Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b

Abstract

"Soft" and "hard" are the two main market classes of wheat (Triticum aestivum L.) and are distinguished by expression of the Hardness gene. Friabilin, a marker protein for grain softness (Ha), consists of two proteins, puroindoline a and b (pinA and pinB, respectively). We previously demonstrated that a glycine to serine mutation in pinB is linked inseparably to grain hardness. Here, we report that the pinB serine mutation is present in 9 of 13 additional randomly selected hard wheats and in none of 10 soft wheats. The four exceptional hard wheats not containing the serine mutation in pinB express no pinA, the remaining component of the marker protein friabilin. The absence of pinA protein was linked inseparably to grain hardness among 44 near-isogenic lines created between the soft variety Heron and the hard variety Falcon. Both pinA and pinB apparently are required for the expression of grain softness. The absence of pinA protein and transcript and a glycine-to-serine mutation in pinB are two highly conserved mutations associated with grain hardness, and these friabilin genes are the suggested tightly linked components of the Hardness gene. A previously described grain hardness related gene termed "GSP-1" (grain softness protein) is not controlled by chromosome 5D and is apparently not involved in grain hardness. The association of grain hardness with mutations in both pinA or pinB indicates that these two proteins alone may function together to effect grain softness. Elucidation of the molecular basis for grain hardness opens the way to understanding and eventually manipulating this wheat endosperm property.

Figure 1

A Gly-46 to Ser-46 sequence change in pinB is common among hard wheat varieties and is absent in soft wheat varieties. Gel photo (negative image) of PCR products obtained by using a serine-specific PCR primer on genomic DNA of soft and hard hexaploid wheat varieties. Soft (S) indicates that the variety has soft-textured grain, and hard (H) indicates hard-textured grain. Lane marked MW is a 1 kb molecular mass marker ladder.

Figure 2

PinA transcripts (Puro A) are absent in hard wheat varieties that lack the pinB Ser-46 mutation. PinB transcripts (Puro B) are present in all wheats irrespective of hardness. Each lane was loaded with 10 μg of total RNA isolated from immature seeds at 14 days after flowering. Replicate blots were probed with either pinA or B. SKHD and Class represent the Single Kernel Characterization System phenotypic grain hardness reading and hardness class, respectively (S, soft class; H, hard class).

Figure 3

Friabilin component pinA is absent in Falcon hard wheat variety but present in the soft wheat varieties Chinese Spring (CS), Heron, and the disomic substitution line of Langdon durum containing the 5D chromosome of Chinese Spring [LGD-CSDS5D(5B), abbreviated L5D(5B)] and the serine-type hard wheat disomic substitution line of Chinese Spring containing the 5D of Cheyenne, a hard wheat [CS-CNN DS5D, abbreviated CS (CNN5D)]. Langdon (LGD, abbreviated L) durum has neither pinA nor pinB. Photo of silver-stained SDS/PAGE gel of 14- to 16-kDa region showing Triton X-114-isolated starch surface proteins. Arrows indicate position of friabilin component proteins pinA and pinB (A and B, respectively).

Figure 4

PinA transcripts (Puro A) are absent in the hard wheat variety Falcon (F) and two hard NILs derived from Falcon and Heron (H) soft wheat, one each, after 7 backcrosses (90267 and 90278). PinA transcripts are present in Heron and the two soft NILs (90259 and 90291). PinB transcripts (Puro B) are present in all wheats irrespective of hardness. Each lane was loaded with 10 μg of total RNA isolated from immature seeds at 14 days after flowering (DAF). Replicate blots were probed with either pinA or pinB. SKHD and Class are as described in Fig. 2.

Figure 5

No evidence for recombination between pinA and grain softness. Phenotypic grain hardness of 44 Falcon/Heron hard/soft NILs as measured by Single Kernel Characterization System (SKCS) vs. NIR hardness. Presence or absence of pinA is as shown (+, pinA present; •, pinA absent; ▴, NILs consisting of physical mixtures of seeds containing and lacking pinA).

Figure 6

Presence of GSP-1 transcript is not associated with grain hardness and is not controlled by chromosome 5D. (A) Transcript levels of GSP-1 in the soft wheat variety Heron, the hard wheat variety Falcon, and two soft and two hard NILs derived, one of each hardness class, from Falcon and Heron after seven backcrosses. (B) Transcript levels of GSP-1 in soft and hard chromosome 5D recombinant lines (SR and HR, respectively) derived from Chinese Spring and the hard disomic substitution line (CS-CNN DS5D), the durum variety Langdon (L), and the soft disomic substitution line LGD-CSDS 5D(5B) [abbreviated, Langdon (L5D(5B)]. Each lane was loaded with 10 μg of total RNA isolated from immature seeds at 14 days after flowering, and the blots were probed with a GSP-1-derived probe. SKHD and Class are as described in Fig. 2.