Multiple loci with cumulative effects on late maturity α-amylase (LMA) in wheat

Main conclusion The cumulative action of combinations of alleles at several loci on the wheat genome is associated with different levels of resistance to late maturity α-amylase in bread wheat. Abstract Resistance to late maturity α-amylase (LMA) in bread wheat (Triticum aestivum L.) involves a complex interaction between the genotype and the environment. Unfortunately, the incidence and severity of LMA expression is difficult to predict and once the trait has been triggered an unacceptably low falling number, high grain α-amylase may be the inevitable consequence. Wheat varieties with different levels of resistance to LMA have been identified but whilst some genetic loci have been reported, the mechanisms involved in resistance and the interaction between resistance loci requires further research. This investigation was focused on mapping resistance loci in populations derived by inter-crossing resistant wheat varieties or crossing resistant lines with a very susceptible line and then mapping quantitative trait loci. In addition to the previously reported locus on chromosome 7B for which a candidate gene has been proposed, loci were mapped on chromosomes 1B, 2A, 2B, 3A, 3B, 4A, 6A and 7D. These loci have limited effects on their own but have a cumulative effect in combination with each other. Further research will be required to determine the nature of the causal genes at these loci, to develop diagnostic markers and determine how the genes fit into the pathway that leads to the induction of α-AMY1 transcription in the aleurone of developing wheat grains. Depending on the target environmental conditions, different combinations of alleles may be required to achieve a low risk of LMA expression. Supplementary Information The online version contains supplementary material available at 10.1007/s00425-023-04131-1.


INTRODUCTION
In the absence of sprouting some wheat cultivars produce aamylase during the later stages of grain ripening sufficient to reduce the fatling number and amylograph peak viscosity to unacceptable levels. In some cultivats, €.g.t Huntsman (UK), this only occurs in some seasons, apparently in response to a specific set of environmental conditions (Gale and Lenton, 1987). Other cultivars by contrast e.g., Spica (Australia) and Lerma 52 (Mexico), produce late maturity a-amylase under a wide range, if not all, growing conditions (Mares and Gale, 1990), althougþ the effect is most dramatic in a cooler, humid ripening environment.,This phenomenon has been examined in some detail using the cultivar Spica (Mares and Gale, 1990). In this cultivar synthesis of late maturity a-amylase begins approximately 40 days post anthesis, involves c-amylase isozymes controlled by genes on the group 6 chromosomes (r.e., high pI isozymes typical of the early stages of germination), is controlled by a single recessive gene and is modulated by the environment and the genetic background. Enzyme was distributed throughout the length of the grain, thus contrasting with germinated grain where there is a marked gradient from the embryo to the distal end.
Recently in Australia new sources of late maturify a-amylase were identified in a number of breeding programs. For two breeding programs this has meant withdrawing potential new cultivars prior to, or following release and necessitated a critical evaluation of the programs' germplasm base.
The aims of this investigation were to catalogue the pedigrees of genotypes exhibiting late maturity a-amylase, to compare the production of amylase in the field and the glasshouse, and to examine segregation for ø-amylase in reciprocal crosses.

MATERIALS AND METHODS
Wheat cultivars were grown in the ñeld at Narrabri until flowering at which time some plants were transplanted to a glasshouse (temperature range L6-25"C). Plants in the field ripened in a hot, dry environment in contrast to the cooler, more humid glasshouse environment. At harvest ripeness (12% moisture) grain was harvested from all plants for falling number and c-amylase determinations. a-Amylase was determined on single grains using a modification of the method of Barnes and Bl4keney (1974)  The most important source of late maturity a-amylase in Australian breeding programs would appear to be the cultivar Mentana (Table I). This cultivar appears in the parentage oflerma Comparison of field-ripened and glasshouse ripened samples a-Amylase activities in grain of control cultivars were similar and low in both ripening environments (Fig. 1a). Cultivars such as Spica and Lerma had high grain a-amylase levels in both environments, however, levels were highest in glasshouse-ripened samples (Fig. 1b By contrast, for the cultivar 8D159, a-amylase levels were generally low and similar to control cultivars in the ñeld material but were elevated, relative to controls in the glasshouse (Fig. 1c). c.

RANKED SINGLE GRAINS
d. t82 The distribution of ø-amylase was also examined in F, grains on Ft plants from the cross Chinese Spring (low cu-amylase) x Spica (high ø-amylase) (Fig. 1d). Again a-amylase levels in individual grains were higher in the glasshouse-ripened sample and there was greater differentiation between high c-amylase and low a-amylase grains.
Distribution of a-amylase in F, grains of reciprocal crosses between Chinese Spring and Spica, and Chinese Spring and Lerma 52 Mean values of ø-amylase in F, grains were similar to those of the low a-amylase parent, Chinese Spring, however, there was a consistent trend towards higher mean d-amylase where the high aamylase parent was the female (Table III). Analysis of variance indicated that the difference was significant (P=0.01) for the reciprocal crosses involving Chinese Spring and Lerma only.

DISCUSSION
Late maturity ø-amylase production represents a serious problem to breeders, grain receival agents and marketing authorities. The safest course of action would be to avoid the use of high amylase germplasm, however, in some instances these lines have a number of áesirãbleagronomicorquality attributes. Selection within breeding populations for low a-amylase is made difficult by the recessive naiure of the character, the influence of the environment' and the genetic background (e.g., expression of high a-amylase gene is very low in warmer environments). In.the cases of genotypes showing extreme sensitivity to the environment, selection may not be possible in many environments.
Glasshouse-ripened samples exhibited higher levels of a-amylase, gave better discrimination between high and low types, and elicited a response in some cultivars that maintained low a-amylase in the field. Whereas this technique offers some hope for breeders, there is nevertheless an urgent need to develop screening techniques (e'9., biochemical or molecular markers) capable of identi$ing grains homozygous for low c-amylase.
The observed trend, within F, grains, for a-amylase to be slightly higher when the high amylase parent was used as the maternal parent is aensistent with control being exercised by the triploid endosperm and aleurone rather than the diploid embryo. This suggestion is supported by previous investigations (Mares and Gale, 1990) which noted that enzyme detected in scraped aleurone tissue was distributed along the length of the grain, rather than concentrated near the embryo as in germinated grains. Chorleywood, Hertfordshire WD3 5SH, England SUMMARY Wax crystals were found in the embryo cavity of long-stored red wheat varieties, but none was found in white varieties stored for the same time. It is hypothesized that the wax from which the crystals developed may have played a role in controlling the entry of water into thè grain during the critical post-ripening stage when pre-harvest sprouting is a hazard. Should the hypothesis be proved the wax Characteristics may provide a marker for sprout resistance in early generation stocks. Wax has been dissected from the old samples of ied grain, and its composition determined, for comparison wjth waxes extrãcted from fresher grains. The waxes comprise about 200 components. Some systematic'differences exist between sproutresistant and sprout-susceptible types.

INTRODUCTION
A chance observation was made under the scanning electron microscope, of tufts of needlelike structures in the embryo cavity of grains of the old English wheat variety Yeoman which had been stored for 25 years.