Crops ›› 2020, Vol. 36 ›› Issue (1): 55-60.doi: 10.16035/j.issn.1001-7283.2020.01.010

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Design and Verification of New Functional Marker of Chilling-Tolerance COLD1 Gene in Rice Seedling Stage

Tian Mengxiang1,Gong Yanlong2,Zhang Shilong1(),He Youxun1,Lei Yue2,Yu Benxun1,Yu Li1,Li Jiali2,Zhang Dashuang2,Ye Yongyin1   

  1. 1Bijie Institute of Agricultural Sciences, Bijie 551714, Guizhou, China
    2Guizhou Rice Research Institute, Guiyang 550006, Guizhou, China
  • Received:2019-07-28 Revised:2019-12-18 Online:2020-02-15 Published:2020-02-23
  • Contact: Shilong Zhang E-mail:bjrice@163.com

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Abstract:

The nucleotide variation of SNP2 in the fourth exon of COLD1 gene associated with the difference of chilling tolerance at the seedling stage between indica and japonica rice. Individuals with japonica COLD1 Jap allele have higher chilling tolerance. Therefore, the COLD1 Jap allele of japonica rice could be introduced via hybridization, thereby enhancing the cold tolerance of indica rice. In order to improve the selection efficiency of COLD1 gene, a new functional marker for detecting the single nucleotide polymorphism SNP2 between COLD1 Jap and COLD1 Indwas designed based on the technical principles of amplification refractory mutation system PCR. Furthermore, the availability of the functional marker was evaluated using five indica varieties, five japonica varieties, one F1 individual and one F2 from the indica × japonica cross. The results showed that the designed functional markers could accurately identify homozygous COLD1 Jap, homozygous COLD1 Ind and heterozygous genotypes, and amplified bands were identical to their corresponding genotypes, suggesting that it was an effective method for identifying COLD1 gene using this marker designed in this study. The method is simple and inexpensive and could overcome the shortcoming of the COLD1 gene functional markers previously designed based on derived cleaved amplified polymorphic sequence. In conclusion, the new functional marker could be widely used in genetic resource identification and molecular marker-assisted selection breeding of rice COLD1 gene.

Key words: Rice, Chilling-tolerance, COLD1 gene, Functional marker

Fig.1

Design strategies for the primers to detect COLD1 gene The underlines indicate the functional polymorphism SNP2 in the COLD1 gene; the ellipses indicate the same base; the arrows indicate the position and amplification direction of the corresponding primers; the letters in the box present the mismatched bases"

Table 1

The primer sequences of functional marker for COLD1 gene"

引物Primer 序列（5′-3′）Sequence (5′-3′)
COLD1-O-F CATTTCCCCATGCCTTCTCC
COLD1-O-R CAACTGTCCCAACGATACGC
COLD1-I-F1 CCTGGCTTACAGGGAAATTGATGAGAT
COLD1-I-F2 CTGGCTTACAGGGAAATTGATGAGAC
COLD1-I-R GAGCTGCCTTTCCAATGTTTTGATGTTCT

Fig.2

Detection of rice materials by functional marker of COLD1 gene M: Ladder H1 (100, 200, 300, 400, 500, 600, 700, 800, 900, 1000bp）,the same below; 1-11 are Nipponbare, 9311, F1 (9311/Koshihikari), Koshihikari, Kongyu 131, Daohuaxiang 2, Songjing 9, Teqing, Minghui 63, Zhenshan 97, and Nanjing 6, respectively"

Fig.3

Identification of the genotypes of SNP2 site at COLD1 in 16 F2 individuals from the cross Nanjing 6/Daohuaxiang 2 using functional marker"

Fig.4

Identification of differential bases of COLD1Ind gene in Indica rice by four primers 1 and 4: Koshihikari; 2 and 5: 9311; 3 and 6: F1 (9311/Koshihikari)"

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