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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (4): 1027-1034.doi: 10.3724/SP.J.1006.2022.14066

• RESEARCH NOTES • Previous Articles    

Creation and identification of peanut germplasm tolerant to triazolopyrimidine herbicides

LIU Jia-Xin1,2(), LAN Yu1,2, XU Qian-Yu1, LI Hong-Ye1, ZHOU Xin-Yu3, ZHAO Xuan1, GAN Yi1, LIU Hong-Bo1, ZHENG Yue-Ping1, ZHAN Yi-Hua1, ZHANG Gang3, ZHENG Zhi-Fu1,2,*()   

  1. 1College of Advanced Agricultural Sciences, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
    2College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
    3Wendeng Jiahe Seed Corporation, Ltd., Weihai 264400, Shandong, China
  • Received:2021-04-18 Accepted:2021-07-12 Online:2022-04-12 Published:2021-08-11
  • Contact: ZHENG Zhi-Fu E-mail:252606752@qq.com;zzheng@zafu.edu.cn
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(31871660)

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Abstract:

At present, the germplasm resources of herbicide-tolerant peanut (Arachis hypogaea) are scarce, which restrict the diversification of peanut-based cropping system. To create peanut germplasm with tolerance to different herbicides, a mutant population consisting of more than 55,000 peanut lines were generated by ethylmethanesulfonate mutagenesis. We screened this population with a variety of different herbicides to obtain multiple mutants with tolerance to different herbicides. One of these lines displayed strong tolerance to florasulam and flumetsulam in the experiments with foliar herbicide spraying under field conditions as well as in various laboratory evaluation for the herbicide tolerance, while the herbicide-tolerant trait did not have adverse effects on peanut yield and quality. To determine whether this trait was associated with a target-site-based resistance to the herbicides, we compared gene sequences and relative expression levels of these two acetohydroxyacid synthases (AHASs) as the herbicide target enzymes between the mutant and wild type. Molecular cloning and sequence analysis revealed that peanut chromosome A10 and B10 each contained a gene, named as AhAHAS1a and AhAHAS1b, which were highly similar to Arabidopsis AHAS. Peanut chromosome A08 and B08 also each carried an AHAS gene, named as AhAHAS2a and AhAHAS2b, respectively. However, compared with the wild type, the genes in the mutants had no nucleotide substitutions that could alter the amino acid sequences. Furthermore, it was evident that there was no significant difference in the relative expression levels of AhAHAS genes between the mutant and wild type. In summary, these results indicate that the herbicide tolerance of the mutant might be caused by non-target-site-based resistance mechanism.

Key words: Arachis hypogaea, ethylmethanesulfonate mutagenesis, triazolopyrimidines, herbicide tolerance, quality trait

Table 1

Primer sequences used in this study"

引物
Primer		 引物序列
Primer sequence (5'-3')		 目的基因
Target gene
LY1/2FP ATGGCTGCCACTGCTTCCAAAC A10或B10染色体的AHAS
AHAS on chromosome A10 or B10
LY1RP TCAATATTTTGTTCTGCCATCGCCT
LY2RP CAATATTTTGTTCTGCCATCACCT
XQY1FP CATTCTCCACCGTATCTCCATC A08或B08染色体的AHAS
AHAS on chromosome A08 and B08
XQY1RP TACATGCCACTGTCTGGTTACTACT
LY31FP ACGAAGCATGCTTACCTTGTTCTTG A10或B10染色体的AHAS
AHAS on chromosome A10 and B10
LY31RP AACATAACTGGTTGATCCCAATTGG
XQY2FP AGCTTGAGGCTTTCGCGAGT A08或B08染色体的AHAS
AHAS on chromosome A08 and B08
XQY2RP CCCTTTTCCTCCAAGATCCCA
XQY4FP CAGGATTTGCCGGTGATGATG β-肌动蛋白
β-Actin
XQY4RP TCTGTTGGCCTTCGGGTTGAG

Fig. 1

Effects of foliar spraying with herbicides on seedling growth in the wild type and mutant peanuts Peanut plants were treated with herbicides at the 3-leaf growth stage. The amount of herbicide “Mai Xi” (450 mL hm-2) used was three times that of normal field application, and photographs were taken after 4 weeks of the treatment. A: the wild type ‘Shanhua 15’; B: the herbicide tolerant mutant H2-20-1 (M7)."

Fig. 2

Effects of foliar spraying with herbicide on growth in the wild type and mutant peanut at maturation stage Peanut plants were treated with herbicides at the 3-leaf growth stage. The amount of herbicide “Mai Xi” (450 mL hm-2) used was three times that of normal field application, and photographs were taken at maturity stage. A: the wild type ‘Shanhua 15’; B: the herbicide tolerant mutant H2-20-1 (M7)."

Fig. 3

Effects of seed soaking with herbicides on root growth of mutant H2-20-1 and wild type ‘Shanhua 15’ The concentrations of flumetsulam (Flu) and florasulam (Flo) were 9.90 g hm-2 and 7.50 g hm-2, respectively. The amount of herbicides used was twice that of normal field application and photographs were taken on the 15th day after treating. WT: wild type."

Fig. 4

Effects of herbicides on root growth of mutant H2-20-1 and wild type ‘Shanhua 15’ under the condition of sand culturing The herbicides were dissolved in water and mixed into sand. The concentrations of flumetsulam (Flu) and florasulam (Flo) were 9.90 g hm-2 and 7.50 g hm-2, respectively. The amount of herbicides used was twice that of normal field application and photographs were taken on the 15th day after treating. WT: wild type."

Fig. 5

Oil content of mature seeds in wild type and peanut mutant H2-20-1 Two batches of seeds from different planting years (in 2020 and 2019) were used for analysis. WT: wild type. t-test statistical analysis revealed that there was no significant difference in seed oil content between the mutant and wild type peanut sampled in two consecutive years."

Fig. 6

Fatty acid composition of mature seeds of wild type and peanut mutant H2-20-1 Two batches of seeds from different planting years in 2020 and 2019 were used for data analysis. WT: wild type. The asterisk (*) represents significant difference at P < 0.05 compared with WT by t-test."

Fig. 7

Amino acid composition of mature seeds in wild type and peanut mutant H2-20-1 WT: wild type. The asterisk (*) represents significant statistical difference at P < 0.05 compared with WT by t-test."

Table 2

Sequence similarity of the AHAS1 between peanut mutant H2-20-1 (MT) and wild type (WT)"

AHAS1a (MT) AHAS1a (WT) AHAS1b (MT) AHAS1b (WT)
AHAS1a (MT) 100 99 99
AHAS1a (WT) 99 99
AHAS1b (MT) 100
AHAS1b (WT)

Table 3

Sequence similarity of the AHAS2 between peanut mutant H2-20-1 (MT) and wild type (WT)"

AHAS2a (MT) AHAS2a (WT) AHAS2b (MT) AHAS2b (WT)
AHAS2a (MT) 100 99 99
AHAS2a (WT) 99 99
AHAS2b (MT) 100
AHAS2b (WT)

Fig. 8

Relative expression patterns of AHAS genes in different tissues of wild-type peanut at different developmental stages Value of gene relative expression level in the stem at seeding stage was set at “1”. DAF: days after flowering."

Fig. 9

Relative expression patterns of AHAS genes between peanut mutant H2-20-1 and wild type The relative expression level of AHAS2 in wild-type leaves was set at “1”. t-test showed there was no significant difference in the relative expression level of AHAS gene between peanut mutant and wild type."

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