ICAR-NRCPB, New Delhi

ICAR-NRCPB, New Delhi

PI: Dr P K Mandal

Co PI: Dr S K Sinha

Amylase trypsin inhibitors in wheat:Grain development in wheat is very important and crucial process in crop life cycle and amylase-trypsin inhibitors (ATI) are a type of defence protein expressed only during grain development.

Expression analysis of ATI genes during grain filling: Previously, relative expression of CM3 ATI in 5 different genotypes was shown considering actin as an internal control in four different stages after anthesis i.e. 2, 3, 4 and 5 WAA (weeks after anthesis). During the reporting period the other different ATIs were also analyzed, but in 2 different varieties/accessions of each genome.

Genotypes including three diploid progenitors of wheat used for ATI expression studies: CM3 shows similar result for the other varieties/accessions of different genotypes (Fig.1). It shows higher expression in T. monococcum (AA), Ae. speltoides (BB), PDW 233 (AABB) at the end of the grain filling as the other genotypes i.e. Ae. squarossa (DD), PBW343 (AABBDD) shows less expression at the end of the grain filling. The analysis shows that hexaploid wheat shows higher expression of ATI in early stages during grain development. The tetraploid wheat shows higher expression in later stages of grain filling.

Relative expression of ATI CM3 among the different genotypes at different growth stages.

A: Comparative expression during development in each genotype separately;

B: Comparative expression among the genotypes in each growth stage separately.

Similarly the other important ATIs was also analyzed and showed different expression pattern in different genotypes at different growth stages.

Relative expression of ATI CM16 among the different genotypes at different growth stages.

A: Comparative expression during development in each genotype separately;

B: Comparative expression among the genotypes in each growth stage separately

Relative expression of ATI 0.19 among the different genotypes at different growth stages. A: Comparative expression during development in each genotype separately; B: Comparative expression among the genotypes in each growth stage separately

Relative expression of ATI CM2 among the different genotypes at different growth stages. A: Comparative expression during development in each genotype separately; B: Comparative expression among the genotypes in each growth stage separately

Relative expression of ATI CM1 among the different genotypes at different growth stages. A: Comparative expression during development in each genotype separately; B: Comparative expression among the genotypes in each growth stage separately.

Amylase and Trypsin inhibition assay:

As there are many other types of inhibitors present in wheat, we had also quantified the level of total amylase inhibition as well as total trypsin inhibition caused by a particular wheat genotype at mature stage.

The result shows higher inhibition of amylase enzyme by wheat extract of DD genotype as the other progenitor’s AA and BB shows less inhibition of amylase enzyme. Among the cultivated wheat hexaploid shows higher inhibition of amylase enzyme as compared to tetraploid wheat. For trypsin inhibition, among the three wheat progenitors wheat extract of AA genotypes shows higher inhibition of trypsin enzyme as compared to the other two i.e. BB and DD. This result shows that among the cultivated wheat hexaploid shows higher inhibition of trypsin enzyme as compared to tetraploid wheat. 

 Trancriptome analysis of cultivated wheat and its progenitors during grain filling:

As grain development is very important physiological process, under this project we performed reference assisted de-novo transcriptome analysis of cultivated wheat and its progenitors during grain filling. On an average of 64.03 million high quality reads were obtained, of which ~79.04% of reads aligned to the reference (Triticum aestivum) genome. Denovo analysis was carried out for the unaligned reads. Transcripts generated for reference based as well as Denovo were merged to create master unigene, which was further used for annotation and differential analysis.

Summary of trimming and read mapping results of the sequences generated from five cDNA libraries of AA: T. monococcum (PKM1); BB: Ae. speltoides (PKM2); DD: A. squarossa (PKM3), durum wheat (AABB: PDW233) (PKM4) and bread wheat (AABBDD: PBW343) (PKM5).