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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (4): 684-690.doi: 10.3724/SP.J.1006.2021.02035

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

Development and application of functional marker for high nitrogen use efficiency and chilling tolerance gene OsGRF4 in rice

SUN Ping-Yong1(), ZHANG Wu-Han1, ZHANG Li2, SHU Fu1, HE Qiang1, PENG Zhi-Rong1, DENG Hua-Feng1,3,*()   

  1. 1State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, Hunan, China
    2Nuclear Agriculture and Space Breeding Research Institute, Changsha 410125, Hunan, China
    3Hunan Academy of Agricultural Sciences, Changsha 410125, Hunan, China
  • Received:2020-05-22 Accepted:2020-08-19 Online:2021-04-12 Published:2020-09-10
  • Contact: DENG Hua-Feng E-mail:zlspy23@126.com;dhf@hhrrc.ac.cn
  • Supported by:
    Natural Science Foundation of Hunan(2019JJ50427);Natural Science Foundation of Hunan(2020JJ5287);Hunan Province Key Research and Development Program Project(2018NK1020-1);Hunan Agricultural Science and Technology Innovation Project(2020CX08);Hunan Agricultural Science and Technology Innovation Project(2019TD06)

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Abstract:

The use of molecular marker-assisted selection (MAS) to breed rice with high nitrogen-use efficiency is one of the most effective methods to reduce the amount of nitrogen fertilizer quantity and to develop the green and sustainable agriculture. The gene growth-regulating factor 4 (OsGRF4) encodes a growth regulatory factor protein, and the mutation in the coding region from the nucleotides TC to AA in 487 and 488 substituted the amino acid serine for lysine, which resulting in enhancing nitrogen-use efficiency, increasing grain yield, and improving chilling tolerance in rice. In order to improve the selection efficiency of OsGRF4 in rice breeding, an allele-specific PCR (AS-PCR) marker combination, PF+DMR+PR and PF+XMR+PR, was developed based on the single nucleotide polymorphism in the functional region of the OsGRF4 alleles. The functional marker was used to identify genotypes of different varieties and an F2 population derived from Chuandali/Jusuidao. The three different genotypes of OsGRF4 locus could be accurately distinguished, which was further confirmed by sequencing. This functional marker is simple to operate and low-cost, and could provide a technical support when using MAS to breed new rice varieties of high nitrogen-use efficiency, high yields, and increased chilling tolerance.

Key words: rice, high nitrogen-use efficiency, chilling tolerance, OsGRF4 gene, AS-PCR

Fig. 1

Strategy of functional markers design for OsGRF4 SNP3 indicates functional polymorphic sites; PF and PR are outer primers; DR and XR are reverse inner primers, which matching the bases AA and TC, respectively. A mismatch base C was introduced in the 3′ end of DR and XR to develop DMR and XMR. The arrows indicate amplification direction of the primers."

Fig. 2

PCR amplification of Chuandali and Jusuidao using different primer pairs The singular lane is ‘Chuandali’, the dual lane is ‘Jusuidao’. M: 100 bp marker; 1, 2: PF+PR; 3, 4: PF+DR; 5, 6: PF+XR; 7, 8: PF+DR+PR; 9, 10: PF+XR+PR; 11, 12: PF+DMR; 13, 14: PF+XMR; 15, 16: PF+DMR+PR; 17, 18: PF+XMR+PR."

Fig. 3

Molecular detection of OsGRF4 genotypes by functional markers in different rice varieties A: PF+DMR+PR; B: PF+XMR+PR; M: 100 bp marker; 1: Chuandali; 2: Jusuidao; 3: NIL-OsGRF4; 4: NIL-Osgrf4; 5-24: Nongxiang 99, Yuzhenxiang, Xiangwanxian 17, Gang 46B, BL122, Jiafuzhan, Nongxiang 18, Minghui 86, Xinyinzhan, Yuzhuxiang, R700, Fengyuan B, Nongxiang 29, Nanyangzhan, R299, 02428, C418, P7144, Nongxiang 16, CY016."

Fig. 4

Molecular detection of genotypes by co-separation marker GL2-11 in F2 population derived from Chuandali/Jusuidao M: 100 bp marker; 1: Chuandali; 2: Jusuidao; 3-24: individual plants isolated from F2 population."

Fig. 5

Molecular detection of OsGRF4 genotypes by functional markers in F2 population derived from Chuandali/Jusuidao M: 100 bp marker; A: PF+DMR+PR; B: PF+XMR+PR; 1: Chuandali; 2: Jusuidao; 3-24: individual plants isolated from F2 population."

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