D.G. Bonnett

The effect of different height reducing genes on the early growth of wheat

Genes that reduce height without compromising seedling vigour or coleoptile length have great potential for wheat improvement. We therefore investigated the effects of various reduced height (Rht) genes on the early stages of plant development, using a combination of near isogenic, recombinant, mutant and wild type comparisons. Gibberellin (GA) insensitivity caused by Rht-B1b or Rht-D1b was associated with reduced leaf elongation rate and coleoptile length. Similar results were found for two other sources of dwarfing, Rht11 and Rht17. We found one class of Rht genes (e.g. Rht8) which had no effect on coleoptile length, leaf elongation rate or responsiveness to GA, indicating that these dwarfing genes may act later in wheat development to reduce height and increase harvest index, without affecting early growth. A third class of Rht genes was found in three durum backgrounds. These had reduced coleoptile lengths and leaf elongation rates, but had a greater response to GA than the corresponding tall varieties. We discuss these results in relation to the possible mechanisms underlying the reduction in height and the suitability of the different Rht genes for wheat improvement.

Strategies for efficient implementation of molecular markers in wheat breeding

Although molecular markers allow more accurate selection in early generations than conventional screens, large numbers can make selection impracticable while screening in later generations may provide little or no advantage over conventional selection techniques. Investigation of different crossing strategies and consideration of when to screen, what proportion to retain and the impacts of dominant vs. codominant marker expression revealed important choices in the design of marker-assisted selection programs that can produce large efficiency gains. Using F2 enrichment increased the frequency of selected alleles allowing large reductions in minimum population size for recovery of target genotypes (commonly around 90%) and/or selection at a greater number of loci. Increasing homozygosity by inbreeding from F2 to F2:3 also reduced population size by around 90% in some crosses with smaller incremental reductions in subsequent generations. Backcrossing was found to be a useful strategy to reduce population size compared with a biparental population where one parent contributed more target alleles than the other and was complementary to F2 enrichment and increasing homozygosity. Codominant markers removed the need for progeny testing reducing the number of individuals that had to be screened to identify a target genotype. However, although codominant markers allow target alleles to be fixed in early generations, minimum population sizes are often so large in F2 that it is not efficient to do so at this stage. Formulae and tables for calculating genotypic frequencies and minimum population sizes are provided to allow extension to different breeding systems, numbers of target loci, and probabilities of failure. Principles outlined are applicable to implementation of markers for both quantitative trait loci (QTL) and major genes.

Quantitative trait loci on chromosome 4B for coleoptile length and early vigour in wheat (Triticum aestivum L.)

The Norin-10 dwarfing genes, Rht-B1b (Rht1) and Rht-D1b (Rht2), are commonly used to reduce plant height and increase grain yield in wheat breeding programs. These dwarfing genes lower sensitivity of vegetative tissue to endogenous gibberellin to reduce cell and subsequent stem elongation. This reduction in cell elongation capacity reportedly results in a concomitant reduction in coleoptile length and early vigour (leaf area) thereby affecting seedling establishment and growth. A detailed genetic map from a cross between tall Halberd (Rht-B1a) and semidwarf Cranbrook (Rht-B1b) wheat cultivars was used to assess genetic factors affecting seedling growth. Parental and 150 doubled haploid progeny lines were characterised for seedling and height-related traits in controlled and field environments. Genotypic variation was large and predominantly under additive genetic control with evidence for transgressive segregation for some traits. Narrow-sense heritabilities were moderate to high (h2 = 0.31–0.91) indicating a strong genetic basis for differences between progeny. Molecular marker analyses identified a number of significant (P < 0.05) quantitative trait loci (QTL) for each trait. A major QTL, mapping directly to the Rht-B1 locus on chromosome arm 4BS, accounted for up to 49% of the genotypic variance in peduncle length and plant height, and 27–45% of the genotypic variance in coleoptile length across different temperatures. Another QTL, located close to the RFLP marker XksuC2 on the long arm of chromosome 4B, accounted for 15–27% of the genotypic variance in coleoptile length. The influence of the XksuC2-linked QTL on coleoptile length was greatest at 19˚C and decreased with cooler temperatures. The same QTL affected reductions in leaf size, and both coleoptile tiller size and presence to affect overall seedling vigour. There was also some evidence for epistatic interactions influencing coleoptile tiller growth. Reductions in plant size at the Rht-B1b and XksuC2 loci were associated with presence of the Cranbrook 4B allele. The negative genetic effect of the Rht-B1b dwarfing gene on early growth of wheat confirms phenotypic evidence of a pleiotropic effect of Rht-B1b on establishment and early vigour. Genetic increases in coleoptile length and early leaf area development are likely to be limited in wheat populations containing the Rht-B1b dwarfing gene.

A QTL on chromosome 6A in bread wheat (Triticum aestivum) is associated with longer coleoptiles, greater seedling vigour and final plant height

Wheat crops with greater early vigour shade the soil surface more rapidly and reduce water loss. Evaporative losses affect water-use efficiency particularly in drier regions where most of the rainfall occurs early in the growing season before canopy closure. Greater seedling leaf area and longer coleoptiles are major determinants of increased vigour and better crop establishment. A previously developed high vigour breeding line ‘Vigour 18’ was used to establish a large recombinant inbred family and framework map to identify a QTL on chromosome 6A that accounted for up to 8% of the variation for coleoptile length, 14% of seedling leaf width and was associated with increased plant height. The SSR marker NW3106, nearest to the 6A QTL, was also associated with greater leaf width in a breeding population that was also derived from a cross involving the high vigour donor line ‘Vigour18’. The association between the NW3106 marker and coleoptile length was validated in a second breeding population which was developed using an unrelated long coleoptile donor line. The ‘Vigour18’ allele of the QTL on chromosome 6A promoted coleoptile length and leaf width during early plant growth but was also associated with increased plant height at maturity. Markers linked to the QTL are being used to increase the frequency of increased vigour and long coleoptile alleles in early generations of breeding populations.

A 192bp allele at the Xgwm261 locus is not always associated with the Rht8 dwarfing gene in wheat (Triticum aestivum L.)

The height-reducing gene Rht8 was introduced into Italian wheats by breeder Nazareno Strampelli from the Japanese landrace Akakomugi, and has been widely used in wheats adapted to southern and eastern European conditions. Following identification of a close linkage to Rht8, microsatellite marker Gwm261 has been used extensively to screen large numbers of diverse international germplasm. A 192bp allele at this locus has been taken as “diagnostic” for Rht8 and used to infer the international distribution of Rht8. In this paper, we report several instances of cultivars and mapping populations that vary for the presence of the 192bp allele at the Xgwm261 locus (Xgwm261192), but with no associated reduction in height, suggesting a lack of association with Rht8. For instance, in the population derived from a cross between Sunco (Rht-B1b, Xgwm261165) and Tasman (Rht-D1b, Xgwm261192), there were significant height differences associated with the segregation of Rht-B1b and Rht-D1b, but no height differences between Xgwm261 genotypes. Similar results were obtained in a population derived from the cross between Molineux (Rht-B1b, Xgwm261192) and Trident (Rht-D1b Xgwm261208). In contrast, the cross between Trident and Chuanmai 18 (Xgwm261192) gave significant height effects at both the Rht-D1 and Xgwm261 loci, with no epistatic interaction between loci. Chuanmai 18 is closely related to the Strampelli wheat Mara (ancestrally derived from Akakomugi) and is therefore likely to carry Rht8. The old Japanese cultivar Norin 10, used by Norman Borlaug to introduce Rht-B1b and Rht-D1b into Mexican wheats, also has a 192bp allele at the Xgwm261 locus, and the sequence of the amplified product is identical to that of Akakomugi. We suggest that the widespread use of Norin 10-derived germplasm during and after the Green Revolution introduced a second haplotype into international germplasm, in which Xgwm261192 has no association with Rht8. Therefore, the presence of Xgwm261192 is only indicative of Rht8 in wheat cultivars that have inherited this allele from Akakomugi or a Strampelli wheat ancestor.

Simultaneous selection of major and minor genes: use of QTL to increase selection efficiency of coleoptile length of wheat (Triticum aestivum L.)

Plant breeders simultaneously select for qualitative traits controlled by one or a small number of major genes, as well as for polygenic traits controlled by multiple genes that may be detected as quantitative trait loci (QTL). In this study, we applied computer simulation to investigate simultaneous selection for alleles at both major and minor gene (as QTL) loci in breeding populations of two wheat parental lines, HM14BS and Sunstate. Loci targeted for selection included six major genes affecting plant height, disease resistance, and grain quality, plus 6 known and 11 “unidentified” QTL affecting coleoptile length (CL). Parental line HM14BS contributed the target alleles at two of the major gene loci, while parental line Sunstate contributed target alleles at four loci. The parents have similar plant height, but HM14BS has a longer coleoptile, a desirable attribute for deep sowing in rainfed environments. Including the wild-type allele at the major reduced-height locus Rht-D1, HM14BS was assumed to have 13 QTL for increased CL, and Sunstate four; these assumptions being derived from mapping studies and empirical data from an actual HM14BS/Sunstate population. Simulation indicated that compared to backcross populations, a single biparental F1 cross produced the highest frequency of target genotypes (six desired alleles at major genes plus desired QTL alleles for long CL). From 1,000 simulation runs, an average of 2.4 individuals with the target genotype were present in unselected F1-derived doubled haploid (DH) or recombinant inbred line (RIL) populations of size 200. A selection scheme for the six major genes increased the number of target individuals to 19.1, and additional marker-assisted selection (MAS) for CL increased the number to 23.0. Phenotypic selection (PS) of CL outperformed MAS in this study due to the high heritability of CL, incompletely linked markers for known QTL, and the existence of unidentified QTL. However, a selection scheme combining MAS and PS was equally as efficient as PS and would result in net savings in production and time to delivery of long coleoptile wheats containing the six favorable alleles.

Evaluation of a reduced-tillering (tin) gene in wheat lines grown across different production environments

Abstract. Post-anthesis water deficit and increasing vapour pressure deficit are common and can result in reduced grain yield and the development of small or shrivelled wheat kernels (screenings) that reduce grain value. Previous studies suggest incorporation of a tiller inhibition (tin) gene to restrict tiller number and thereby slow water use and promote the development of larger, fertile spikes to increase kernel weight. This paper reports on the influence of the tin gene on grain yield and screenings in multiple wheat genetic backgrounds assessed in field experiments in 2005 and 2006. Across environments, grain yield ranged from 0.90 to 5.50 t/ha and screenings from 4 to 20%. The effect of tin on grain yield and screenings varied with environment and genetic background. Grain yield was unchanged in tin lines derived from varieties Brookton, Chara, and Wyalkatchem assessed in southern Australian environments. However, there was a 31 and 10% advantage of free-tillering over tin-containing Silverstar lines for the 2005 western and 2006 northern experiments, respectively, resulting in an average 12% reduction in grain yield of Silverstar tin lines. In northern experiments, where screenings ranged from 4 to 12%, Silverstar-based tin lines produced significantly fewer screenings than free-tillering sister lines. Reduction in screenings was associated with a higher kernel weight (+10%) and a tendency for lower grain yield, although individual Silverstar tin progeny with grain yield equivalent to the parent were readily identified. The incorporation of the tin gene has considerable potential to reduce the incidence of screenings in commercial wheat crops. Variation in grain yield associated with the tin gene was dependent on genetic background, with potential for selection of higher yielding tin progeny for commercial line development.

Borlaug, Strampelli and the worldwide distribution of Rht8.

The height-reducing gene Rht8 was introduced by breeder Nazareno Strampelli from the Japanese landrace ‘Akakomugi’ and has been widely used in wheats adapted to southern and eastern Europe ∈dent Definitive identification of Rht8 is difficult because it does not make seedlings insensitive to gibberellin application and its height effect may be confounded with other genes affecting plant height or flowering time. Following identification of a close linkage between microsatellite WMS261 and Rht8, the marker has been used extensively to screen large numbers of diverse international germplasm. A 192bp allele at the WMS261 locus was assumed to be ‘diagnostic’ for Rht8 and its international distribution was inferred from the marker data ∈dent We report several instances of cultivars and mapping populations which vary for the presence of the Xgwm261-192bp allele, but with no associated reduction in height, suggesting a lack of linkage with Rht8 ∈dent Our studies have identified that Norin10, used by Norman Borlaug to introduce Rht-B1b and Rht-D1b into Mexican wheats, also carries WMS261-192bp, and this allele was retained in some of his varieties. The widespread use of Norin10-derived germplasm during and after the green revolution represents a second source of WMS261-192bp microsatellite allele in international germplasm, with no linkage to Rht8. Therefore, the presence of this allele can only be indicative of Rht8 in wheat varieties with a clear pedigree relationship to Akakomugi or Strampelli

EFFICIENT INTEGRATION OF MOLECULAR AND CONVENTIONAL BREEDING METHODOLOGIES

Molecular markers make it possible for breeders to combine desirable alleles at a greater number of loci and at earlier generations than is possible with conventional breeding methodologies. With an increasing number of markers for important traits, marker assisted selection (MAS) strategies that minimize population sizes and assay numbers while ensuring important alleles are not lost become increasingly important. In practical breeding, strategies will often have to balance MAS with the need to retain variability for important traits that are selected partly or wholly by conventional means This paper will discuss strategies to maximize the number of traits that can be combined in a single breeding cycle taking account of practical limits such as reasonable population sizes that can be managed in a breeding program. Examples from our breeding program and mapping populations will be used to illustrate the effects of different MAS strategies on population size and levels of residual genetic variation at unselected loci. It is these unselected loci that become important where conventional selection follows MAS