Targeted Deletion of the First Intron of the Wxb Allele via CRISPR/Cas9 Significantly Increases Grain Amylose Content in Rice

Background The rice Waxy (Wx) gene plays a major role in seed amylose synthesis and consequently controls grain amylose content. Wx gene expression is highly regulated at the post-transcriptional level. In particular, the GT/TT polymorphism at the 5′splicing site of its 1st intron greatly affects this intron’s splicing efficiency and defines two predominant Wx alleles, Wxa and Wxb. Wxa rice often harbours intermediate to high amylose contents, whereas Wxb rice exhibits low to intermediate amylose contents. By deleting the Wx 1st intron using CRISPR/Cas9 technology, we generate a completely novel Wx allele and further investigate how intron removal affects Wx gene expression and rice grain amylose content. Results CRISPR/Cas9-mediated targeted deletion of the Wx 1st intron was performed on 4 rice inbred lines: KY131 (Wxb), X32 (Wxb), X35 (Wxa) and X55 (Wxlv). Deletion of the 1st intron occurred in 8.6–11.8% of the primary transformants of these 4 inbred lines. Compared to wild-type plants, amylose content was significantly increased from 13.0% to approximately 24.0% in KY131 and X32 mutant lines, which both carried the Wxb allele. However, no significant difference in amylose content was observed between wild-type plants and X35 and X55 mutant lines, which carried the Wxa and Wxlv alleles, respectively. Wx gene expression analysis of wild-type plants and mutants yielded results that were highly consistent with amylose content results. KY131 and X32 mutants accumulated increased levels of steady mRNA transcripts compared with wild-type plants, whereas steady mRNA levels were not altered in X35 and X55 mutants compared with wild-type plants. Grain quality, including appearance quality and eating and cooking quality, which are tightly associated with amylose content, was also assessed in wild-type and mutant plants, and data were presented and analysed. Conclusions This study presents a novel and rapid strategy to increase amylose content in inbred rice carrying a Wxb allele. Our data strongly suggest that the 1st intron of the Wx gene regulates Wx gene expression mainly at the post-transcriptional level in rice. This finding is in contrast to a previous hypothesis suggesting that it influences Wx gene transcription. In addition, removal of the first intron generates a completely novel Wx allele. Further studies on this new Wx allele will provide invaluable insights into the regulation of Wx gene expression, which will help researchers engineer new Wx alleles to facilitate the breeding of rice cultivars with better eating and cooking quality.

DNA Marker Based Diversity Across Rice Genotypes and Advanced Breeding Lines Bred for Temperate India

Background: Characterization and evaluation of plant genetic resources play an important role for their utilization in the crop improvement programmes. Methods and results: This study entails the agro-morphological, cooking quality and molecular characterization of 51 genotypes / advance breeding lines of rice from Kashmir Himalayas. Significant variability was observed for all agro-morphological and cooking quality traits among all the studied genotypes. Cluster analysis using UPGMA method divided the genotypes into two major clusters having 15 and 36 genotypes. Thirty eight genotypes screened using 24 SSR markers detected 48 alleles with 2.0 alleles per locus and an average polymorphism information content (PIC) of 0.37. High polymorphism information content (PIC) values was observed for the primers RM263 (0.67), RM159 (0.59) and RM333 (0.50). Furthermore, out of 38 SSR markers screened on 192 temperate rice germpalsm lines, R4M17 accurately differentiated indica and temperate japonica genotypes amplifying 220 bp and 169bp, respectively. Accordingly, 15 genotypes were reported as indica and 28 temperate japonica in addition to 149 genotypes as intermediate types. Conclusion: The information on marker-based diversity and performance based on cooking quality and agronomic traits helped to select the most divergent lines for crossing and also the analysis was useful to generate information on indica - japonica classification of our germplasm.

Protein formation and nutritional advantages of promising pea lines in the forest-steppe of the middle Volga region

The experiments were conducted at Samara Research Institute of Agriculture, a branch of the SamSC RAS in 2018–2020. The purpose of the research. Evaluation of promising lines of peas from the competitive variety trial nursery for the nutritional quality of seeds to create new varieties for the Middle Volga region. Methodology and methods of research. The material for the study was 6 promising lines of peas. The nutritional quality of the lines were evaluated according to the following characteristics: the protein content and composition in the seeds, the boiling time of the seeds, the seed cooking coefficient, and the taste of the boiled seeds. The peas grew and developed in dry (2018–2019) and moderate (2020) weather conditions. Results. The protein content in the seeds of the lines was determined by external environmental factors, especially the weather conditions during the bean filling phase. The seeds accumulated more protein when the weather was dry during the bean filling. The protein content of seeds had a positive correlation (r = 0.944) with the average daily air temperature, and a negative correlation (r = 0.986) with precipitation during the bean filling phase. On average, the lines had the same protein content as the Samarius standard but corresponded to the standard value of high-quality varieties (not less than 24.0 %). The following lines had the same seed protein content as the standard (25.6 %): B3737/2-2 (25.2 %), Kt6575 (25.8 %), Kt6358 (24.9 %). These lines contained a large amount of water-soluble protein fraction in comparison with the standard: Kt6575 (16.9 %), Kt6358 (16.6 %), and B3737/2-2 (16.9 %). B3737/2-2 and Kt6575 showed high cooking quality (seed boiling time 110–122 minutes, seed cooking coefficient 2.5–2.6). All lines had a good taste of boiled seeds equal to 4–5 points. Scientific novelty. B3737/2-2, Kt6575, Kt6358 have been suggested to be used as sources of new varieties for their high cooking quality.

Factors affecting the cooking quality of stored carioca beans (Phaseolus vulgaris)

The culinary quality of carioca beans is related to their market value and consumer acceptability. The depreciation of the cooking/technological quality of the product occurs mainly because of the integument browning and the longer cooking time of the grains, which are influenced by the storage time and conditions. The loss of culinary quality reduces the market value of carioca beans because consumers reject darkened grains that are attributed to a longer cooking time. As a result, cooking time (resistance to cooking), the color of the integument, and the texture of the cooked beans are determinant factors in the acceptance of carioca bean cultivars. The browning of the grain integument and the cooking time mainly depends on the environmental conditions, storage time, the tegument of each genotype, and the chemical and physical properties of the cotyledons. Therefore, this review aims to survey the scientific literature on the extrinsic and intrinsic factors that affect the culinary quality of carioca beans.