Analysis of gene expression in the developing seed with the quality of wheat

Abstract

Wheat is the leading crop in the temperate world with its ability to adapt to different environments allowing it to be grown in regions with diverse climatic and environmental conditions. Wheat quality improvement is one of the major objectives of wheat breeding. Breeding for the desired combination of quality attributes is most effective when genes regulating the desired attributes are known.The major objective of this study was to associate gene expression in the developing seed with the quality of wheat: grain hardness, flour yield and the nutritive value of the grain. RNA-sequencing data obtained from 35 diverse worldwide wheat genotypes at two stages of seed development, 14 and 30 days post anthesis (DPA), were used in this study. One of the challenges in next generation sequencing (NGS) data analysis, in polyploid plants such as wheat, is the difficulties in accurate identification of the allele-specific gene expression. To overcome this challenge, a protocol was developed to identify allele-specific/sub-genome-specific gene expression.Sub-genome-specific gene expression of BADH2 was identified to demonstrate this protocol. It was observed that differential patterns of sub-genome-specific gene expression changes over time as the expression patterns at 14 DPA were different than that at 30 DPA. Sub-genome-specific gene expression patterns at a particular time point were similar for most genotypes but not for all.A highlight of this study was the identification of a gene which showed strong association with flour yield, namely, the putative gene encoding fasciclin-like arabinogalactan protein (FLA). This gene was identified from the differential gene expression analysis between good milling (flour extraction rate ≥77) and poor milling (flour extraction rate ≤ 77) wheat genotypes. FLA contains the ancient cell-adhesion domain which may play a role in the structural strength of the grain. At both 14 and 30 DPA, all the good-milling genotypes showed down-regulation of FLA-8, whereas, up-regulation was observed in all the poor millers.Direct selection for genotypes with appropriate expression of these genes will greatly accelerate wheat breeding and ensure high recoveries of flour from the wheat. Although grain texture has a significant impact on the milling performance of wheat, no correlation was observed between grain hardness (SKCS HI) and flour yield (Flour extraction rate, Laboratory Buhler mill). In this study, statistically significant differentially expressed genes between soft and hard wheat genotypes were also identified, at 14 and 30 DPA. Puroindoline (Pin) genes were the most statistically significant differentially expressed genes. Pina and Pinb genes are known to control the majority of variation in wheat grain hardness, but not all. Soft wheats usually contain wild-type Pin alleles, whereas, hard wheats contain mutations in either of the Pin alleles.Unexpectedly, variation in some of the hard wheat genotypes with wild-type Pin alleles was explained by differential expression levels of Pina and Pinb. Pinb was expressed at higher levels than Pina in these genotypes whereas the opposite was found in other genotypes. Gene expression of GSP-1 and Pinb-2 showed no association with grain hardness. Expression of Pina and Pinb explained more than 60% of the variation in grain hardness.Another objective was to study the nutritional quality of the wheat. People in developed and developing world are moving from energy-rich food to nutrition-rich food to increase the health benefits. The nutritional value of wheat can be improved by producing grain with a higher amount of aleurone tissue. Aleurone is the richest source of nutrients in the wheat grain. But less is known about the genetic regulation of aleurone-layer development. In this study, expression of wheat transcription factors (TF) (wheat Transcription factor database) was identified in developing (6, 9 and 14 DPA) wheat seed tissues (the aleurone and the endosperm) for the first time, using the Australian cultivar Banks. TFs from the MIKC family were highly up-regulated in the aleurone, whereas, the TFs from the PLATZ family were highly up-regulated in endosperm. Increased numbers of TFs were expressed at 14 DPA in the aleurone but the endosperm showed decreases in numbers. The same trend was observed for the global expression of wheat genes in an earlier study by other researchers, using the same sequencing data. Expression was also identified for the dek1, cr4 and sal1 genes which have been associated with aleurone layer development in maize. Multiple alleles of these genes were identified in wheat; dek1 on chromosome 6AL, 6BL and 6DL, and cr4 and sal1 on 7AL, 7BL and 7DL. Expression of sal1 was higher, at any developmental stage, compared to dek1 and cr4. Comparable expression of dek1 was observed in both the aleurone and the endosperm but cr4 expression was more specific to aleurone. The research findings of this study will provide a new set of tools for wheat breeders that should reduce constraints of selection for these aspects of wheat quality.