

Fig. 4

Molecular detection of partial rice varieties by CAPs5-1M: DL2000; 1-24: Digu, Nipponbare, Nanjing 1, Mianhui 501, Zhehui 7954, Yanhui 559, Zhenhui 084, BG90-2, Gui 630, Minghui 63, Qingsi’ai 16B, Fuhui 718, Yangdao 6, Gang 46B, Lucaihao, Minghui 78, IRBB21, Shuhui 527, Duohui 1, Guichao 2, Mianhui 725, Yuehui 188, Guanghui 128, Zhenshan 97B."

Fig. 5

Gradient PCR amplification products of Digu and Nipponbare using allelic-specific PCR markersM: DL2000; A: Gradient PCR amplification products by 1Bsr-d1; B: Gradient PCR amplification products by 1bsr-d1; C: Gradient PCR amplification products by 2Bsr-d1; D: Gradient PCR amplification products by 2bsr-d1; E: Gradient PCR amplification products by 3Bsr-d1; F: Gradient PCR amplification products by 3bsr-d1; 1-12: amplification products of Didu by different primers in annealing temperatures of 45.1°C, 45.3°C, 45.9°C, 46.8°C, 47.9°C, 49.1°C, 50.4°C, 51.7°C, 52.8°C, 53.8°C, 54.5°C, 54.8°C; and 13-24: amplification products of Nipponbare by different primers in annealing temperatures of 45.1°C, 45.3°C, 45.9°C, 46.8°C, 47.9°C, 49.1°C, 50.4°C, 51.7°C, 52.8°C, 53.8°C, 54.5°C, 54.8°C."

Fig. 6

PCR amplification products using allelic-specific PCR markers 3Bsr-d1 and 3bsr-d1M: DL2000; 1-6: Digu, Nipponbare, Nanjing 9108, Guanglu’ai 4, Wuyujing 3, Nanjing 45."

Fig. 7

Molecular detection of partial rice varieties by 3Bsr-d1 and 3bsr-d M: DL2000; 1-24: Digu, Nipponbare, Nanjing1, Mianhui 501, Zhehui 7954, Yanhui 559, Zhenhui 084, BG90-2, Gui 630, Minghui 63, Qingsi,ai 16B, Fuhui 718, Yangdao 6, Gang 46B, Lucaihao, Minghui 78, IRBB21, Shuhui 527, Duohui 1, Guichao 2, Mianhui 725, Yuehui 188, Guanghui 128, Zhenshan 97B."

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