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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (03): 324-331.doi: 10.3724/SP.J.1006.2018.00324

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

Development and Application of the Functional Marker for Imidazolinone Herbicides Resistant ALS Gene in Rice

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

  • Received:2017-07-19 Accepted:2017-11-21 Online:2018-03-12 Published:2017-12-11
  • Contact: Jie YANG E-mail:wfqjaas@163.com;yangjie168@aliyun.com
  • Supported by:
    This study was supported by the National Key Research and Development Program (2017YFD0100400-3), the Jiangsu Province Key Research and Development Program (Modern Agriculture, BE2015355), the Exploratory Project of the Jiangsu Academy of Agricultural Sciences (ZX(17)2014), the Natural Science Foundation of Jiangsu Province of China (BK20171326), and the Special Fund for Scientific Research on Public Causes (201303102).

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Abstract:

Breeding and utilization of herbicide resistant rice are significant to rice production. By screening the rice germplasm, we found the herbicide resistant material “Jinjing 818”. An SNP mutation G to A was present in Acetolactate synthase (ALS) gene at 1880 bp position, leading to the alteration from serine (S, AGT) to asparagine (N, AAT), which confers herbicide resistance. In this study, 11 allelic-specific PCR (AS-PCR) primers were designed based on the functional mutation. After optimized these primers, we obtained two primer combinations F1N (S1/S9) and F1M (S1/S10), named AS-ALS marker. Using this marker detected the genetic population, its parents, F1 hybrid, F2 and also rice collections, inbred lines, showing that the herbicide susceptibleness allelic ALS-G could be amplified by F1N, the herbicide resistance allelic ALS-A by F1M, and heterozygous genotype by F1N and F1M simultaneously. The genotype of those tested materials perfectly matched with the phenotype of herbicide resistance or susceptibleness. Aided by AS-ALS marker selection, the homozygous ALS-A pedigrees in multi-generation backcross or self-cross showed stable herbicide resistance. Therefore, the allelic-specific PCR functional marker AS-ALS can be used in herbicide breeding efficiently, also screening herbicide resistant rice germplasm. In conclusion, the AS-ALS marker developed in this research is inexpensive and effective in breeding practice.

Key words: rice (Oryza sativa L.), herbicide, acetolactate synthase, ALS gene, functional marker

Table 1

Molecular markers design of ALS gene"

编号
No.		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
S1 AS-ALS-F1 GAGGCAATCATCGCTACTGG
S2 AS-ALS-R1-1 ACAAACCTAGACAGCAGGAAG
S3 AS-ALS-R1-2 CTCTTTATGGGTCATTCAGGTC
S4 AS-ALSn-F2-1 TGTGCTGCCTATGATCCtgAG
S5 AS-ALSn-F2-2 TGTGCTGCCTATGATCCCtAG
S6 AS-ALSm-F2-1 TGTGCTGCCTATGATCCCgAA
S7 AS-ALSm-F2-2 CATGTGCTGCCTATGATCtCgAA
S8 AS-ALSn-R2-1 TGTCCTTGAATGaGCCCCtAC
S9 AS-ALSn-R2-2 TGTCCTTGAATGCGCCCCtAC
S10 AS-ALSm-R2-1 TGTCCTTGAATGCGCCCCtAT
S11 AS-ALSm-R2-2 TGTCCTTGAATGCGCCCttAT

Fig. 1

Sequencing alignment of the ALS gene The ALS gene sequence of Nipponbare has a GenBank number NC_008395.2, the ALS gene sequence of StrawhullS has a GenBank number AY885673.1."

Fig. 2

PCR products of Nipponbare and Jinjing 818 using different primer pairs A: Nipponbare; B: Jinjing 818; M: DL2000 marker (from up to down, 2000, 1000, 750, 500, 250, and 100 bp). Lanes 1 to 11 represent S4/S2, S4/S3, S5/S2, S5/S3, S6/S2, S6/S3, S7/S2, S7/S3, S1/S8, S1/S9, and S1/S10, respectively."

Fig. 3

PCR products after improving annealing temperature A: Nipponbare; B: Jinjing 818; M: DL2000 marker (from up to down, 2000, 1000, 750, 500, 250, and 100 bp). Lanes 1 to 6 represent S4/S2, S4/S3, S5/S2, S5/S3, S1/S9, and S1/S10, respectively."

Fig. 4

Detection of parts of F2 populations using AS-ALS marker and the correspondence with phenotypes M: DL2000 marker (from up to down, 2000, 1000, 750, 500, 250, and 100 bp); P1, Jinjing 818; P2: Nanjing 9108; F1: Jinjing 818/Nanjing 9108; 1-21: the plants of the F2 populations; R: resistant to herbicide; S: susceptible to herbicide."

Fig. 5

Phenotypes of F2 populations after spraying herbicide 1: Jinjing 818; 2: Nanjing 9108; 3: Jinjing 818/Nanjing 9108; 4: the plant with ALS-A genotype; 5: the plant with ALS-G/A genotype; 6: the plant with ALS-G genotype."

Fig. 6

Detection of parts of the high generation lines using AS-ALS marker and the correspondence with phenotypesM, DL2000 marker (from up to down, 2000, 1000, 750, 500, 250, and 100 bp); P1: Jinjing 818; P2: Nanjing 9108; 1-22: the plants of the BC3F3 populations; R: the plants resistant to herbicide; S: the plants susceptible to herbicide."

Fig. 7

Screening rice resources using AS-ALS marker M: DL2000 marker (from up to down, 2000, 1000, 750, 500, 250, and 100 bp). Lanes 1-24 represent Jinjing 818, Nipponbare, Nanjing 40, Nanjing 41, Nanjing 44, Nanjing 45, Nanjing 46, Nanjing 49, Nanjing 9108, Nanjing 5055, Huaidao 5, Suxiu 867, Wuyunjing 21, Wuyunjing 24, Wuyunjing 27, Xudao 3, Xudao 8, Zhendao 88, Zhendao 99, Lianjing 7, Changnongjing 7, 9311, IR36, and Nanjing 16."

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