Establishment of a Multi‐Index Classification System for Oat Grain Hardness Based on Phenotypic, Molecular, and Physicochemical Indicators

ABSTRACT Background Grain hardness is a simple yet critical quality trait that governs raw material selection and processing performance in the oat industry. While wheat hardness has been thoroughly characterized with a mature and widely accepted grading system, oat grains are inherently softer than wheat, and the long‐standing lack of an independent evaluation system and in‐depth mechanistic insights into oat grain hardness has severely hindered the industrial standardization of oat products globally. Aims This study aimed to screen robust and reliable indicators for oat grain hardness, elucidate the key biochemical determinants underlying oat hardness formation, and establish a standardized quantitative classification system for precise oat hardness grading. Methods A total of 40 hulled and naked oat varieties with wide‐ranging hardness levels, sourced from China and Australia, were selected as experimental materials. A multi‐scale systematic approach was adopted, including physical property measurements (texture profile analyzer [TPA], single kernel characterization system [SKCS], grinding hardness index [HI]), solvent retention capacity (SRC) assay, and molecular marker analysis via Vin gene expression quantification. Correlation analysis was performed to validate core hardness indicators, and K‐means clustering was used to construct the classification system. Results SKCS values were validated as robust indicators of oat grain texture, which exhibited significant correlations with TPA parameters, HI measurements, Vin1 gene expression levels, as well as calcium chloride SRC and sodium carbonate SRC values. Notably, a significant positive correlation ( r = 0.43) was revealed between CaCl 2 SRC and β‐glucan content, indicating that the cross‐linking between calcium ions and cell wall macromolecules is a key biochemical determinant of oat grain hardness. By integrating the core validated indicators, a three‐dimensional classification system for oat hardness, comprising SKCS values, Vin1 expression levels, and CaCl 2 SRC, was established via K‐means clustering. This system achieved precise categorization of oats into three grades: hard type (SKCS > 26.21, Vin1 < 0.41, CaCl 2 SRC > 110.97%), intermediate type (SKCS 20.41–26.21, Vin1 0.41–1.21, CaCl 2 SRC 90.13%–110.97%), and soft type (SKCS < 20.41, Vin1 > 1.21, CaCl 2 SRC < 90.13%). Conclusions This study fills critical gaps in the mechanistic research and standardized grading system for oat grain hardness. The established three‐dimensional classification system provides a unified, quantitative framework for international oat quality grading, industrial processing parameter optimization, and molecular marker‐assisted breeding of oat varieties with target hardness traits.