G. J. Rebetzke

Perfect markers for the Rht-B1b and Rht-D1b dwarfing genes in wheat

Abstract.PCR-based markers were developed to detect the point mutations responsible for the two major semi-dwarfing genes Rht-B1b (Rht1) and Rht-D1b (Rht2) in wheat. These markers were validated by testing 19 wheat varieties of known Rht genotype. They included Rht-B1b and Rht-D1b dwarfs, double-mutant varieties and tall wheats. These were correctly genotyped with the Rht-B1b and Rht-D1b-specific primers, as well as markers specific for the tall alleles Rht-B1a and Rht-D1a. Using a family of doubled-haploid lines segregating for Rht-B1b and Rht-D1b, the markers were mapped to the expected homoeologous regions of chromosomes 4B and 4D, respectively. Both markers were strongly correlated with a reduction in height, accounting for 23% (Rht-B1b) and 44% (Rht-D1b) of the phenotypic variance in the population. These markers will have utility in marker-assisted selection of the Rht-B1b and Rht-D1b genes in wheat breeding programs.

Traits and selection strategies to improve root systems and water uptake in water-limited wheat crops

Wheat yields globally will depend increasingly on good management to conserve rainfall and new varieties that use water efficiently for grain production. Here we propose an approach for developing new varieties to make better use of deep stored water. We focus on water-limited wheat production in the summer-dominant rainfall regions of India and Australia, but the approach is generally applicable to other environments and root-based constraints. Use of stored deep water is valuable because it is more predictable than variable in-season rainfall and can be measured prior to sowing. Further, this moisture is converted into grain with twice the efficiently of in-season rainfall since it is taken up later in crop growth during the grain-filling period when the roots reach deeper layers. We propose that wheat varieties with a deeper root system, a redistribution of branch root density from the surface to depth, and with greater radial hydraulic conductivity at depth would have higher yields in rainfed systems where crops rely on deep water for grain fill. Developing selection systems for mature root system traits is challenging as there are limited high-throughput phenotyping methods for roots in the field, and there is a risk that traits selected in the lab on young plants will not translate into mature root system traits in the field. We give an example of a breeding programme that combines laboratory and field phenotyping with proof of concept evaluation of the trait at the beginning of the selection programme. This would greatly enhance confidence in a high-throughput laboratory or field screen, and avoid investment in screens without yield value. This approach requires careful selection of field sites and years that allow expression of deep roots and increased yield. It also requires careful selection and crossing of germplasm to allow comparison of root expression among genotypes that are similar for other traits, especially flowering time and disease and toxicity resistances. Such a programme with field and laboratory evaluation at the outset will speed up delivery of varieties with improved root systems for higher yield.

Genetic variation for improving the salt tolerance of durum wheat

Durum wheat (AB genomes) is more salt-sensitive than bread wheat (ABD genomes), a feature that restricts its expansion into areas with sodic or saline soils. Salt tolerance in bread wheat is linked with a locus on the D genome that results in low Na+ uptake and enhanced K+/Na+ discrimination. In order to introduce salt tolerance into current durum wheats from sources other than the D genome, a search for genetic variation in salt tolerance was made across a wide range of tetraploids representing 5 Triticum turgidum sub-species (durum, carthlicum, turgidum, turanicum, polonicum). Selections were screened for low Na+ uptake and enhanced K+/Na+ discrimination. This was assessed in seedlings grown in 150 mМ NaCl with supplemental Ca2+, by measuring the Na+ and K+ accumulated in the blade of a given leaf over 10 days. Large and repeatable genetic variation was found. Low Na+ accumulation and high K+/Na+ discrimination of similar magnitude to that of bread wheat was found in the sub-species durum. These selections have the potential for improving salt tolerance in durum wheat breeding programs.

Quantitative trait loci for water-soluble carbohydrates and associations with agronomic traits in wheat

Several environmental factors including drought and disease can reduce leaf area and photosynthesis during grain-filling to decrease grain yield and kernel weight of cereal crops. Water-soluble carbohydrates (WSC) accumulated around anthesis can be mobilised to assist in filling of developing grains when post-anthesis assimilation is low. Cultivar differences support opportunities to select for high WSC but little is known of the extent or nature of genetic control for this trait in wheat. Three wheat mapping populations (Cranbrook/Halberd, Sunco/Tasman, and CD87/Katepwa) were phenotyped for WSC and other agronomic traits across multiple environments. The range for WSC concentration (WSC-C) was large among progeny contributing to moderate-to-high narrow-sense heritabilities within environments (h2 = 0.51–0.77). Modest genotype × environment interaction reduced the correlation of genotype means across environments (rp = 0.37–0.78, P < 0.01) to reduce heritability on a line-mean (h2 = 0.55–0.87) basis. Transgressive segregation was large and genetic control complex, with 7–16 QTLs being identified for WSC-C in each population. Heritability was smaller (h2 = 0.32–0.54) for WSC mass per unit area (WSC-A), reflecting large genotype × environment interaction and residual variance with estimating anthesis biomass. Fewer significant QTLs (4–8) were identified for this trait in each population, while sizes of individual genetic effects varied between populations but were repeatable across environments. Several genomic regions were common across populations including those associated with plant height (e.g. Rht-B1) and/or anthesis date (e.g. Ppd1). Genotypes with high WSC-C were commonly shorter, flowered earlier, and produced significantly (P < 0.01) fewer tillers than those of low WSC-C. This resulted in similar yields, lower final biomass, and fewer grains per m2, but greater dry weight partitioning to grain, kernel weight, and less grain screenings in high compared with low WSC-C genotypes. By contrast, lines high for WSC-A produced more fertile tillers associated with similar or greater anthesis and maturity biomass, grain number, and yield, yet similar kernel weight or size compared with genotypes with low WSC-A. The data support an important role for WSC-A in assuring stable yield and grain size. However, the small effects of many independent WSC QTLs may limit their direct use for marker-aided selection in breeding programs. We suggest using molecular markers to enrich populations for favourable height and anthesis date alleles before the more costly phenotypic selection among partially inbred families for greater WSC-A.

Genetic control of sodium exclusion in durum wheat

Salt tolerance in the genus Triticum is associated with low accumulation of Na+ in leaves. Durum and other tetraploid wheats generally have high accumulation of Na+ relative to bread wheat, and are salt sensitive, but a durum wheat landrace, Line 149, was found to have unusually low leaf Na+ accumulation. Populations were developed from crosses between 149 and the high Na+ accumulation variety Tamaroi, as well as between 149 and a durum wheat landrace with very high Na+ accumulation, Line 141. The third leaf of parental lines, F1, F2, and low- and high-selected F2:3 progeny was assayed for Na+ uptake when grown in 150 mM NaCl. Sodium concentrations were significantly (P 0.50n.s. for 149/Tamaroi and 149/141, respectively), indicating duplicate dominance epistasis arising from segregation of 2 interacting dominant genes. Small yet significant (P < 0.01) genotypic variation was also observed for minor genes affecting Na+ accumulation. Realised heritabilities were moderate to high (h2R = 0.43–0.90) across populations, indicating good response to selection for low Na+ accumulation in the F2 generation. The simple genetic control of Na+ accumulation suggests relative ease of selection of lines with low Na+ accumulation. However, presence of dominance will require selection to be delayed until after 1 or 2 generations of inbreeding, or after progeny-testing of selected low Na+ accumulation families.

Breeding long coleoptile, reduced height wheats

Semidwarf wheats have the potential to produce high yields when sown and managed under optimal conditions. However, farm yields often fall below this potential because of poor seedling establishment and low early vigour associated with gibberellic acid (GA)-insensitive reducing-height ( Rht) genes contained in these wheats. Australian and overseas wheats containing major and minor Rht genes sensitive to GA were intercrossed to develop three populations. Seedlings sensitive to GA and therefore lacking Rht-B1b ( Rht1) and Rht-D1b ( Rht2) plant height genes were selected for further study. GA- sensitive F4-derived lines were sown in field and glasshouse environments to determine plant height, and then sown at four temperatures to determine coleoptile length. Genetic variation in plant height and coleoptile length was large and significant ( P<0.01) among lines within each population with a number of lines identified as producing plant heights as short as current semidwarf varieties. Transgressive segregation for coleoptile length produced progenies with coleoptiles significantly ( P < 0.05) longer than the longest coleoptile parent in each population. Genotype × temperature interactions for coleoptile length were small thereby resulting in high line-mean heritabilities (h2 = 85–89) for this character. Larger plant-to-plant variation reduced single-plant estimates of heritability for plant height (h2 = 29–31) but heritability was increased (h2 = 68–78) with replication within and over environments. High narrow-sense heritabilities indicate that phenotypic selection should produce modest genetic gain for both characters. Variation in coleoptile length was poorly related to differences in plant height (r2 = 0.00 to 0.04 ns) while selection differentials for plant height were not associated with any change in coleoptile length of the selected groups. When considered together, height and coleoptile length appeared to be largely under independent genetic control among GA-sensitive wheats. These results suggest that GA-sensitive Rht genes could be used to select shorter height, longer-coleoptile wheats with improved establishment and seedling vigour.

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.

Identification of quantitative trait loci for traits conferring weed competitiveness in wheat (Triticum aestivum L.)

As weeds develop resistance to a broad range of herbicides, wheat (Triticum aestivum L.) cultivars with superior weed competitive capacity are needed to complement integrated weed management strategies. In this study, agronomic and morphological traits that enable wheat to compete effectively with weeds were identified. Halberd, Cranbrook, and 161 Cranbrook x Halberd doubled haploid (DH) lines were examined in field experiments conducted over two growing seasons. The weed species Lolium rigidum L. (annual ryegrass) was sown in strips perpendicular to the direction of wheat seeding. Various traits were measured during each season with competitive ability determined by both percent loss in wheat grain yield and suppression of ryegrass growth. Width of leaf 2, canopy height, and light interception at early stem elongation (Z31), and tiller number, height at maturity, and days to anthesis were important for competitive ability in 1999. In the previous year, length of leaf 2 and size of the flag leaf contributed to competitiveness. Seasonal effects appeared to have some impact on the relative contribution of crop traits to competitive ability. The morphological traits involved in maintaining grain yield differed from those that contributed to the suppression of ryegrass growth. Development of the Cranbrook x Halberd chromosomal linkage map enabled the putative identification of quantitative trait loci (QTL) associated with competitive ability in the DH population. Many of the QTL were mapped to similar positions in both years. Further, several traits, including time to anthesis, flag leaf size, height at stem elongation, and the size of the first 2 leaves, were mapped to similar positions on chromosomes 2B and 2D. Narrow-sense heritabilities on an entry-mean basis were typically high within each year for traits associated with weed competitive ability. However, large genotype x year interactions reduced these heritabilities, making genetic gain through phenotypic selection difficult. The identification of QTL repeatable over seasons indicates the potential for marker-assisted selection in a wheat breeding program selecting for improved grain yield and weed competitiveness.

A wheat genotype developed for rapid leaf growth copes well with the physical and biological constraints of unploughed soil

Conventional wheat (Triticum aestivum L.) cultivars grow slowly in unploughed soil because of physical and biological constraints. Here a conventional cultivar (Janz) is compared with a novel experimental line (Vigour 18), bred for high leaf vigour, to explore the hypothesis that a vigorous wheat grows better in unploughed soil. Roots of both genotypes in unploughed soil were three times more distorted with 30% shorter apices and 60% shorter expansion zones than roots in ploughed soil, because of voids between blocky peds and packed sand particles that impeded root apices. More than half the root length contacted dead, remnant roots. Vigour 18 roots grew 39% faster, were thicker and distorted less than Janz roots in unploughed soil, but developed similarly in ploughed soil. Vigour 18 shoots grew 64% faster in unploughed soil, but 15% faster in ploughed soil. Fumigation of unploughed soil improved the growth of Janz only. We suggest that faster root growth, different exudates promoting a more beneficial rhizosphere microflora, or modified shoot responses are possible mechanisms to explain Vigour 18s superior growth. Vigorous genotypes may present a new opportunity for increased productivity with conservation farming.

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.