Cloning and characterization of special HMW glutenin subunit genes from Aegilops longissima L. and their potential for wheat quality improvement

Abstract

Identification and cloning of new glutenin genes from wheat-related species can provide candidate gene resources for quality improvement of wheat. In this study, ten special high-molecular-weight glutenin subunits (HMW-GS), including five x-type (1S^l2x, 1S^l16x, 1S^l17x, 1S^l23x, and 1S^l25x) and five y-type (1S^l2y, 1S^l6y₁, 1S^l16y, 1S^l17y, and 1S^l23y) from Aegilops longissima L. (S^lS^l, 2n = 2x = 14) were identified, and their complete encoding genes were cloned by allelic-specific polymerase chain reaction (AS-PCR). The deduced amino acid (aa) residues of the x-type subunit genes ranged from 821 aa (2469 bp) to 941 aa (2829 bp), while those of the y-type subunit genes varied from 749 aa (2250 bp) to 771 aa (2361 bp). These special HMW-GS had a longer central repetitive domain with more glutamine repeats and glutamine residues compared to the previously characterized HMW-GS in common wheat, which provided a structural basis for superior gluten quality formation. The authenticity of the four cloned genes were verified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Abundant single-nucleotide polymorphism (SNP) and insertion/deletion (InDel) variations among these genes were identified, which would benefit for developing specific molecular markers used for wheat gluten quality improvement. Phylogenetic analysis revealed that the 1S^l-encoded HMW-GS had close relationships with those from bread wheat, which were divergent from Triticum species at 2.10-10.00 million years ago. Our results indicate that the 1S^l genome contains superior candidate glutenin genes that have potential application values for the improvement of wheat bread making quality.

Keywords: AS-PCR; Aegilops longissima L.; Gluten quality; HMW-GS; MALDI-TOF/TOF–MS; Phylogenetics.

Fig. 1

Identification of new HMW-GS in Ae. longissima L. by SDS-PAGE. Lines 1–8: China Spring (CS), PI542196, PI604108, PI604129, PI604130, PI604136, PI604142, and CS-1S^l/1B)

Fig. 2

AS-PCR amplification of x-type and y-type HMW-GS genes from 1S^l genome of Ae. longissima L. with primers SxF + SxR and SyF + SyR. a PCR amplification of x-type HMW-GS from 1S genome of Ae. longissima L., 1, 2: 1S¹2x; 3, 4: 1S¹16x; 5, 6: 1S¹17x; 7, 8: 1S¹23x; 9, 10: 1S¹25x; M: DNA makers. b PCR amplification of y-type HMW-GS from 1S genome of Ae. longissima L., 1, 2: 1S¹2y; 3, 4: 1S¹6y₁; 5, 6: 1S¹16y; 7, 8: 1S¹17y; 9, 10: 1S¹23y; M: DNA markers

Fig. 3

Mutliple sequence alignment of the deduced amino acid sequences of 1S^lx and 1Bx types HMW-GS and five newly cloned subunit genes in this work in Ae. longissima. The red box indicates the cysteine residues

Fig. 4

Multiple sequence alignment of the derived amino acid sequences of 1S^ly and 1By types HMW-GS and five newly cloned subunit genes in this work in Ae. longissima. The red box indicates the cysteine residues

Fig. 5

The phylogenetic tree of 30 HMW-GS based on complete amino acid sequences. 1S¹2x* and 1S¹16y* is cloned from the S genome of Chinese Spring chromosome substitution line CS-1Sl/1B, in which the 1B chromosome was substituted by 1S^l from Ae. longissima