Height reduction and agronomic performance for selected gibberellin-responsive dwarfing genes in bread wheat (Triticum aestivum L.)

Field Crops Research 126 (2012) 87–96

Contents lists available at SciVerse ScienceDirect

Field Crops Research journal homepage: www.elsevier.com/locate/fcr

Height reduction and agronomic performance for selected gibberellin-responsive dwarfing genes in bread wheat (Triticum aestivum L.) G.J. Rebetzke ∗ , M.H. Ellis 1 , D.G. Bonnett 1 , B. Mickelson, A.G. Condon, R.A. Richards CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia

a r t i c l e

i n f o

Article history: Received 30 November 2010 Received in revised form 25 September 2011 Accepted 26 September 2011 Keywords: Breeding Heritability Harvest index Establishment Early vigour Coleoptile Germplasm Dwarfing genes

a b s t r a c t Improved ability to establish well when sowing at depth, into crop residues or hard soils should lead to increased yields in these situations. The semi-dwarfing Rht-B1b and Rht-D1b genes reduce plant height and increase grain number and yield in favourable environments. However, these genes are associated with reduced coleoptile length and leaf length extension to slow leaf area and biomass accumulation especially when seed are sown deep. Preliminary evidence indicates the potential of Rht4, Rht5, Rht8, Rht12 and Rht13 gibberellin-responsive (GAR) dwarfing genes to reduce plant height without affecting seedling vigour. Four large, inbred populations were generated varying for presence of one or more GARdwarfing genes. Lines were genotyped with molecular markers linked to each dwarfing gene and grown in multiple environments to evaluate seedling growth and agronomic performance. Genotypic variation was large for plant height, aerial biomass, grain yield and its components, grain number and size. Height reduction was greatest for Rht5 (−55%), Rht12 (−45%), Rht13 (−34%), Rht4 (−17%), and to a lesser extent Rht8 (−7%). In comparison, height reductions associated with Rht-B1b averaged 23%. Reduced height was genetically correlated with reduced lodging score (rg = 0.84–0.93), increased dry-matter partitioning to grain (i.e. harvest index; rg = −0.46** to −0.86**) and increased grain number (rg = −0.22* to −0.73**). Most dwarfing genes were associated with increased grain number: Rht13 (+27%), Rht4 (+19%), Rht12 (+19%), and Rht-B1b (+9%). Rht8 had little effect on grain number (−1%) whereas later maturity associated with Rht5 contributed to reduced grain number (−66%). The influence of dwarfing genes on aerial biomass was negligible, with some Rht4, Rht12 and Rht13 semi-dwarf lines identified combining greater partitioning and aerial biomass to increase grain yield. Compared to tall siblings, coleoptile lengths and seedling leaf breadths were largely unaffected by GAR-dwarfing genes but leaf length was on average smaller in lines containing Rht5 or Rht12. These studies demonstrate the potential of GAR-dwarfing genes for increasing grain number and yield without compromising aerial biomass or coleoptile length in bread wheat. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction Reduced plant height is a key objective of wheat breeding programs worldwide (Mathews et al., 2006). Global adoption of the Rht-B1 and Rht-D1 dwarfing genes has been associated with reduced lodging and increased grain number to promote greater crop yields. Yields are particularly high with favourable growing conditions such as occurs with large nutritional inputs (Pearman et al., 1978). Yield benefits associated with these dwarfing genes tend to be greatest in well-managed, irrigated environments and particularly at levels approaching maximal yields (Mathews et al.,

Abbreviations: GAI, gibberellic acid-insensitive; GAR, gibberellic acidresponsive; RILs, recombinant inbred lines. ∗ Corresponding author. Tel.: +61 262465153; fax: +61 262465399. E-mail address: [email protected] (G.J. Rebetzke). 1 Current address: CIMMYT Int. Apdo. Postal 6-641, 06600 México, DF, Mexico.

2006). However, the yield advantage becomes less pronounced in lower-yielding environments especially when productivity is at 3 tonnes per ha or less, and/or where high temperature or drought limit productivity (Richards, 1992a; Butler et al., 2005; Chapman et al., 2007). The influence of the Rht-B1b and Rht-D1b alleles on reductions in plant height varies with environment and genetic background, and is commonly reported at around 20–25% of the wild-type allele (e.g. Hoogendoorn et al., 1990; Butler et al., 2005; Mathews et al., 2006). The Rht-B1b and Rht-D1b alleles reduce stem internode length to reduce overall plant height through a decreased sensitivity of vegetative tissues to endogenous gibberellin (Hoogendoorn et al., 1990; Keyes et al., 1989). Cell length and width is reduced and this is believed to contribute to reductions in internode length and subsequent plant height. Although these alleles reduce all internodes by the same relative amount, their greatest absolute reduction is on the longest internode, the peduncle (Hoogendoorn et al., 1990). A shorter peduncle requires relatively less assimilate for elongation

0378-4290/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2011.09.022

88

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

thereby freeing-up assimilate for florets growing at the same time (Youssefian et al., 1992). Greater assimilate availability increases floret survival to increase potential grain number. In many rainfed cropping regions including those characterised as ‘Mediterranean environments’, much of the water available for crop growth is supplied by in-season rainfall. Rainfall events in these environments may be frequent but small in size (commonly 5–10 mm) and much of the water cannot be used effectively by the crop as it quickly evaporates from the soil surface reducing water-use efficiency (Sadras and Rodriguez, 2007). This has been demonstrated for crops growing in southern Australia where it is estimated that between 70 and 110 mm of in-crop rainfall is lost through soil evaporation (Sadras and Rodriguez, 2007). Improved canopy management can reduce soil water loss but as much as 50% of in-crop rainfall can still be lost in well-managed crops (Condon et al., 2002). More rapid leaf area development soon after sowing reduces soil evaporation to increase crop water-use efficiency, ˜ biomass and grain yield (López-Castaneda and Richards, 1994; Botwright et al., 2002). Greater early vigour should also improve weed-competitiveness (Coleman et al., 2001), and increase root growth to improve nitrogen capture early in the season (Palta et al., 2007). Furthermore, increasing focus on environmental sustainability and fuel prices have heightened interest in conservation farming methods and especially reduced tillage. However, hard soils and retained stubble common to conservation farming slow early growth to reduce crop biomass. Greater early vigour has potential to overcome constraints imposed by reduced tillage (Watt et al., 2005). Reductions in cell size associated with both Rht-B1b and RhtD1b gibberellic acid-insensitive (GAI) alleles effects reductions in seedling leaf size and coleoptile tiller production to reduce seedling leaf area (Allan, 1989; Richards, 1992b; Rebetzke et al., 2001; Botwright et al., 2005; Ellis et al., 2004). These genes reduce subcrown internode and coleoptile lengths (Allan, 1989; Ellis et al., 2004; Rebetzke et al., 2001, 2007a). Shorter coleoptiles can delay sowing despite adequate soil moisture beyond conventional sowing depth. Availability of long-coleoptile, semi-dwarf wheats would allow wheat growers to sow into soil moisture lying below the drying topsoil or where moisture conservation tillage practises such as stubble retention and dust-mulching occurs. Longer coleoptiles may permit crops to be sown at the optimal time to increase biomass and yield (Shackley and Anderson, 1995). Deep sowing of short coleoptile Rht-B1b and Rht-D1b wheats commonly results in few, later-emerging seedlings with small relative growth rates, leaf area and biomass (Rebetzke et al., 2007b), and ultimately smaller final biomass and yield (Mahdi et al., 1998; Rebetzke et al., 2007b). Further, Allan (1989) and Botwright et al. (2001) suggested that the slower rates of emergence associated with Rht-B1b and Rht-D1b can increase the likelihood of soil-crusting prior to emergence. There is an increasing interest in the development of wheat cultivars with greater seedling vigour and the capacity to emerge from deep sowing (Rebetzke and Richards, 2000; Schillinger et al., 1998). Replacement of the Rht-B1b and Rht-D1b GAI-dwarfing alleles with alternate gibberellic acid-responsive (GAR) dwarfing genes shows potential for reducing plant height without compromising seedling vigour (Rebetzke and Richards, 2000; Rebetzke et al., 1999, 2004a; Ellis et al., 2004). Indeed, studies have already demonstrated the potential of Rht8 in the development of semi-dwarf, long-coleoptile wheats targeted at sowing depths exceeding 100 mm (Schillinger et al., 1998; Rebetzke et al., 2007b). Rht8 has a smaller effect on height reduction (ca. 8–12%) than the GAI-dwarfing genes Rht-B1b, Rht-B1c and Rht-D1b (Rebetzke and Richards, 2000; Ellis et al., 2004, 2005). Despite this, Rht8 has been identified in commercial wheat varieties (e.g. China; Zhang et al., 2006), highlighting the potential of the GAR-dwarfing genes for use in breeding of commercial varieties. The Rht8 allele has been shown to reduce plant height

and increase carbon-partitioning to grain to increase grain number and yield (Rebetzke and Richards, 2000). In addition to Rht8 there is a suite of major GAR, dwarfing genes (e.g. Rht4, Rht5, Rht12, Rht13, Rht14, and Rht18) that reduce plant height by as much as 50% when compared with tall-parental or near-isogenic controls (Konzak, 1987; Loskutova, 1998; Ellis et al., 2004, 2005) but are seemingly neutral in their effects on coleoptile length and seedling leaf size (Ellis et al., 2004). The aim of this paper was to extend the work of Ellis et al. (2004) by examining the influence of a subset of GAR-dwarfing genes on plant height and agronomic performance in targeted wheat populations. Specifically, we report on the influence of large-effect dwarfing alleles Rht4, Rht5, Rht8, Rht12 and Rht13 on genotypic variation in plant height, examine genetic relationships between plant height and early growth, and the effect of height on total biomass, grain number and yield. 2. Materials and methods 2.1. Populations and lines Four wheat populations were developed varying for GARdwarfing genes that reduce plant height without affecting seedling growth (Ellis et al., 2004): Vigour 18/Burt ert 937 (V/B); Vigour 18/Marfed M1 (V/Ma); Vigour 18/Mercia Rht12 (V/Me); and Chuanmai 18/Magnif M1 (C/M). Genotypes were inbred having been developed as either double-haploid or recombinant inbred lines. Vigour 18 is a standard height, high vigour breeding line containing no known major dwarfing genes whereas Burt ert 937, Marfed M1, Chuan-mai 18, Mercia Rht12 and Magnif M1 contain the Rht4, Rht5, Rht8, Rht12 and Rht13 dwarfing alleles, respectively (Ellis et al., 2004, 2007). Vigour 18 is an early flowering genotype whereas the dwarfing gene donors varied from very quick to very slow in their development. Progeny-lines were essentially random with the exception of the V/Me population where a small number of lines (ca. 15%) were removed owing to later flowering. Overall, between 124 and 172 lines per population were genotyped for presence of each dwarfing gene using linked molecular markers [Xwmc317 (Rht4), Xbarc102 (Rht5), Xgwm261 (Rht8), Xwmc410 (Rht12) and Xwms577 (Rht13)] as described in Ellis et al. (2005). Two sets of experiments reflecting chronological development of populations were grown: set 1 contained the V/B, V/Ma and V/Me populations evaluated in three to five environments; and set 2 evaluated the C/M population in five environments. Control entries were grown in all experiments including the parents, commercial semi-dwarf (H45, Hartog, Janz, Westonia) and tall (Halberd) varieties. In addition, sowings were made of APD and KCD near-isogenic, spring-habit wheat lines varying for the Rht-B1b and Rht-D1b dwarfing alleles (Richards, 1992a). 2.2. Agronomic assessment Lines of the four populations were sown into 6-m long, 0.17-m spaced, 5-row plots across multiple years and sites (Table 1). Sowing dates varied from May 22 to June 19 in each environment. All entries were sown at an optimal 4- to 5-cm sowing depth as single, unreplicated plots in an augmented design containing multiple replicates of the control varieties. Short and tall lines were grouped separately to minimise competition effects through shading. Seeding rate varied between 180 (Stockinbingal, New South Wales) and 200 [Ginninderra Experiment Station (GES), Australian Capital Territory and Griffith, New South Wales] seeds per m2 . Nutrients were supplied at sowing as Starter 15® applied at 103 kg/ha and supplemented with additional nitrogen as required. Plots were

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

89

Table 1 Genotype means for agronomic characteristics measured on semi-dwarf (Janz) and tall (Vigour 18) genotypes in each year and location under study. Environ (year/site) 2002 Stockinbingal 2003 ACT 2004 ACT Griffith Stockinbingal 2005 Stockinbingal

Entry Janz Vigour 18

Plant height (cm) 68 98a

Grain yield (t/ha)

Aerial biomass (t/ha)

Harvest index

Grain number (no/m2 )

2.7 2.1

7.8 6.9

0.35 0.30

7841 5188

Grain weight (mg) 35 40

Janz Vigour 18

80 108

5.1 3.4

12.4 10.9

0.41 0.31

13,064 6589

40 41ns

Janz Vigour 18 Janz Vigour 18 Janz Vigour 18

92 104 80 103 81 110

6.5 3.4 5.1 3.6 3.5 2.1

16.1 9.8 14.6 11.6 10.4 7.5

0.40 0.35 0.35 0.31 0.33 0.28

16,385 8287 10,384 7214 9525 4646

40 41ns 49 50ns 36 41

Janz Vigour 18

85 127

2.9 1.8

8.1 6.2

0.36 0.29

7838 4298

37 41

ns: Janz and Vigour 18 were not statistically different at P = 0.05 a Entry means within each year × location were statistically different at P = 0.05 except where indicated.

reliant on pre- and growing-season rainfall, and in some cases, supplemented with irrigation at flowering and grain-filling (GES and Griffith). Sowings were maintained free of weeds and diseases with the application of appropriate herbicide and fungicide control measures as necessary. For each plot, plant development was recorded near anthesis using the Zadoks decimal score (Zadoks et al., 1974). Plant height was determined at maturity as the distance from the soil surface to the top of the ear (awns excluded) of the tallest culms for each plot. For all plots, between 80 and 120 culms were hand-cut at ground level at maturity using a 30-cm wide quadrat oriented across three inside rows. Samples were air-dried at 35 ◦ C for three days and weighed before and after threshing, and harvest index calculated as the ratio of grain to total above-ground biomass. Plots were end-trimmed to ca. 5.4-m length and the outside border rows removed before machine harvesting. Grain yields were subsequently expressed at ca. 12% moisture. Grain weight was measured for a representative 200-grain sample collected from each harvest index sample, and grain number (per m2 ) calculated from grain weight and plot yields. Lodging was scored at maturity using a rating from 1 (all spikes lying prostrate) to 9 (all spikes perpendicular to the soil surface).

2.4. Statistical analysis Combined analysis of variance and covariance over all environments were performed for all characters using the SAS mixed linear models procedure MIXED (Littell et al., 1996). Variance and covariance components for genotype and genotype × environment interaction effects were estimated for each population assuming genotypes and environments were random effects. Narrow-sense heritabilities (and their standard errors) were estimated on a single-plot and entry-mean basis. Analysis of the effect of each dwarfing gene on plant height and agronomic performance was assessed using single-degree of freedom contrasts for populations varying for a single gene (i.e. Rht4, Rht5 and Rht12), and protected least significant difference (l.s.d.) or orthogonal contrasts for the C/M population varying for alleles at two major (Rht8 and Rht13) dwarfing loci. Genotype classes were based on presence of the linked molecular markers for each allele. Controls were compared after estimation of a protected least significant difference (l.s.d.). Genetic correlations and standard errors were estimated between plant height and agronomic characteristics for each population using the SAS procedure MIXED (after Holland, 2006). 3. Results

2.3. Early vigour assessment Early vigour and coleoptile length assessment was undertaken for control varieties, parents, the GAI-dwarfing gene NILs, and random lines from each population. Good quality seeds free of any visible damage and weighing between 35 and 40 mg were obtained for each entry. These were sown into an unreplicated, augmented design in wooden seedling trays (600 mm × 300 mm × 120 mm) containing a fertile, compost-based potting mix. A subset of parents, check lines and progeny were replicated twice per environment so that the average replication per experiment was ca. 1.4. For coleoptile length, seeds were sown 2-cm below the soil surface and placed into darkened growth cabinets set at constant temperatures of 15 and 21 ◦ C. Seedlings were removed from the dark after ca. 200 degree-days (assuming a base temperature of 0 ◦ C) and coleoptile lengths determined with a ruler as the distance from the scutellum to the tip of the coleoptile. Entries were also sown at 1-cm depth and then placed into a well-lit growth cabinet with temperature settings of 16/19 ◦ C (day/night). Seedlings were watered when necessary and assessed for leaf 1 breadth and length using a digital Vernier calliper when 90% of seedlings reached ca. 3.5 leaves.

Experiments were undertaken across a broad and contrasting range of environments. Set 1 studies were undertaken at Stockinbingal in 2002 and 2004, GES in 2003 and 2004, and Griffith in 2004 with the latter two sites being irrigated. Set 2 studies were undertaken in the same environments except the Stockinbingal 2002 study was not sown and replaced with a sowing at Stockinbingal in 2005. In-crop rainfall varied from a below-average 151 mm for Stockinbingal 2002 to 275 mm for GES 2004. Similarly, mean daily maximum temperature varied across environments, being coolest (15.3 ◦ C) at GES in 2003 and warmest (20.0 ◦ C) at Stockinbingal in 2005. Mean daily minimum temperatures were correspondingly cooler at GES 2003 (3.4 ◦ C) and Stockinbingal 2005 (7.5 ◦ C), respectively. The sampled field environments provided a broad range of conditions over which genotypic differences in plant height, grain yield and other agronomic characteristics could be assessed. These differences were demonstrated via comparative performance of the semi-dwarf Rht-B1b variety Janz and tall Rht-B1a breeding line Vigour 18 (Table 1). In all environments it was possible to discriminate plant height differences between the two genotypes. Grain yield and number differences were greatest in higher rainfall and irrigated sites (Griffith and GES) whereas harvest index differences were greatest in the cooler GES 2003 sowing. Grain size was similar

90

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

Table 2 Variance components, genotypic coefficients of variation (GCV) and narrow-sense heritability (h2 ) for plant height measured on random lines in four populations varying for major dwarfing alleles evaluated across multiple environments. Parameter

 genotype  2 genotype × environment  2 residual GCV (%) h2 (%) 2

a b

Population Vigour 18/Burt ert 937 (Rht4)

Vigour 18/Marfed M1 (Rht5)

Vigour 18/Mercia Rht12 (Rht12)

Chuan-mai 18/Magnif M1 (Rht8, Rht13)

158 ± 21 56 ± 8 16 ± 3 15 90 (69)b

389 ± 34 6±6 27 ± 6 28 96 (92)

402 ± 37 14 ± 6 33 ± 6 24 98 (90)

604 ± 64 41 ± 4 34 ± 3 31 98 (89)

a

Variance component ± standard error. Line-mean heritability (single-plot heritability in parenthesis).

for all environments except Griffith 2005, where irrigation provided more available water through grain-filling. Genotypic variation was large and statistically significant (P < 0.05) for all traits measured on each population. This was strongly evidenced for plant height where the genotypic variance was ca. 3–50 times greater than the genotype × environment interaction variance across the four height-varying populations (Table 2). In turn, both single-plot and line-mean heritabilities for plant height were very high, commonly exceeding 80%. Genetic variance in plant height was smallest for the V/B and largest for the V/Me and C/M populations, respectively. Similarly, genotypic coefficients of variation were smallest for V/B and largest for the C/M population, with the latter varying for two dwarfing genes, Rht8 and Rht13. The V/Ma population showed large genotypic variance for plant height (Table 2), and also anthesis date (data not shown).

Across populations, the V/Ma population was the most variable for anthesis date, with development among lines in other populations being reasonably similar (data not shown). The range in plant height among control entries was large and consistent with expectation. For example, the range in height among the APD NILs strongly reflected their dwarfing genotypes so that height of the standard height APD0 was 28% greater than that of the single dwarf APD1, and 68% taller than the double-dwarf APD2 when assessed in the set 1 experiments (Table 3). Relative height differentials were 30% and 69% for APD1 and APD2, respectively in the second set of experiments, demonstrating the robustness of these height differences across environments and studies (Table 4). Similarly, the KCD set produced height reductions of 31 and 61% for the Rht-B1b and Rht-B1b+D1b near-isogenic pairs, respectively, relative to the tall control (data not shown). The heights of the

Table 3 Means (in bold) and ranges for different agronomic characters measured on lines in populations varying for different major dwarfing alleles and evaluated across multiple environments. Height genotype classes were defined by presence of alleles at linked molecular markers. Population/genotype class (a) Vig18/Burt ert 937 Rht4 Min. Max. rht4 Min. Max. (b) Vig18/Marfed M1 Rht5 Min. Max. rht5 Min. Max. (c) Vig18/MerciaRht12 Rht12 Min. Max. rht12 Min. Max. Controls APD0 (Rht-B1a) APD1 (Rht-B1b) APD2 (Rht-B1b,D1b) Burt ert 937 (Rht4) Marfed M1 (Rht5) Sunco Rht12 (Rht12) Vigour 18 (tall) HM14bS (Rht8) Hartog (Rht-D1b) Janz (Rht-B1b) Westonia (Rht-D1b) l.s.d. line means

No. lines (n)

Plant height (cm)

Grain yield (t/ha)

Aerial biomass (t/ha)

Harvest index

Grain number (no/m2 )

Grain weight (mg)

Anthesis score (Zadoksa )

68

74 62 91 89** 73 102

3.5 2.4 5.4 3.1* 1.7 4.8

10.8 8.4 15.9 10.8 7.8 14.0

0.34 0.25 0.41 0.30* 0.23 0.38

8940 4912 8552 7540** 2024 7774

37 30 47 41** 32 52

57 47 66 61* 46 66

39 25 55 87** 60 110

0.7 0.1 2.1 2.3** 0.5 3.4

– – – – – –

– – – – – –

2341 569 5126 6972** 4076 9873

30 29 33 37** 32 47

41 35 45 60** 35 70

53 43 74 97** 71 115

4.3 2.9 5.6 3.9* 2.0 5.5

11.9 8.0 15.8 12.7 8.0 16.1

0.37 0.27 0.45 0.31** 0.21 0.39

10,871 5434 13,750 9234** 6051 12,877

38 30 45 43** 33 50

54 50 67 64** 54 70

104 81 62 77 35 52 109 83 79 84 83

4.5 4.9 3.5 2.9 0.3 4.5 3.6 4.9 4.5 5.2 5.9

13.5 13.7 9.6 9.9 1.4 10.8 12.1 13.8 12.5 14.4 13.9

0.34 0.37 0.43 0.28 0.21 0.42 0.29 0.33 0.37 0.36 0.42

10,732 12,781 9998 9486 733 11,031 8734 11,401 12,623 12,624 14,698

42 38 35 27 24 41 44 42 35 41 37

66 67 59 41 37 61 70 69 65 65 65

6

1.3

4.1

0.04

2109

4

7

61

79

94

43

79

– not measured. *, ** denotes dwarfing and wild-type allele means are statistically different at P = 0.05 and 0.01, respectively. a A score of 65 is given when 50% of spikes in the plot have reached anthesis.

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

91

Table 4 Means (in bold) and ranges for different agronomic characters measured on lines in the Chuan-mai 18/Magnif M1 population varying for the Rht8 and Rht13 dwarfing genes and evaluated across multiple environments. Height genotype classes were defined by presence of alleles at linked molecular markers. Grain weight (mg)

Anthesis score (Zadoksa )

11,178 8005 14,214 11,995 8600 14,804 9419 4715 14,272 9426 4269 15,145

41 33 48 41 35 46 45 35 50 44 37 51

65 55 69 62 56 69 66 57 69 65 59 69

0.01

505

1

2

13.1 12.9 9.8 13.0 13.7 13.5 13.1 12.9 13.8 17.9

0.35 0.38 0.40 0.39 0.35 0.40 0.39 0.43 0.45 0.24

11,202 13,601 11,404 12,098 11,446 13,067 11,996 14,511 14,985 10,210

39 36 34 40 42 40 42 38 41 42

65 63 58 68 63 66 67 69 64 70

1.7

0.04

1989

4

4

Genotype class

No. lines (n)

Plant height (cm)

Grain yield (t/ha)

Aerial biomasss (t/ha)

Harvest index

Rht8+Rht13 Min. Max. Rht13 Min. Max. Rht8 Min. Max. rht8+rht13 Min. Max.

49

63 44 95 73 56 120 104 75 129 112 80 144

4.4 2.9 5.1 4.7 3.2 5.7 4.0 2.7 5.3 4.0 1.9 5.5

11.7 7.2 13.4 13.1 9.1 14.5 11.8 8.3 15.7 12.3 7.1 15.3

0.38 0.28 0.44 0.37 0.31 0.43 0.35 0.27 0.41 0.33 0.25 0.40

2

0.2

0.5

107 82 63 86 82 98 80 84 81 121

4.4 4.9 3.9 5.1 4.8 5.3 5.1 5.5 6.2 4.3

8

0.4

l.s.d. Rht means Controls APD0 (Rht-B1a) APD1 (Rht-B1b) APD2 (Rht-B1b+D1b) Chuan-mai 18 (Rht8) Magnif M1 (Rht13) Halberd (tall) HM14bS (Rht8) H45 (Rht-B1b) Janz (Rht-B1b) Vigour 18 (tall) l.s.d. line means a

36

49

53

Grain number (no/m2 )

A score of 65 is given when 50% of spikes in the plot have reached anthesis.

GAI, semi-dwarf varieties Hartog, Janz and Westonia were 20–25% shorter than the standard height APD0 and Vigour 18, while plant heights of the GAR-Rht8 genotypes Chuan-mai 18 and HM14bS were approximately 10–30% smaller than the tall controls APD0 and Vigour 18 (Table 4). Lines from each population were genotyped with molecular markers linked to dwarfing genes varying in each population. Tables 3 and 4 show trait means and ranges for sister-lines genotyped for the different dwarfing alleles Rht4, Rht5, Rht8, Rht12 and Rht13. Plant heights were consistently shorter for lines carrying the dwarf allele at each of the dwarfing loci. When compared to the mean of tall sister lines (those carrying the wild-type allele), the largest reduction in plant height was associated with Rht5 (−55%), and then Rht12 (−45%), Rht13 (−34%), Rht4 (−17%), and smallest for Rht8 (−7%)(Fig. 1). These height reductions were as great, or greater, than the 22–24% height reduction associated with presence of Rht-B1b in the two NIL sets. Further, the height reductions associated with Rht5 and Rht12 were similar to those of the RhtB1b+Rht-D1b NIL double-dwarfs (Table 3). There was some overlap in the distributions of plant heights for the different dwarfing alleles in each population (Tables 3 and 4). This overlap was particularly large for the V/B and C/M populations where alleles of smaller effect independent of the major dwarfing alleles (i.e. ‘background effects’) contributed to changes in plant height. For example, lines homozygous for the Rht13 dwarfing linked-marker (Xwms577), and lacking the Rht8-linked Xgwm261-192bp allele, ranged in plant height from 56 to 126 cm. There was little evidence for transgressive segregation in any population except the C/M population where 41% of lines were shorter in height than parent Magnif M1 and 53% were taller than CM-18. The range in grain yield and total biomass was large in all tested populations (Tables 3 and 4). Further, some higher-yielding progeny-lines achieved grain yields equal to, and sometimes exceeding, the commercial controls. For example, a number of Rht4, Rht12 and Rht13 lines had equivalent yield performance to commercial varieties Hartog, Janz and Westonia. Similarly, total biomass of some lines was as great, and sometimes greater, than

that of the commercial checks. When averaged for each dwarfing gene, dwarfing alleles were commonly associated with significantly (P < 0.01) greater grain yield. This increase in yield reflected increased harvest index and a similar or greater total biomass (e.g. Rht13) (Fig. 1; Tables 3 and 4). An exception was Rht5 where presence of the dwarfing allele was associated with significantly (P < 0.01) delayed flowering (i.e. smaller Zadoks score) and reduced grain number to decrease harvest index and grain yield (Fig. 1, Table 3). For most other dwarfing genes, presence of the reduced height allele was associated with increased grain number but a compensatory reduction in grain weight (Fig. 1). An exception was Rht8 where the small reduction in height had little effect on either grain number or size. The influence of the GAR-dwarfing genes on grain yield components was consistent with the influence of RhtB1b on the same components. For example, in the APD NIL set, presence of a single GAI-dwarfing allele (i.e. APD1) was associated with increased grain number to increase harvest index and yield despite a reduction in grain weight (Fig. 1). Increasing the frequency of GAI-alleles from 2 to 4 in the development of APD2 was associated with a reduction in grain yield despite the increased harvest index (Tables 3 and 4). This reduction reflected fewer, smaller grains and a substantial reduction in total biomass. Lodging was significantly greater in all non-Rht progeny (Fig. 1). Genotypic correlations were estimated for plant height and selected agronomic traits in each population. Genotypic reductions in plant height were associated with delayed flowering in the V/Ma (i.e. Rht5), and to a lesser extent, the V/B (i.e. Rht4) and V/Me (i.e. Rht12) populations, respectively (Table 5). Reduced height associated with presence of Rht8 was correlated with earlier flowering whereas presence of Rht13 was independent of development score. Reductions in plant height were commonly positively correlated with genotypic increases in grain number, harvest index, and in turn, grain yield (Table 5). An exception was the Rht5-varying population where an association between reduced height and later flowering contributed to reductions in grain yield for shorter lines. Grain number and harvest index were themselves genetically correlated (rg = 0.35–0.88, P < 0.01), indicating that increases in grain

92

Percent change relative to wild type (tall)

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

75

50

Rht1an 2 d Rht-B1b (b) (a)Rht4, Rht4,Rht5 Rht5,anRdht12

(b) Rht8 and Rht13 Rht8 Rht13 Rht8+Rht13

Rht4 Rht5 Rht12 Rht-B1b

25

0

-25

-50

-75

Plant height

Grain yield

Aerial Harvest Grain biomass index number

Grain weight

Lodging score

Plant Grain height yield

Aerial Harvest Grain Grain Lodging biomass index number weight score

Fig. 1. Mean performance of lines homozygous for major dwarfing alleles relative to performance of lines homozygous for the wild-type (tall) allele for agronomic characteristics measured in different populations grown in multiple environments. Bars represent (left to right): (a) Rht4, Rht5, Rht12 and Rht-B1b; and (b) Rht8, Rht13 and Rht8+Rht13. Height genotype classes were defined by presence of alleles at linked molecular markers.

number were related to genetic increases in harvest index. Genotypic increases in plant height were associated with increased grain size and small albeit significant increases or decreases in total biomass (Table 5). Among the different dwarfing genes assessed, Rht8 tended to have the smallest effect on grain yield and harvest index. This contrasted strongly with the modest-sized genetic effect of Rht4 and large genetic effect of Rht12 and Rht13 on grain yield and yield components. Genotypic reductions in plant height were strongly correlated with reduced susceptibility to plant lodging across all populations (Table 5). In the growth cabinet experiments, differences among entries were large and significant (P < 0.01) for size of leaf one and coleoptile length (Table 6). Among control varieties, leaf area and coleoptile length were commonly smallest for GAI, semi-dwarf varieties Janz and Wyalkatchem, and largest for the GAR-parental and tall controls. Mean plant height (field means) and coleoptile lengths of tall lines in each population were similar to that of the tall APD and KCD NILs (Fig. 2). Increasing frequency of the GAI-dwarfing alleles was associated with linear reductions in coleoptile length in both APD (b = −1.27, r2 = 0.93) and KCD (b = −1.13, r2 = 0.98) NILs (Fig. 2). That is, coleoptile length was reduced ca. 1.1–1.3-mm for each 1 cm reduction in plant height with selection of Rht-B1b and/or Rht-D1b. Thus for GAI-wheats, average height reductions of ca. 25-cm were associated with coleoptile length reductions of

28–33-mm. This reduction contrasts with the Rht4, Rht8, Rht12 and Rht13, and to a lesser extent Rht5, dwarfing genes where height reduction was independent of changes in coleoptile length. For example, presence of Rht12 was associated with reduction in average plant height of 44-cm yet mean coleoptile lengths were approximately the same for dwarf and tall progeny alike (Table 6, Fig. 2). Similarly, mean coleoptile length for double-dwarf Rht8+Rht13 lines was similar to that of the tall rht8+rht13 siblings. For most GAR-dwarfing alleles, the genotypic range in coleoptile length was independent of plant height and consistent with the range in the corresponding tall sister lines. In turn, long coleoptile Rht5 lines could be readily identified (e.g. Fig. 2) despite the average reduction in coleoptile length associated with this dwarfing allele (Table 6). 4. Discussion Breeders select for many traits in their efforts to improve adaptation. A key consideration is agronomic type whereby initial selection is for an ideotype where plant height and flowering are optimal for the target environment(s) (Richards et al., 2002). Accumulation of minor alleles in selection for reduced height had slowly albeit successfully reduced plant height leading up to the green revolution. Identification and deployment of Rht-B1b and Rht-D1b have since provided breeders with height-reducing genes of large effect.

Table 5 Genetic correlation coefficients for plant height and a range of agronomic traits measured on random inbred lines evaluated in multiple environments and varying for presence of one or more major gibberellin-responsive dwarfing genes. Character

Anthesis score Grain yield Total biomass Harvest index Grain number Grain weight Lodging score

V/B

V/Ma

V/Me

C/M

(Rht4)

(Rht5)

(Rht12)

(Rht8a )

(Rht13b )

0.20* −0.67** 0.10 −0.62** −0.64** 0.90** –

0.83** 0.63** – – −0.22* 0.34** 0.88**

0.59** −0.54** 0.02 −0.86** −0.48** 0.68** 0.84**

−0.27** −0.28** −0.22* −0.46** −0.29** 0.81** 0.88**

0.04 −0.61** −0.18* −0.81** −0.73** 0.68** 0.93**

*,** denotes correlation significantly different from zero at P = 0.05 and 0.01, respectively. a Genotypic correlation for all lines in the Chuan-mai 18/Magnif M1 population except those containing the Rht13-linked, Xwms577 dwarfing allele. b Genotypic correlation for all lines in the Chuan-mai 18/Magnif M1 population except those containing the Rht8-linked, Xgwm261 dwarfing allele.

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

93

Table 6 Dwarfing gene means for coleoptile length and seedling vigour characteristics measured in populations varying for one or more major dwarfing genes. Means are also given for control varieties and germplasm grown in the same experiments. Height genotype classes were defined by presence of alleles at linked molecular markers. Population/genotype class

Early vigour Leaf 1 breadth (mm)

105 105ns

4.4 4.6

123 116

4.5 4.3

101 116**

4.4 4.8*

118 139**

4.2 5.4**

121 116ns

4.7 4.8

119 134**

4.5 5.1**

101 114 99 104

4.6 4.5 4.5 4.8

116 118 107 121

4.2 4.3 3.9 4.7

l.s.d. C-M18/Magnif M1 means Controls APD0 (Rht-B1a) APD1 (Rht-B1b) APD2 (Rht-B1b+D1b) Chuan-mai 18 (Rht8) Janz (Rht-B1b) Wyalkatchem (Rht-D1b) Magnif M1 (Rht13) V/Me S2 (Rht12) Vigour 18 (tall) Halberd (tall)

7

0.3

16

0.5

111 79 64 110 74 65 102 110 113 118

4.6 4.5 4.5 4.7 3.2 4.5 4.8 4.8 5.3 5.0

125 108 70 108 83 112 116 112 130 120

4.6 3.9 2.6 4.1 2.1 4.0 4.5 4.3 5.5 4.8

l.s.d. control means

10

0.4

27

1.2

(a) Vig18/Burt ert 937 Rht4 (semi-dwarf) rht4 (tall) (b) Vig18/Marfed M1 Rht5 (semi-dwarf) rht5 (tall) (c) Vig18/Mercia Rht12 Rht12 (semi-dwarf) rht12 (tall) (d) CM-18/Magnif M1 Rht8+Rht13 (double-dwarf) Rht13 (semi-dwarf) Rht8 (semi-dwarf) rht8b+rht13 (tall)

Leaf 1 length (mm)

Leaf 1 area (cm2 )

Coleoptile length (mm)

rht8+rht13

Rht8

rht12

rht5 rht4

Rht13 Rht4

150

Rht8+Rht13

Rht5

Rht12

*, ** denotes dwarfing and wild-type allele means are statistically different at P = 0.05 and 0.01, respectively.

Mean coleoptile length (mm)

140 130 120 T

110 T 100 90 S

80 70 D 60

S

D

50 35

45

55

65

75

85

95

105

115

Mean plant height (cm) Fig. 2. Box plot of coleoptile length genotype means and ranges (cabinet measured) plotted against mean plant height (field measured) for lines homozygous for different GA-responsive and corresponding wild-type (tall) alleles. The heavy horizontal line in each box represents the mean for all lines homozygous for the dwarfing or wild-type allele, the lighter horizontal line the median, the lower and upper edges of the box the 25th and 75th percentiles, and the ‘error bars’ the 10th and 90th percentiles. ‘D’, ‘S’ and ‘T’ are means for the double-dwarf, semi-dwarf and tall APD () and KCD (䊉) Rht-B1 and Rht-D1 near-isolines evaluated in the same study. Height genotype classes were defined by presence of alleles at linked molecular markers.

This has facilitated selection in early generations and the recovery of wheats of semidwarf stature within smaller population sizes. This study assessed plant height and correlated traits across large populations contrasting for different, alternative dwarfing genes. The set under investigation included the most promising five

of 16 reported GAR-dwarfing genes for use in bread wheat improvement (Ellis et al., 2004). Further, large-sized, height-varying populations were assessed using linked markers to genotype lines for presence or absence of the different dwarfing alleles identified in Ellis et al. (2005). Genes for plant height accounted for large proportions of the phenotypic variance although it must be noted that recombination between the linked marker and dwarfing allele may lead to an underestimation of the true size and proportion of variance associated with alleles at each locus (Ellis et al., 2005). Despite this, the use of genotyped populations can remove potential confounding of plant height phenotype with genetic background and has been used previously for plant height (e.g. Butler et al., 2005; Ellis et al., 2005; Rebetzke et al., 2001, 2007a). Heritabilities for plant height were large for all populations, indicating a strong correlation of phenotype with genotypic value, and reflecting large genetic and small genotype × environment interaction variances. The high heritabilities for plant height were similar to estimates obtained for populations varying for GAIalleles (e.g. H = 0.94; Quail et al., 1989) but greater than for previous estimates in populations varying for both Rht8 and/or minor reduced-height alleles (i.e. H = 0.68–0.73; Rebetzke et al., 1999). The high heritabilities in the current study reflected height variation arising from large effect dwarfing genes. Smaller genotype × environment interaction and residual variances reflect greater penetrance for alleles at individual loci and subsequently smaller sampling error. Selection of alleles of large effect increases confidence when targeting reduced height during early generations of family testing. Further, association with linked-molecular markers (e.g. Ellis et al., 2005) increases the utility of the major GAR-dwarfing genes in wheat breeding. Comparisons among populations demonstrated large genetic effects on height reduction associated with dwarfing alleles Rht5 (−55%), Rht12 (−45%) and Rht13 (−34%), moderate height reduction for Rht4 (−17%), and a small height reduction with Rht8 (−8%).

94

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

The strong dwarfism associated with Rht5, Rht12 and Rht13 was similar to the ca. 40% height reduction of the Rht-B1b+Rht-D1b GAIdouble-dwarfs, whereas height reduction for Rht4 was similar to the ca. 20% reduction reported for GAI-Rht-B1b and Rht-D1b alleles herein and elsewhere (e.g. Flintham et al., 1997; Nizam Uddin and Marshall, 1989). The GAR-dwarfing alleles assessed here are genetically independent (Ellis et al., 2005), and effects at two independent loci, Rht8 and Rht13, were additive in effect indicating potential to produce GAR, double- or sesqui-dwarf genotypes with possibly greater harvest index and final yield. Agronomic performance is a key consideration in assessment of new alleles for use in breeding. Considerable genotypic variation was observed for all agronomic traits measured in each population. With exception of the later-flowering Rht5, the different GAR-dwarfing alleles reduced plant height to increase grain yield, grain number and harvest index, consistent with reported effects of height reduction in lines and varieties containing GAIdwarfing alleles. Presence of the GAI-Rht-B1b and Rht-D1b dwarfing genes is commonly associated with greater grain number per spike to increase grain number per unit area (Fischer and Stockman, 1986; Nizam Uddin and Marshall, 1989). Because ear and peduncle growth coincide developmentally in wheat, genetic reductions in height reduce stem growth, thereby reducing competition for assimilate between the elongating stem and developing ear (Youssefian et al., 1992). Ear mass and fertile floret number at anthesis is subsequently greater for semi-dwarf wheats when compared with their tall, near-isogenic counterparts (Miralles et al., 1998). Selection for increased grain yield makes use of this reallocation in assimilate as evidenced by greater harvest index and more grain per m2 in breeding programs releasing wheats with the Rht-B1b and Rht-D1b genes (Fischer and Quail, 1990; Waddington et al., 1986). The greater harvest index and grain number for the GAR-dwarfing alleles is consistent with the effects of the GAIdwarfing alleles, and supports the model of reduced competition between growing florets and elongating stems. Total biomass at maturity was usually unaffected by dwarfing alleles whereas height reductions associated with Rht13 were associated with small albeit significant increases in total biomass. In turn, grain yield increases in GAR-dwarfing lines reflected greater harvest index, biomass or both. A key component to adaptation is flowering and the synchronisation of development with environmental conditions prevailing during crop growth. There was a general trend for reduced height to be associated with later maturity across populations. Phenotyping development in short lines can be challenging particularly in dry environments where cell and subsequent peduncle extension may be limited by reduced soil water availability. In turn, the determination of accurate anthesis dates can be very difficult with shorter lines appearing much slower in their development. Further, reduced grain yields and other agronomic performance may appear linked to shorter height through such a sampling association. Lines containing Rht5 and Rht12 dwarfing alleles were later-flowering with the Rht5-containing dwarfs being particularly slow in development. Despite the large population sizes assessed for Rht5, we identified few earlier-flowering, semi-dwarfs suggesting close linkage of Rht5 with alleles for later-maturity. This association may reflect proximity to an earliness per se (Eps) locus located near the centromere on chromosome 3BL (Pánková et al., 2008). The association of Rht12 with later-flowering is wellestablished (e.g. Addisu et al., 2009) and reflects a close linkage of the dwarfing gene with Vrn-A1 on chromosome 5AL (Worland et al., 1994). The Rht4 and to a lesser extent Rht13 dwarfing alleles were largely independent of anthesis date. The Rht8-linked marker allele was associated with increasingly earlier development scores consistent with previous reports (e.g. Addisu et al., 2009). The Rht8 donor in this study, CM-18, is earlier flowering and

contains the photoperiod-insensitive, Ppd-D1a development allele (Rebetzke et al., in press). Reduced-height lines containing Rht8 were earlier flowering, and it is unclear whether the reduction in plant height associated with Rht8 was partly due to their quicker development. Optimum plant height for maximising grain yield varies according to potential grain yield of the environment (Fischer and Quail, 1990; Richards, 1992a). In some irrigated environments, where yield potentials are high, single dwarf GAI-wheats may not be sufficiently reduced in height. Excessive height contributes to reduced harvest index and to greater lodging, reducing yields and grain quality, and contributing to difficulties in harvesting (Stapper and Fischer, 1990). Further, where crop duration is extended (e.g. as occurs with early breaks in southern Australia), extra duration for vegetative growth contributes to a greater plant height. Consequently single dwarf GAI-wheats grow tall, commonly lodge and have reduced harvest index (Gomez-Macpherson and Richards, 1995). Plant growth regulators may be used to reduce plant heights but there is additional cost and increasing resistance to their use. There is also the potential to grow GAI Rht1+Rht2 doubleddwarfs, and stronger GAI-dwarfs such as Rht-B1c (Rht3) and Rht-D1d (Rht10) but these have short coleoptiles contributing to poor establishment (Addisu et al., 2009) and have slower leaf area development (Rebetzke et al., 2004b) to reduce crop biomass. Stronger effect GAR-dwarfing alleles including Rht12 and Rht13 may have potential for increasing harvest index and total biomass in higheryielding environments without compromising establishment and early growth. In lower-yielding environments where biomass accumulation may limit potential crop yields (Richards, 1992a) the large height-reducing effect of Rht5 and Rht12 may reduce their utility. In such environments Rht13 and the moderate strength Rht4 and Rht8 dwarfing alleles show promise for maintaining greater biomass while increasing harvest index, grain number and yield. The GAI-dwarfing genes Rht-B1b and Rht-D1b partly contribute to yield increase in favourable environments through reduced plant lodging (Berry et al., 2007; Stapper and Fischer, 1990). Erect, upright plants are easier to harvest and experience good light transmission and reduced mutual shading through grain-filling. For the studies reported herein, shorter plants produced smaller lodging scores. Similarly, lodging and grain yield were themselves genetically correlated across populations (rg = −0.35–0.62) with the exception of Rht5 where shorter height and later flowering reduced grain yields. The drier, shorter-season conditions experienced in the reported studies reduced the potential for plant lodging and its subsequent influence on yield. Thus dwarfing gene effects on height and yield largely reflected increased partitioning (greater harvest index) to grain. That said, further studies under very favourable conditions are recommended where lodging is controlled (e.g. through use of canopy supports) to better understand how these alternative dwarfing genes influence grain yield independent of lodging. The shorter coleoptile length, and smaller seedling leaf area and biomass observed here of GAI-genotypes Janz, APD1 and APD2 were consistent with previous reports of reduced seedling vigour for GAI-semi-dwarf wheats (e.g. Allan, 1989; Richards, 1992b; Rebetzke et al., 2001, 2007a; Whan, 1976). The GAI-dwarfing alleles reduce cell elongation to reduce cell size (Keyes et al., 1989; Hoogendoorn et al., 1990). Smaller cells contribute to shorter plants but also shorter coleoptiles and smaller seedling leaf area. The agronomic implications arising from reduced seedling vigour are well established. For example, Rht-B1b and Rht-D1b wheat near-isolines developed in four genetic backgrounds established poorly to produce fewer emerged seedlings when compared with tall isolines sown at a depth of 8 cm (Allan, 1989). Likewise, Rebetzke et al. (2007b) found Rht-B1b and Rht-D1b genotypes produced shorter coleoptiles and were slower to emerge at 8-cm and particularly 11cm sowing depth. In the same study seedling numbers for tall and

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

Rht8 semidwarfs were largely unaffected with deep sowing despite being slower to emerge. Coleoptile length and plant height were positively correlated for the APD and KCD NILs, consistent with previous reports of populations varying for presence of GAI-dwarfing alleles (Allan et al., 1961; Fick and Qualset, 1976; Rebetzke et al., 2007a). For the GAR-dwarfing alleles assessed herein, plant height and coleoptile length were largely independent. For example, lines containing Rht12 were an average 45% shorter in height but equal to coleoptile lengths of tall sister lines. The neutral effect of Rht12 on coleoptile length is consistent with previous observations for Rht12 lines assessed across contrasting populations and mating designs (e.g. Ellis et al., 2004; Rebetzke et al., 2004a). Similarly, Addisu et al. (2009) demonstrated that the coleoptiles of Rht12 and tall NILs were of similar length but significantly longer than those of the corresponding GAI-dwarfing gene-containing, sister NILs. Extreme dwarfism from Rht5 was associated with a slightly shorter average coleoptile length. However, substantial coleoptile length variation within Rht5-containing lines indicated potential for selection of background alleles in development of long-coleoptile, reducedheight Rht5 lines. Similarly, slightly shorter seedling leaves reduced leaf area for Rht5- and Rht12-containing lines. Leaf breadths were largely unaffected and potential exists to identify longer-leafed, dwarf progeny to increase leaf area. Coleoptile lengths associated with Rht8 and Rht13 were similar to those of tall sister lines whether considered singly or together in Rht8+Rht13 double-dwarf genotypes. There was effectively no reduction in coleoptile length or in seedling leaf area for all the GAR-dwarfing alleles considered here, and irrespective of the size of their dwarfing effect. This highlights the potential of these alleles in selection for greater seedling vigour, and extends previous reports (Schillinger et al., 1998; Ellis et al., 2004; Rebetzke et al., 1999, 2004a; Rebetzke and Richards, 2000). In conclusion, this study reports on the use of large populations to confirm and extend previous work using small numbers of comparisons supporting use of GAR-dwarfing alleles for selection of vigorous, semidwarf bread wheats. The large populations allowed robust assessment of both average- and among-line genetic effects for potentially useful alternative dwarfing genes. Dwarfing genes varied in the extent of height reduction, and with the exception of the later-flowering, extreme-dwarf Rht5, were associated with increased grain number, harvest index and grain yield. The deployment of these novel GAR-dwarfing alleles in wheat breeding programs is already well underway for the weaker effect Rht8. Availability of linked molecular markers sees potential to extend selection to Rht4, Rht5, Rht12 and Rht13 for targeted environments. Later maturity associated with Rht5 and Rht12 may require large populations in initial crosses so as to identify earlier-flowering, reduced-height recombinants, particularly if selecting for shorterseason environments. Acknowledgments We would also like to thank staff at the CSIRO Ginninderra Experiment Station, ACT, and Bernie Hart of Junee Reefs NSW for assistance and use of the Stockinbingal site. Thanks also to two anonymous reviewers for their thoughtful comments. References Addisu, M., Snape, J.W., Simmonds, J.R., Gooding, M.J., 2009. Reduced height (Rht) and photoperiod insensitivity (Ppd) allele associations with establishment and early growth of wheat in contrasting production systems. Euphytica 166, 249–267. Allan, R.E., Vogel, O.A., Burleigh, J.R., Peterson Jr., C.J., 1961. Inheritance of coleoptile length and its association with culm length in four winter wheat crosses. Crop Sci. 1, 328–332. Allan, R.E., 1989. Agronomic comparisons between Rht1 and Rht2 semidwarf genes in winter wheat. Crop Sci. 29, 1103–1108.

95

Berry, P.M., Sylvester-Bradley, R., Berry, S., 2007. Ideotype design for lodgingresistant wheat. Euphytica 154, 165–179. Botwright, T.L., Rebetzke, G.J., Condon, A.G., Richards, R.A., 2001. The effect of rht genotype and temperature on coleoptile growth and dry matter partitioning in young wheat seedlings. Aust. J. Plant Physiol. 15, 417–423. Botwright, T.L., Condon, A.G., Rebetzke, G.J., Richards, R.A., 2002. Field evaluation of early vigour for genetic improvement of grain yield in wheat. Aust. J. Agric. Res. 53, 1137–1145. Botwright, T.L., Rebetzke, G.J., Condon, A.G., Richards, R.A., 2005. Influence of the gibberellin-responsive Rht8 dwarfing gene on leaf epidermal cell dimensions and early vigour in wheat (Triticum aestivum L.). Ann. Bot. 95, 631–639. Butler, J.D., Byrne, P.F., Mohammadi, V., Chapman, P.L., Haley, S.D., 2005. Agronomic performance of Rht alleles in a spring wheat population across a range of moisture levels. Crop Sci. 45, 939–947. Chapman, S.C., Mathews, K.L., Trethowan, R.M., Singh, R.P., 2007. Relationships between height and yield in near-isogenic spring wheats that contrast for major reduced height genes. Euphytica 157, 391–397. Coleman, R.K., Gill, G.S., Rebetzke, G.J., 2001. Identification of quantitative trait loci (QTL) for traits conferring weed competitiveness in wheat (Triticum aestivum L.). Aust. J. Agric. Res. 52, 1235–1246. Condon, A.G., Richards, R.A., Rebetzke, G.J., Farquhar, G.D., 2002. Improving intrinsic water-use efficiency and crop yield. Crop Sci. 42, 122–131. Ellis, M.H., Rebetzke, G.J., Azanza, F., Richards, R.A., Spielmeyer, W., Richards, R.A., 2005. Molecular mapping of GR dwarfing genes in bread wheat. Theor. Appl. Genet. 111, 423–430. Ellis, M.H., Rebetzke, G.J., Chandler, P., Bonnett, D.G., Spielmeyer, W., Richards, R.A., 2004. The effect of different height reducing genes on the early growth of wheat. Funct. Plant Biol. 31, 583–589. Ellis, M.H., Bonnett, D.G., Rebetzke, G.J., 2007. A 192bp allele at the gwm261 locus is not always diagnostic for Rht8 in bread wheat. Euphytica 157, 209–214. Fick, G.N., Qualset, C.O., 1976. Seedling emergence, coleoptile length, and plant height relationships in crosses of dwarf and standard-height wheat. Euphytica 25, 679–684. Fischer, R.A., Stockman, Y.M., 1986. Increased kernel number in Norin 10-derived dwarf wheat: evaluation of the cause. Aust. J. Plant Physiol. 13, 767–784. Fischer, R.A., Quail, K.J., 1990. The effect of major dwarfing genes on yield potential in spring wheats. Euphytica 46, 51–56. Flintham, J.E., Borner, A., Worland, A.J., Gale, M.D., 1997. Optimizing wheat grain yield: effects of Rht (gibberellin-insensitive) dwarfing genes. J. Agric. Sci. 128, 11–15. Gomez-Macpherson, H., Richards, R.A., 1995. Effect of sowing time on yield and agronomic characteristics of wheat in south-eastern Australia. Aust. J. Agric. Res. 46, 1381–1399. Holland, J.B., 2006. Estimating genotypic correlations and their standard errors using multivariate restricted maximum likelihood estimation with SAS Proc MIXED. Crop Sci. 46, 642–654. Hoogendoorn, J., Rickson, J.M., Gale, M.D., 1990. Differences in leaf and stem anatomy related to plant height of tall and dwarf wheat. J. Plant Phys. 136, 72–77. Keyes, G.J., Paolillo, D.J., Sorrells, M.E., 1989. The effects of dwarfing genes Rht1 and Rht2 on cellular dimensions and rate of leaf elongation in wheat. Ann. Bot. 64, 683–690. Konzak, C.F., 1987. Mutations and mutation breeding. In: Heyne, E.C. (Ed.), Wheat and Wheat Improvement. American Society of Agronomy, Madison, WI, pp. 428–443. Littell, R.C., Milliken, G.A., Stroup, W.W., Wolfinger, R.D., 1996. SAS System for Mixed Models. SAS Institute Inc., Cary, NC. ˜ López-Castaneda, C., Richards, R.A., 1994. Variation in temperate cereals in rainfed environments III water use and water-use efficiency. Field Crops Res. 39, 85–98. Loskutova, N.P., 1998. The influence of Rht 1–5, Rht 8–9 and Rht 13 genes on morphological characters and yield productivity of wheat. In: Slinkard, A.E. (Ed.), Proceedings of the 9th International Wheat Genetics Symposium. University Extension Press, University Saskatchewan, Saskatoon, pp. 283–284. Mahdi, L., Bell, C.J., Ryan, J., 1998. Establishment and yield of wheat (Triticum turgidum L.) after early sowing at various depths in a semi-arid Mediterranaen environment. Field Crops Res. 58, 187–196. Mathews, K.L., Chapman, S.C., Trethowan, R., Singh, R.P., Crossa, J., Pfeiffer, W., van Ginkel, M., DeLacy, I., 2006. Global adaptation of spring bread and durum wheat lines near-isogenic for major reduced height genes. Crop Sci. 46, 603–613. Miralles, D.J., Katz, S.D., Colloca, A., Slafer, G.A., 1998. Floret development in near isogenic wheat lines differing in plant height. Field Crops Res. 59, 21–30. Nizam Uddin, M., Marshall, D.R., 1989. Effects of dwarfing genes on yield and yield components under irrigated and rainfed conditions in wheat (Triticum aestivum L.). Euphytica 42, 127–134. Palta, J.A., Fillery, I.R.P., Rebetzke, G.J., 2007. Restricted-tillering wheat does not lead to greater investment in roots and early N uptake. Field Crops Res. 104, 52–59. Pánková, K., Milec, Z., Simmonds, J., Leverington-Waite, M., Fish, L., Snape, J.W., 2008. Genetic mapping of a new flowering time gene on chromosome 3B of wheat. Euphytica 164, 779–787. Pearman, I., Thomas, S.M., Thorne, G.N., 1978. Effect of nitrogen fertilizer on growth and yield of semi-dwarf and tall varieties of winter wheat. J. Agric. Sci. 91, 31–45. Quail, K.J., Fischer, R.A., Wood, J.T., 1989. Early generation selection in wheat I yield potential. Aust. J. Agric. Res. 40, 1117–1133. Rebetzke, G.J., Richards, R.A., Fischer, V.M., Mickelson, B.J., 1999. Breeding long coleoptile, reduced height wheats. Euphytica 106, 159–168.

96

G.J. Rebetzke et al. / Field Crops Research 126 (2012) 87–96

Rebetzke, G.J., Richards, R.A., 2000. Gibberellic acid-sensitive dwarfing genes reduce plant height to increase kernel number and grain yield of wheat. Aust. J. Agric. Res. 51, 235–245. Rebetzke, G.J., Appels, R., Morrison, A., Richards, R.A., McDonald, G., Ellis, M.H., Spielmeyer, W., Bonnett, D.G., 2001. Quantitative trait loci on chromosome 4B for coleoptile length and early vigour in wheat (Triticum aestivum L.). Aust. J. Agric. Res. 52, 1221–1234. Rebetzke, G.J., Richards, R.A., Sirault, X.R.R., Morrison, A.D., 2004a. Genetic analysis of coleoptile length and diameter of wheat. Aust. J. Agric. Res. 55, 733–743. Rebetzke, G.J., Botwright, T.L., Moore, C.S., Richards, R.A., Condon, A.G., 2004b. Genotypic variation in specific leaf area for genetic improvement of early vigour in wheat. Field Crops Res. 88, 179–189. Rebetzke, G.J., Ellis, M.H., Bonnett, D.G., Richards, R.A., 2007a. Molecular mapping of genes for coleoptile growth in bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 114, 1173–1183. Rebetzke, G.J., Richards, R.A., Fettell, N.A., Long, M., Condon, A.G., Botwright, T.L., 2007b. Genotypic increases in coleoptile length improves wheat establishment, early vigour and grain yield with deep sowing. Field Crops Res. 100, 10–23. Rebetzke, G.J., Ellis, M.H., Bonnett, D.G. Combining gibberellic acid sensitive and insensitive dwarfing genes in breeding of higher-yielding, sesqui-dwarf wheats. Field Crops Res., in press. Richards, R.A., 1992a. The effect of dwarfing genes in spring wheat in dry environments I Agronomic characteristics. Aust. J. Agric. Res. 43, 517–527. Richards, R.A., 1992b. The effect of dwarfing genes in spring wheat in dry environments. II. Growth, water use and water use efficiency. Aust. J. Agric. Res. 43, 529–539. Richards, R.A., Rebetzke, G.J., Condon, A.G., van Herwaarden, A.F., 2002. Breeding opportunities for efficient water-use and yield. Crop Sci. 42, 111–121.

Sadras, V.O., Rodriguez, D., 2007. The limit to wheat water-use efficiency in eastern Australia. II. Influence of rainfall patterns. Aust. J. Agric. Res. 58, 657–669. Schillinger, W.F., Donaldson, E., Allan, R.E., Jones, S.S., 1998. Winter wheat seedling emergence from deep sowing depths. Agron. J. 90, 582–586. Shackley, B.J., Anderson, W.K., 1995. Responses of wheat cultivars to time of sowing in the southern wheatbelt of Western Australia. Aust. J. Exp. Agric. 35, 579–587. Stapper, M., Fischer, R.A., 1990. Genotype, sowing date and plant spacing influence on high-yielding irrigated wheat in southern New South Wales 2. Growth, yield and nitrogen use. Aust. J. Agric. Res. 41, 1021–1041. Waddington, S.R., Ransom, J.K., Osmanzai, M., Saunders, D.A., 1986. Improvement in the yield potential of bread wheat adapted to northwest Mexico. Crop Sci. 26, 698–703. Watt, M., Kirkegaard, J.A., Rebetzke, G.J., 2005. A wheat genotype with high inherent shoot vigour can adapt to the physical and biological properties of unploughed soil. Funct. Plant Biol. 32, 1–12. Whan, B.R., 1976. The association between coleoptile length and culm length in semi-dwarf and standard wheats. J. Aust. Inst. Agric. Sci. 42, 194–196. Worland, A.J., Sayers, E.J., Börner, A., 1994. The genetics and breeding potential of Rht12, a dominant dwarfing gene in wheat. Plant Breed. 113, 187–196. Youssefian, S., Kirby, E.J.M., Gale, M.D., 1992. Pleiotropic effects of the gibberellininsensitive Rht dwarfing genes in wheat II. Effects on leaf, stem, ear and floret growth. Field Crops Res. 28, 191–210. Zadoks, J.C., Chang, T.T., Konzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415–421. Zhang, X., Yang, S., Zhou, Y., He, Z., Xia, X., 2006. Distribution of the Rht-B1b, Rht-D1b and Rht8 reduced height genes in autumn-sown Chinese wheats detected by molecular markers. Euphytica 152, 109–116.