Development of functional markers for peroxidase activity genes TaPod-7AS and TaPod-4AL in wheat

Abstract

Grain peroxidase (POD) has a pivotal role in determining wheat flour color and end-use products. High POD activity in wheat grains can improve the whiteness and commercial value of flour. The POD genes TaPod-7AS and TaPod-4AL have high effects on POD activity in wheat. Function marker for POD genes can be used to improve flour color attributes accurately and efficiently in wheat molecular breeding. Here, wheat POD genes TaPod-7AS and TaPod-4AL were sequenced and characterized, gene expression analysis indicated that TaPod-7AS, TaPod-4AL, TaPod-D1 genes were highly expressed at 21 days after flowering. Function markers POD-7AS1 and POD-7AS2 were developed for TaPod-7AS, amplifying 424-bp and 464-bp PCR fragments in the cultivars with low and high POD activities, respectively. The markers POD-4AL1 and POD-4AL2 for TaPod-4AL amplified 541-bp and 391-bp fragments in the cultivars with low and high POD activities, respectively. The functional markers were validated in a diverse panel of 268 winter wheat cultivars and advanced lines, indicating significant associations between genotypes and POD activity (P < 0.05). The genotypes with favorable alleles TaPod-7ASb/TaPod-4ALb/TaPod-D1b had higher POD activity (749.9 U.g^-1.min^-1) than those with alleles TaPod-7ASa/TaPod-4ALa/TaPod-D1a (mean POD activity 619.4 U.g^-1.min^-1).

Keywords: TaPod-4AL; TaPod-7AS; Triticum aestivum; Functional marker; Grain POD gene.

Fig. 1

POD gene structure in Arabidopsis thaliana, rice Oryza sativa and Triticum aestivum

Fig. 2

Deduced amino acid sequence alignment of TaPod-7AS, TaPod-4AL, TaPod-D1 from wheat

Fig. 3

Chromosome distribution and analysis of the 4th and 7th homologous groups of POD activity in wheat

Fig. 4

Phylogenetic analysis of 171 POD proteins from 4th and 7th homology groups of POD activity in wheat

Fig. 5

POD domain diagram structure and motif composition in Arabidopsis thaliana, rice Oryza sativa and Triticum aestivum

Fig. 6

Alignment of partial DNA sequences of the alleles TaPod-7ASa and TaPod-7Asb on chromosome 7AS, and TaPod-4ALa and TaPod-4ALb on chromosome 4AL. The sequences of the forward primers and complementary sequences of the reverse primers for POD-7AS1 and POD-7AS2, POD-4AL1 and POD-4AL2 are boxed

Fig. 7

Polymorphisms of polymerase chain reaction fragments amplified by POD-7AS1 (a) and its complementary marker POD-7AS2 (b) in six cultivars with lower peroxidase (POD) activities and six with higher POD activities, respectively. M DNA ladder DL2000; 01 Fengchan3; 02 Bainong3217; 03 An1331; 04 Zhongmai895; 05 11 Xinong1376; 06 Aca 801; 07 Norin 67; 08 Wennong14; 09 Yumai49; 10 Neixiang188; 11 Yumai34;12Beijing0045

Fig. 8

Polymorphisms of polymerase chain reaction fragments amplified by POD-4AL1 (a) and its complementary marker POD-4AL2 (b) in six cultivars with lower peroxidase (POD) activities and six with higher POD activities, respectively. M DNA ladder DL2000; 01 Xiaozhan54; 02 Shi4185; 03 Luohan2; 04 Shan715; 05 Zhoumai23; 06 Heng7228; 07 Yangxiaomai; 08 Yanzhan4110; 09 Zhengzhou3; 10 Lumai9; 11 Mantol; 12 Barra

Fig. 9

Average POD activities of wheat varieties with different allele combinations. Note: different letters for mean POD activities indicate significant differences at P <0.05

Fig. 10

Expression patterns of 12 wheat cultivars of TaPod-7AS. Note: different letters for mean POD activities indicate significant differences at P <0.05

Fig. 11

Expression patterns of 12 wheat cultivars of TaPod-4AL. Note: different letters for mean POD activities indicate significant differences at P <0.05

Fig. 12

Expression patterns of 12 wheat cultivars of TaPod-D1

Fig. 13

The expression pattern of POD genes in different tissues and different stages of flowering