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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (11): 2080-2090.doi: 10.3724/SP.J.1006.2021.01089

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

Relationship between the allelic variations at the Ppo-A1 and Ppo-D1 loci and pre-harvest sprouting resistance in wheat

HUANG Yi-Wen1(), DAI Xu-Ran1, LIU Hong-Wei1, YANG Li1, MAI Chun-Yan2, YU Li-Qiang3, YU Guang-Jun3, ZHANG Hong-Jun1,*(), LI Hong-Jie1,*(), ZHOU Yang1,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / National Engineering Laboratory for Crop Molecular Breeding, Beijing 100081, China
    2Center for Technological Innovation of Dwarf-male-sterile Wheat Breeding (Xinxiang), Xinxiang 453731, Henan, China
    3Zhaoxian Experiment Station, Shijiazhuang Academy of Agricultural and Forestry Sciences, Zhaoxian 051530, Hebei, China
  • Received:2020-11-20 Accepted:2021-03-19 Online:2021-11-12 Published:2021-04-01
  • Contact: ZHANG Hong-Jun,LI Hong-Jie,ZHOU Yang E-mail:18838916683@163.com;zhanghongjun01@caas.cn;lihongjie@caas.cn;zhouyang@caas.cn
  • Supported by:
    Science and Technology Project for Modern Seed Industry of Hebei(19226351D);National Key Research and Development Program of China(2017YFD0101000);Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences

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Abstract:

Ppo-A1 and Ppo-D1 are the major genes that control the activity of polyphenol oxidase (PPO) in wheat. It has been reported that the activity of polyphenol oxidase affects pre-harvest sprouting (PHS) resistance, but the effect of different alleles/allelic combinations at the Ppo-A1 and Ppo-D1 loci on PHS resistance remains unclear. The current study was carried out to elucidate the effects based on the germination index obtained from 248 Chinese wheat cultivars in a three-year trial in combination with genotypic data at the Ppo-A1 and Ppo-D1 loci. Analysis of variation for the Ppo-A1 and Ppo-D1 loci showed that year, Ppo-A1 locus and Ppo-A1 × Ppo-D1 interaction had significant effects on germination index. At locus Ppo-A1, germination index of cultivars carrying the allele Ppo-A1b of low PPO activity was 5.22% lower than that carrying the allele Ppo-A1a of high PPO activity on average. In contrast, the cultivars carrying the allele Ppo-D1a of low PPO activity had higher germination index than that carrying the allele Ppo-D1b of high PPO activity at locus Ppo-D1, but no significant differences between two alleles. Among the four allelic combinations, the cultivars with the Ppo-A1bPpo-D1b had the lowest germination index. The relationship between the alleles at locus Ppo-A1 and PHS resistance had been verified in the Lunxuan 13 × Jimai 20 F2 and F2:3 segregation populations. There were significantly positive correlations between PPO activity / relative expression level of Ppo-A1 gene and germination index. This study suggests that functional markers of allele Ppo-A1b can be effectively applied in marker-assisted selection for PHS resistance.

Key words: Triticum aestivum, the breeding for resistance to pre-harvest sprouting, polyphenol oxidase, Ppo-A1b allele, molecular marker-assisted selection

Table 1

Information on the functional markers for identifying different alleles at the Ppo-A1 and Ppo-D1 loci"

位点
Locus		 标记
Marker		 引物序列
Primer sequence (5′-3′)		 等位基因
Allele		 扩增片段
Amplified fragment (bp)		 退火温度
Annealing temperature (℃)		 参考文献
Reference
Ppo-A1 PPO18 F: AACTGCTGGCTCTTCTTCCCA Ppo-A1a 685 60 [25]
R: AAGAAGTTGCCCATGTCCGC Ppo-A1b 876
Ppo-D1 PPO16 F: TGCTGACCGACCTTGACTCC Ppo-D1a 713 60 [26]
R: CTCGTCACCGTCACCCGTAT
PPO29 F: TGAAGCTGCCGGTCATCTAC Ppo-D1b 490
R: AAGTTGCCCATGTCCTCGCC

Table 2

Primers for qRT-PCR"

基因
Gene		 上游引物序列
Forward primer sequence (5′-3′)		 下游引物序列
Reverse primer sequence (5′-3′)
Ppo-A1 GCAACTGCCAAACGCCCGAGC CAGCACGAAGTCGGTGATGCC
Ppo-D1 CCGGACCTTGAGATCCAGGTG. GAAGGTGTCGTCGCCGATGAG
Actin GTTTCCTGGAATTGCTGATCGCAT CATTATTTCATACAGCAGGCAAGC

Table 3

Analysis of variance of germination index of 248 wheat cultivars from 2018 to 2020"

变异来源Source of variation 自由度DF 均方Mean square
基因型 Genotypes (G) 247 0.26**
年份 Years (Y) 2 10.91**
基因型 × 年份互作G × Y interaction 494 0.03**
重复 Replications 2 0.04
误差 Error 1486 0.01

Fig. 1

Correlation analysis of germination index between years in the Chinese wheat cultivars examined **Significance at P < 0.01."

Table 4

Mean germination index, standard deviation (SD), variation range, and coefficient of variation from 248 wheat cultivars and Lunxuan 13 × Jimai 20 population (%)"

品种/群体
Cultivar/population		 年份
Year		 均值±标准差
Mean ± SD		 变幅
Range		 变异系数
Coefficient of variation
248份小麦品种 2018 47.46 ± 19.48 5.33-93.71 40.97
248 wheat cultivars 2019 58.39 ± 17.61 17.14-92.19 30.10
2020 34.21 ± 19.05 2.95-88.71 55.59
平均 Mean 46.70 ± 17.03 11.55-88.70 36.41
轮选13 × 济麦20群体 2019 (F2) 59.49 ± 16.59 18.10-90.76 27.88
Lunxuan 13 × Jimai 20 population 2020 (F2:3) 46.94 ± 13.22 19.14-79.53 28.18

Fig. 2

Comparison of pre-harvest sprouting resistance between parents Lunxuan 13 (A) and Jimai 20 (B) after germination for three days"

Fig. 3

Detection of alleles at the Ppo-A1 and Ppo-D1 loci in selected wheat cultivars using markers PPO18, PPO16, and PPO29 M: marker DNA ladder 2000; 1: Jing 411; 2: Luohan 7; 3: Shannong 20; 4: Shi 4185; 5: Shimai 12; 6: Xinmai 20; 7: Kaimai 20; 8: Xunong 5; 9: Huaimai 20; 10: Xingmai 13."

Table 5

Analysis of variance of germination index for 248 wheat cultivars at the Ppo-A1 and Ppo-D1 loci over three years"

变异来源
Source of variation		 自由度
DF		 均方
Mean square
年份 Years (Y) 2 3.543**
Ppo-A1 1 0.512**
Ppo-D1 1 0.009
Ppo-A1 × Ppo-D1互作Ppo-A1 × Ppo-D1 interaction 1 0.173*
Ppo-A1 × 年份互作 Ppo-A1 × Y interaction 2 0.010
Ppo-D1 × 年份互作 Ppo-D1 × Y interaction 2 0.001
Ppo-A1 × Ppo-D1 × 年份互作 Ppo-A1 × Ppo-D1 × Y interaction 2 0.010
误差 Error 732 0.035

Fig. 4

Comparison of germination index between alleles at the Ppo-A1 (A) and Ppo-D1 (B) loci in the wheat cultivars examined ** represents significant at P < 0.01. Dot: outlier."

Fig. 5

Comparison of germination index among allelic combinations at the Ppo-A1 and Ppo-D1 loci in the wheat cultivars examined Different letters indicate significance at P < 0.01 in germination index between alleles. Dot: outlier."

Fig. 6

Comparison of germination index among genotypes at the Ppo-A1 locus in the Lunxuan 13 × Jimai 20 population Different letters indicate significant difference at P < 0.01 in germination index between alleles."

Fig. 7

Comparison of PPO activity among wheat cultivars (A)/F2:3 lines (B) in the Lunxuan 13 × Jimai 20 population with different genotypes Different letters indicate significant difference at P < 0.01 in germination index (GI) / PPO activity among genotypes."

Fig. 8

Quantitative real time-PCR expression analysis of Ppo-A1 and Ppo-D1 genes in wheat cultivars (A and B) and F2:3 lines (C) in the Lunxuan 13 × Jimai 20 population Different letters indicate significant difference at P < 0.01 in germination index (GI) / relative expression level among genotypes."

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