Upland rice grown in soil-filled chambers and exposed to contrasting water-deficit regimes

Field Crops Research 76 (2002) 25±43

Upland rice grown in soil-®lled chambers and exposed to contrasting water-de®cit regimes II. Mapping quantitative trait loci for root morphology and distribution A.H. Pricea,*, K.A. Steeleb, B.J. Mooreb, R.G.W. Jonesb a

Department of Plant and Soil Science, University of Aberdeen, Aberdeen AB24 3UU, UK b Centre for Arid Zone Studies, University of Wales, Bangor, Gwynedd LL57 3UW, UK Accepted 16 January 2002

Abstract Root morphological characteristics are known to be important in the drought resistance of some rice (Oryza sativa L.) varieties. The identi®cation of quantitative trait loci (QTLs) associated with root morphology and other drought resistancerelated traits should help breeders produce more drought resistant varieties. Stability in the expression of root growth QTL across rooting environments is critical for their use in breeding programs. A greenhouse experiment in which a mapping population of 140 recombinant inbred lines and the parental varieties Bala and Azucena were grown in glass-sided soil chambers and evaluated for root growth and water uptake was conducted. In each of 2 years, two treatments were used; an early water-de®cit (WD0) in which seeds were sown into wet soil but received no more water, and a late water-de®cit (WD49) in which the plants were watered for 49 days and then received no water for a week. The major differences between treatments and years in dry matter partitioning and root growth traits are reported elsewhere. Here, the identi®cation of QTLs for root growth traits by composite interval mapping is described. At LOD > 3:2, there were six QTLs for the weight of roots below 90 cm and maximum root length, 11 for root to shoot ratio, 12 for the number of roots past 100 cm, and 14 for root thickness. A total of 24 regions were identi®ed as containing QTLs (these regions often contained several QTLs identi®ed for different root traits). Some were revealed only in individual experiments and/or for individual traits, while others were common to different traits or experiments. Seven QTLs, on chromosomes 1, 2, 4, 7, 9 (two QTLs) and 11, where considered particularly noteworthy. The complex results are discussed in the context of previously reported QTLs for root growth in other populations, the interaction between QTL with the environment and the value of QTLs for breeding. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Root growth; Drought resistance; Genotype  environment interaction; Soil moisture

1. Introduction Breeding rice varieties with drought resistance is a priority for much of the non-irrigated rice growing *

Corresponding author. Tel.: ‡44-1224-272690; fax: ‡44-1224-272703. E-mail address: [email protected] (A.H. Price).

regions of the world (which account for 43% of the world rice area). Several mechanisms of drought resistance are known to be exploited by rice varieties and the subject has been recently reviewed (Fukai and Cooper, 1995; Nguyen et al., 1997; Price and Courtois, 1999). Potentially, one of the most promising of traits for improving drought resistance is the possession of a

0378-4290/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 2 9 0 ( 0 2 ) 0 0 0 1 0 - 2

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A.H. Price et al. / Field Crops Research 76 (2002) 25±43

deep and thick root system that allows access to water at depth. While there is substantial variation in root system morphology within rice germplasm, the genetics are complex and profoundly in¯uenced by the environment which makes the use of traditional selection methods dif®cult. Rice scientists have begun using molecular genetics to link genes contributing to these polygenic traits to molecular markers with some success. Studies identifying quantitative trait loci (QTLs) for root morphorphological characteristics have been conducted (Champoux et al., 1995; Yadav et al., 1997; Price and Tomos, 1997), indicating the potential usefulness of this approach. To date, no studies have investigated the effect of varying the rooting environment on root growth QTLs. However, soil physical or chemical properties are known to in¯uence root growth. The theory propounded by Thornley (1972), which has more recently been expanded (e.g. by Dewar, 1993), describes source/sink relationships and shows how carbon is allocated proportionately to plant organs responsible for the acquisition of a limiting resource. Accordingly, growth of roots will be favoured over shoots under conditions of low soil nutrient availability. Under drought, however, the interaction between water-deficit, nutrient availability and photosynthesis, combined with spatial heterogeneity of soil water status makes the prediction of carbon allocation dif®cult. Yamauchi et al. (1996) has shown, using plants grown in thin chambers, that drought reduces root growth. This can be compared with evidence from ®eld-grown plants that drying soils can promote root growth measured as total root mass (O'Toole, 1982; Ingram et al., 1994) or rooting depth (Mambani and Lal, 1983). The contradictions probably re¯ect differences in spatial and chronological interactions between the plant and the soil, in which soil properties are important (e.g. exploitable soil volume, hydraulic conductivity, penetration resistance and water release characteristics). While differences in root systems are commonly observed between rice varieties (e.g. Price et al., 1997), there is only one report of varietal differences in root response to drying (Mambani and Lal, 1983). It must be concluded that the extent of varietal differences in the response of roots to altered soil water status and the underlying mechanism are poorly studied and understood.

A programme is being conducted aimed at identifying QTLs for root morphological characteristics that are of potential bene®t in breeding for more drought resistant rice varieties. Previously, hydroponic methods were employed to assess root growth and identify QTLs (Price et al., 1997). However, the impact of the rooting environment on root growth QTLs is not understood. This phenomenon can be investigated using soil-®lled chambers to control the rooting environment. In a companion paper (Price et al., 2002) a root screening system, and the profound changes in root distribution which occur under contrasting environmental conditions, were described. In this paper, we describe the identi®cation of QTLs associated with plant mass, root to shoot ratio, mass of roots below 90 cm, maximum visible root length, adventitious root thickness and the number of roots below 100 cm measured in those experiments. 2. Materials and methods A full description of the experimental procedure is given in the companion paper (Price et al., 2002). Here only a brief description is given in order to allow much of the signi®cance of the work to be appreciated without reference to the other publication. 2.1. Plant material A mapping population of 205 recombinant inbred (F6) seeds was produced from a cross of the variety Bala and Azucena (original parental seed obtained from the International Rice Research Institute) by single-seed descent as described in Price et al. (2000). 2.2. 1997 experiment of Bala, Azucena and the mapping population A total of 140 recombinant inbred lines and ®ve replicates of Azucena and Bala were grown in thin soil chambers under two treatment conditions: a longterm, early water-de®cit (WD0(97)) which started with fully saturated soil but received no more water, and a well-watered treatment in which water was withheld after day 49 (during the ®nal, eighth week of the experiment) (WD49(97)). The chambers were made by taping together two sheets of 4 mm thick

A.H. Price et al. / Field Crops Research 76 (2002) 25±43

27

glass (1:2 m  0:3 m width  length) 15 mm apart and ®lling the space with soil (sandy loam, pH 5.5). The chambers had a 5 mm diameter drainage hole at the bottom of each side. Before sowing, each chamber was saturated with Yoshida's nutrient solution, pH 5.5 (Yoshida et al., 1976). Five seeds were sown in each chamber. After seedling emergence, the seedlings were thinned to two per chamber. One set of chambers did not receive further water or nutrient (WD0). The other chambers were given 1.0 l of nutrient solution three times a week for the ®rst 4 weeks. Thereafter, they received 1.0 l nutrient every day, and after 6 weeks received 1.0 l of water in addition to 1.0 l of nutrient every day. After 7 weeks, no water or nutrients were applied, giving a late water-de®cit (WD49) treatment for the ®nal week. Sheets of expanded polystyrene were placed behind all stacks of chambers to prevent heat loss. Within each treatment, chambers were arranged randomly in stacks of six chambers, but the two treatments were positioned next to each other. Plants were grown for 8 weeks in a greenhouse (minimum temperature 25 8C) in Bangor during the summer of 1997, under daylight supplemented by 150 mmol m 2 s 1 PAR. Twice a week, the position of the chambers within a stack was rotated to remove position-within-stack effects. On a weekly basis and for both treatments, shoot growth was monitored as height of the plant (length from the soil to the tip of the longest leaf), while the length of the longest visible root and the number of roots past 25, 50, 75 and 100 cm was recorded. At the end of the experiment (after 8 weeks), shoots were removed in a single day. Over a 4-week period, each chamber was opened and soil and roots placed on a nail-board of 15 mm nails arranged 2 cm apart in a grid to allow the soil to be washed from the roots by water spray. A short section of three of the thickest roots was removed from each root system both near the base of the shoot and at 90 cm depth and placed in 50% ethanol. These sections were stored at 4 8C and subsequently used to measure root thickness. The washed root systems were separated into four depth classes: 0±30, 30±60, 60±90 and 90± 120 cm and dry weights were recorded.

early water-de®cit treatment (WD0(98)) and late water-de®cit treatment (WD49(98)) did not have small drainage holes at the bottom of the chambers. In the WD49(98) treatment the chambers received nutrient or water at a rate of only 800 ml per chamber.

2.3. 1998 experiment of the mapping population

2.5. Markers added to the molecular map

The experiment was repeated in 1998 with modi®cations in the drainage system of the chambers. Both

A total of six microsatellite markers (pre®xed RM) were screened on the mapping population using the

2.4. Data handling Data was tested for normality before statistical analysis. The means of the both the parents and the F6 in 1997, and only the F6 in 1998 are presented in Table 1. Since there was no replication for the F6 and both treatment and year had a signi®cant and interacting effect on most traits, it is not sensible to calculate broad-sense heritability based on F-values from analysis of variance. Broad-sense heritability was therefore calculated as 100  …1 the minus the ratio of the average variance of the non-segregating genotypes over the variance of the F6 population). These are presented in Table 2. In 1997, the nonsegregating genotypes were Azucena and Bala. In 1998, they were Bala and four other Indian upland varieties that were included in the experiment (data not shown). Total plant mass was the sum of shoot and root dry weight after harvest on day 56. Root to shoot ratio was the root dry weight divided by the shoot dry weight. Maximum root length was calculated as the average of measurements of the longest visible root on days 28 and 35. All traits were normally distributed except for dry weight of root below 90 cm in WD49(97) and WD49(98), root thickness at both base and at 90 cm for WD0(98) and root to shoot ratio in all treatments, for which the square root was used for QTL analysis. QTLs were calculated for individual experiments and for the average of the two treatment types and for the overall average, in order to identify regions with a broad environmental stability from those that are perhaps environment speci®c. For calculating average values (and for visible root lengths for each experiment) data were ®rst normalised by dividing the value for each individual by the population mean for that trait, before a mean was calculated.

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A.H. Price et al. / Field Crops Research 76 (2002) 25±43

Table 1 Mean values of whole plant and root growth traits for Azucena, Bala and F6 in 1997 and for F6 in 1998 under two treatment regimes, WD0 (drought commenced on day 0) and WD49 (drought commenced on day 49†  standard deviation Trait

WD0

WD49

Az97 …n ˆ 5† TDWa (g) R:SDWRb RDW90±120c (g) RT0d (mm) RT90e (mm) MRL28f (cm) MRL35g (cm) NR > 100 cm42h

5.20 1.39 0.58 0.64 0.75 96.6 107.0 15.2

       

Ba97 …n ˆ 5† 0.72 4.67  0.37 0.73  0.23 0.37  0.05 0.54  0.19 0.32  7.4 98.7  9.1 111.7  12.2 19.5 

F697 …n ˆ 133† 0.93 5.36  0.18 1.15  0.20 0.55  0.07 0.65  0.08 0.55  11.5 95.9  6.7 108.6  11.0 17.3 

F698 …n ˆ 115†

0.89 4.48  0.33 0.38  0.21 0.38  0.12 0.55  0.19 0.58  9.2 92.2  8.0 106.7  9.7 3.15 

Az97 …n ˆ 5†

1.57 20.5  0.21 0.40  0.23 0.65  0.16 1.19  0.19 1.00  9.1 88.0  9.8 104.7  1.56 12.7 

Ba97 …n ˆ 5† 1.0 0.05 0.11 0.06 0.08 12.3 4.7 2.9

14.2 0.30 0.18 0.97 0.56 83.0 97.0 11.6

       

3.0 0.06 0.15 0.09 0.12 14.1 10.3 7.0

F697 …n ˆ 133†

F698 …n ˆ 129†

17.9 0.36 0.36 1.08 0.74 84.8 101.5 11.7

20.0 0.49 0.49 1.06 0.93 75.4 99.9 3.57

       

4.1 0.08 0.23 0.14 0.18 12.9 9.2 6.5

       

4.2 0.23 0.23 0.14 0.20 10.6 10.9 1.43

a

Total dry weight at harvest (day 56). Root to shoot dry weight ratio. c Root dry weight between 90 and 120 cm. d Root thickness at base of plant. e Root thickness at 90 cm depth. f Maximum root length on day 28. g Maximum root length on day 35. h The number of roots past 100 cm depth at day 42. b

ampli®cation methodology described by Chen et al. (1997). PCR products were analysed on 3% high resolution agarose gels (SFR agarose, Anachem, UK) with ethidium bromide staining. These were added to the existing RFLP and AFLP map described by Price et al. (2000) using MapMaker 3.0 (Lander et al., 1987; Lincoln et al., 1992). Marker RM246 on chromosome 1 allowed two linkage groups to be Table 2 Broad-sense heritabilities (%) calculated as 1 the ratio of the average variance of the non-segregating genotypes over the variance of the F6 populationa Trait

TDW (g) R:SDWR RDW90±120 (g) RT0 (mm) RT90 (mm) MRL28 (cm) MRL35 (cm) NR > 100 cm42 a

WD0

WD49

1997

1998

1997

1998

15 23 0 74 64 0 0 0

70 69 35 61 57 51 51 0

73 53 67 70 68 0 17 21

31 90 18 31 71 31 47 35

In 1997, the non-segregating genotypes were Azucena and Bala. In 1998, they were Bala and four other Indian upland varieties that were included in the experiment.

joined. The inclusion of three microsatellite markers on chromosome 4 (RM252, RM348 and RM349) allowed one previously unlinked RFLP marker (C1016) to be added to the map and repositioned marker RG620, slightly changing the order of markers on chromosome 4 from that published previously in Price et al. (2000). Additional microsatellite markers RM234 on chromosome 7 and RM247 on chromosome 12 did not signi®cantly alter the map order. The result was a map with 102 RFLP, 34 AFLP and six microsatellite markers on 15 linkage groups with a total length of 1779 cm. There were major segregation distortions only at the bottom of chromosome 7 between RM234 and e12m36.12 in favour of Azucena alleles and at the top of chromosome 10 at marker RG257 in favour of Bala alleles. 2.6. QTL analysis The identi®cation of QTLs was conducted by composite interval mapping using the programme QTLCartographer version 1.15 (C.J. Basten, B.S. Weir and Z.-B. Zeng, Department of Statistics, North Carolina State University). Background markers for composite interval mapping were selected by `forward stepwise regression with backward elimination' using

A.H. Price et al. / Field Crops Research 76 (2002) 25±43

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Table 3 QTLs for traits associated with total dry weight revealed by composite interval mapping (average of experiments in both years is denoted by A after the treatment) Experiment in which trait was detected

Chromosome

WD49(97)

1 3 1 7 No QTL 1 1 4 8 9 11 1 10 9 1 9

WD49(98) WD0(97) WD0(98)

WD49A WD0A Average

QTL position in cM

LOD

R2 (%)

Additive effect

Donor of positive QTL

Above ( ) or below (‡) nearest marker

From the top of linkage group

C86 3 G164 2 C949 1 RG650 ‡ 2

199 211 220 83

5.3 4.1 7.3 3.3

18.1 11.7 19.3 8.4

1.92 1.49 1.92 1.22

A A A A

RM212 C949 RG449 ‡ 4 e18m43.4 G1085 C189 5 C949 4 C701 G1085 C949 9 G1085

179 222 37 33 111 92 219 1 111 213 111

4.2 5.2 4.9 4.1 7.3 6.7 7.6 3.2 5.1 3.5 3.7

8.9 10.9 14.1 7.9 16.0 21.3 21.1 6.9 12.5 9.9 7.6

0.29 0.34 0.35 0.26 0.38 0.44 0.096 0.056 0.053 0.043 0.037

A B B B A A A A A A A

the default threshold. The default window size of 10 cm was used. Permutation testing (using QTLCartographer) on some of this data indicated that a LOD score of 3.2 is suitable as the genome-wide 5% signi®cance threshold for this set of data. The results are

presented in Tables 3±8. They are also presented graphically on the linkage map (AFLPs are indicated but not labelled on maps unless they are referred to in the text) in Figs. 1±4 where the bar equals the one LOD con®dence interval and the maximum LOD

Table 4 QTLs for traits associated with root to shoot dry weight ratio revealed by composite interval mapping (average of experiments in both years is denoted by A after the treatment) Experiment in which trait was detected

Chromosome

WD49(97)

6 8 No QTL 1 2 4 9 1 1 9 8 9 9 8 9

WD49(98) WD0(97)

WD0(98) WD49A WD0A Average

LOD

R2 (%)

2 72

5.3 5.6

14.5 19.2

0.033 0.043

A B

154 20 92 55 191 221 65 33 63 113 90 55

5.5 5.2 3.3 3.2 3.3 3.2 5.5 3.5 4.8 3.8 3.3 5.4

8.9 9.2 6.7 5.2 14.7 8.3 28.0 8.2 18.0 8.8 7.6 10.6

0.103 0.090 0.089 0.077 0.068 0.063 0.092 0.064 0.145 0.106 0.063 0.070

A B A A A B A A A A B A

QTL position in cM Above ( ) or below (‡) nearest marker e12m36:18 ‡ 2 G187 4 G393 1 RG83 RG163 ‡ 4 G385 C86 11 C949 G385 ‡ 10 G187 2 G385 ‡ 8 G1085 ‡ 2 R202 1 G385

From the top of linkage group

Additive effect

Donor of positive QTL

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A.H. Price et al. / Field Crops Research 76 (2002) 25±43

Table 5 QTLs for traits associated with dry weight of roots below 90 cm revealed by composite interval mapping (average of experiments in both years is denoted by A after the treatment) Experiment in which trait was detected

Chromosome

WD49(97)

1 7 9 2 4 No QTL 8 1 No QTL No QTL

WD49(98) WD0(97) WD0(98) WD49A WD0A Average

QTL position in cM

LOD

R2 (%)

Additive effect

Donor of positive QTL

Above ( ) or below (‡) nearest marker

From the top of linkage group

C86 ‡ 4 C451 6 G1085 C601 C513 7

206 32 111 159 63

4.6 4.2 5.8 3.9 3.2

12.8 13.5 13.1 10.8 12.3

0.081 0.081 0.083 0.087 0.079

A A A A A

R2676 C949 3

78 219

4.3 4.6

16.0 11.8

0.089 0.168

B A

Table 6 QTLs for traits associated with root thickness revealed by composite interval mapping Experiment in which trait was detected Thickness at base WD49(97) WD49(98) WD0(97) WD0(98)

Thickness at 90 cm WD49(97)

WD49(98) WD0(97) WD0(98)

Average

Chromosome

QTL position in cM

LOD

R2 (%)

Additive effect

Donor of positive QTL

Above ( ) or below (‡) nearest marker

From the top of linkage group

6 4 4 1 3 9 12

e12m37.7 C513 RM348 ‡ 2 RM212 8 RG191 C506 4 G124

34 70 142 219 41 133 24

7.0 3.6 3.7 3.2 3.3 3.5 4.7

18.2 7.7 11.3 9.4 7.0 10.0 11.3

0.063 0.042 0.038 0.048 0.040 0.051 0.052

A A A B A A B

1 9 10 11 2 9 9 9 6 8 9 9

C86 G1085 C701 C189 7 C601 6 G385 ‡ 6 G385 ‡ 8 G1085 R2654 2 R2676 G385 ‡ 2 G1085 1

203 111 1 90 155 61 63 111 8 78 57 110

3.2 5.0 3.3 3.8 5.0 4.9 5.0 6.9 6.2 3.6 4.3 3.8

7.2 11.5 7.5 11.9 15.8 18.1 18.1 14.6 18.3 10.4 13.0 10.9

0.051 0.063 0.051 0.064 0.083 0.088 0.082 0.076 0.080 0.061 0.072 0.054

A A B B A A A A A B A A

1 4 6 7 9 9 12

G393 RM348 e12m36:18 ‡ 2 C507 G385 G1085 e12m37.13

154 140 2 116 55 111 16

4.4 6.9 4.0 4.7 7.2 9.7 7.7

5.4 8.9 6.0 10.9 9.2 13.7 10.3

0.034 0.045 0.036 0.088 0.045 0.055 0.048

A A A A A A B

A.H. Price et al. / Field Crops Research 76 (2002) 25±43

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Table 7 QTLs for traits associated with maximum root length revealed by composite interval mapping (average of experiments in both years is denoted by A after the treatment) Experiment in which trait was detected

Chromosome

WD49(97)

9 10 No QTL 8 9 9 2 9 2 4 7 9

WD49(98) WD0(97) WD0(98) WD49A WD0A Average

QTL position in cM

LOD

R2 (%)

Additive effect

Donor of positive QTL

Above ( ) or below (‡) nearest marker

From the top of linkage group

e12m36.13 C701

108 1

3.5 3.9

9.7 9.0

0.036 0.034

A B

RG598 6 G1085 ‡ 2 G1085 ‡ 4 C601 6 G1085 C601 ‡ 6 RG190 3 RG650 3 G1085 ‡ 2

105 113 115 153 111 165 12 76 113

3.4 3.9 4.4 3.7 6.4 4.3 4.5 3.9 5.4

13.1 12.9 11.2 15.5 17.4 17.0 11.1 11.0 13.0

0.027 0.033 0.034 0.027 0.030 0.031 0.025 0.025 0.028

B A A A A A B A A

Table 8 QTLs for traits associated with number of roots past 100 cm at day 42 revealed by composite interval mapping (average of experiments in both years is denoted by A after the treatment) Experiment in which trait was detected

Chromosome

WD49(97)

1 3 5 1 No QTL 3 6 7 8 9 11 1 5 8 1 2 2 6 11 12 5 8

WD49(98) WD0(97) WD0(98)

WD49A WD0A

Average

QTL position in cM

LOD

R2 (%)

Additive effect

Donor of positive QTL

Above ( ) or below (‡) nearest marker

From the top of linkage group

C949 7 RG409 RG346 RZ14 ‡ 16

215 29 4 238

3.8 3.9 3.4 3.2

11.9 8.9 7.7 22.8

2.4 2.1 1.8 2.0

A B A A

G164 R2654 ‡ 4 RG650 e18m43:4 ‡ 2 G1085 1 RG2 C949 5 RG346 R902 ‡ 8 C949 1 e18m43:8 ‡ 2 C601 ‡ 12 R2654 RG2 ‡ 4 RM247 RG346 e12m36:7 7

213 12 81 35 110 76 217 4 8 221 117 171 8 80 20 4 8

3.2 4.5 3.5 3.3 5.9 3.5 5.2 3.4 3.3 3.8 4.4 4.5 4.1 4.6 4.1 3.9 3.9

6.4 11.6 6.6 9.1 11.6 6.6 17.6 8.5 13.9 6.9 9.9 16.4 6.8 11.0 6.6 9.3 14.1

0.051 0.065 0.055 0.059 0.079 0.054 0.217 0.140 0.182 0.126 0.168 0.169 0.112 0.150 0.111 0.112 0.137

B A A B A A A A A B B A A A A A A

32 A.H. Price et al. / Field Crops Research 76 (2002) 25±43 Fig. 1. Molecular linkage map with RFLP and microsatellite markers indicated (AFLP marker names omitted unless mentioned in the text) showing QTLs for plant mass in each individual experiment, the calculated average of the two types of treatment and the calculated overall average. The boxes represent the 1 and ‡1 LOD interval and the number above or below the box indicates the LOD score for the QTL. A positive LOD value indicates that Azucena alleles increase the value of the trait. The dashed circles indicate regions containing QTLs above the LOD 3.2 threshold.

A.H. Price et al. / Field Crops Research 76 (2002) 25±43 Fig. 2. Molecular linkage map (without marker names) showing QTLs for root:shoot dry weight ratio (above) and dry weight of root below 90 cm (below) (see Fig. 1 for full explanation of graphic). The dashed circles indicate regions containing QTLs above the LOD 3.2 threshold and each is marked with the QTL code used in Table 9. 33

34 A.H. Price et al. / Field Crops Research 76 (2002) 25±43 Fig. 3. Molecular linkage map (without marker names) showing QTLs for root thickness at 90 cm, at the base of the plant and the calculated overall average (above) and for maximum visible root length averaged after normalisation from observation on days 28 and 35 (below) (see Fig. 1 for full explanation of graphic). The dashed circles indicate regions containing QTLs above the LOD 3.2 threshold and each is marked with the QTL code used in Table 9.

A.H. Price et al. / Field Crops Research 76 (2002) 25±43 Fig. 4. Molecular linkage map (without marker names) showing QTLs for the number of roots visible passed 100 cm measured on day 42 (see Fig. 1 for full explanation of graphic). The dashed circles indicates regions containing QTLs above the LOD 3.2 threshold and each is marked with the QTL code used in Table 9.

35

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A.H. Price et al. / Field Crops Research 76 (2002) 25±43

Table 9 Summary of genomic regions containing root morphology QTL of LOD  3:2 indicating the traits for which QTLs were detected, the donor of the positive allele and the populations in which additional evidence exists for QTL effecting root growth in the same region (see below for references)a QTL code (chromosome and position)

Nearest marker(s)

Trait and experiment in which a QTL was detected (putative QTLs of LOD 2.5±3.1 in italics)

Donor of ‡ ve allele The populations in which other root growth QTLs have been identified (with trait in italics)

1-1

G393±R2417

1-2

RM212±C86

1-3

C86±RZ14

2-1 2-2

RG509±RG83 C601

3-1

RG409

4-1

RG190

RSR; WD097 RT; OA MRL; WD4998 RSR; WD098 RT; WD098(90) RSR; WD098, WD49A DRW; WD4997, WD49A RT; WD098(B), WD4997(90) MRL; WD098, WD4997 NR100; WD098, WD0A, WD4997, WD4998, WD49A RSR; WD097 RSR; WD4998 DRW; WD4998, WD49A RT; WD4998(90) MRL; WD098, WD0A, OA NR100; WD0A RT; WD098(B) NR100; WD4997, WD49A RT; OA MRL; WD097, WD0A, OA

A A B A A BA AA BA BA BBAA A B A A A A A A B B B

4-2

RG449±C513

4-3

RG163

4-4

RM349±RM348

DRW; WD4998 RT; WD098(90), WD4998(B) NR100; WD0A RSR; WD097 DRW; WD49A RT; WD097(B), WD4997(B), OA

A BA B A A A

5-1

RG119±RG346

6-1

R2654±RZ682

7-1 7-2 7-3

G338 G20±C451 RG650±RM234

8-1

R902±G1010

8-2

C225±G2132

8-3

G187

DRW; WD49A MRL; WD4997, WD49A NR100; WD0A, WD4997, WD49A, OA RSR; WD4997, WD49A RT; WD098(90), WD4997(B), WD4998(B), OA NR100; WD098, WD0A RSR; WD4998 DRW; WD4997 RT; OA MRL; OA NR100; WD098 DRW; WD4997 NR100; WD4998, WD49A, OA RSR; WD49A NR100; WD098 RSR; WD4997, WD49A, DRW; WD098 RT; WD098(90)

A A A A A A B A A A A A A A B B B B

Co  Mo RT IR64  Az DRW MRL RT IR58:  IR52: RP None IR64  Az DRW RT CT  IR62: RPF

IR58:  IR52: RP(L) Ba  Az RP IR64  Az MRL? Co  Mo MRL RP CT  IR62: RP IR58:  IR52: RP IR64  Az DRW RP RT IR64  Az DRW CT  IR62. RP? Co  Mo RSR Co  Mo DRW IR64  Az RP(T) IR64  Az RP(T) Co  Mo RSR RT RP CT  IR62: RPF RP None CT  IR62: RD IR58:  IR52: RP(T) None None IR64  Az DRW MRL CT  IR62: RD IR64  Az RP None Co  Mo RSR RT

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Table 9 (Continued ) QTL code (chromosome and position)

Nearest marker(s)

Trait and experiment in which a QTL was detected (putative QTLs of LOD 2.5±3.1 in italics)

Donor of ‡ ve allele The populations in which other root growth QTLs have been identified (with trait in italics)

8-4

R202±RG598

9-1

G385

9-2

G1085

RSR; WD097, OA MRL; WD097 RSR; WD097, WD098, WD0A, OA RT; WD097(90), WD098(90), WD4998(90), OA RSR; WD0A, WD097 DRW; WD098, WD4997, OA RT; WD097(90), WD098…B ‡ 90†, WD4997(90), WD4998(90), OA MRL; WD098, WD0A, WD4997, WD49A, OA NR100; WD098 RT; WD4997(90), OA MRL; WD4997 RT; WD4997…B ‡ 90† NR100; WD098, WD0A

B B A A A A A A A B B B A

DRW; WD097, WD0A RT; WD097(90), WD098(B), OA NR100; WD0A

B B B

10-1

C701

11-1

C189

12-1

CD0127±RM247

IR64  Az MRL RT Co  Mo RT IR64  Az DRW IR  Az DRW MRL RP(T) Co  Mo MRL RSR RT

Co  Mo RSR, RT IR58:  IR52: RP RP(T) Ba  Az RP Co  Mo MRL RSR RT RP CT  IR62: RPF? None

a OA: overall average; RSR: root to shoot ratio; DRW: deep root weight; MRL: maximum root length; RT: root thickness; NR100: number of roots past 100 cm; RPF: root pulling force; RP: root penetration; RP(T): thickness of penetrated roots; RP(L): length of penetrated roots; RD: rooting depth. Ba  Az: Bala  Azucena; reference Price et al. (2000). IR  Az: IR64  Azucena; references Yadav et al. (1997), Hemamalini et al. (2000) and Zheng et al. (2000) (RP only). Co  Mo: Co39  Moroberekan, references Champoux et al. (1995) and Ray et al. (1996) (RP only). CT  IR62.: CT9993-5-10-1-M  IR62266-42-6-2, reference Zhang et al. (1999); IR58:  IR52:: IR58821-23-B-1-21  IR5261-UBN-1-1-2, reference Ali et al. (2000); ?: indicates signi®cant uncertainty in comparative mapping position of the QTLs.

value is indicated above or below the bar. The sign of the LOD score indicates the effect of the Azucena allele (i.e. if the LOD value is positive, the Azucena allele increased the trait value). Putative QTLs revealed at LOD 2.5±3.1 are also shown in the ®gures because they could be true QTLs. These are not listed in Tables 3±8, and are only discussed in the text and listed in Table 9 if they cluster with other QTLs adding support to their validity. 3. Results 3.1. Plant mass Table 1 shows that plant mass was much reduced in the WD0 treatment compared to the WD49 treatment, that in 1997 Azucena was larger than Bala in both treatments and that the F6 was slightly different

between years in both treatments. Broad-sense heritability varied from low to high (Table 2). Results of composite interval mapping are presented in Table 3 and Fig. 1. A total of eight regions contained QTLs of LOD  3:2, all of which represent positive effects of Azucena in the WD49 treatment, but some of which represent positive effects from Bala in the WD0 treatment. On chromosome 1, there were two close QTLs of opposing effects. Between markers C86 and C949, a number of QTLs for plant mass were clustered which were substantial in effect only under the WD49 treatment (R2 values of 21% for the WD49average) where Azucena alleles increase plant mass. There was a large negative effect QTL in the same place for WD0(98) suggesting that at this locus the Azucena alleles have a large positive effect on plant size only under well-watered conditions. Nevertheless, there is a signi®cant QTL effect for overall average plant size (averaged over all experiments)

38

A.H. Price et al. / Field Crops Research 76 (2002) 25±43

presumable due to the magnitude of the effect under WD49 treatments. Between 20 and 30 cm above this, at marker RM212, an opposing QTL was detected in which Azucena alleles increased plant size in WD0(98) and putatively decreased plant size in WD49(98). There was a region on the bottom of chromosome 9 at G1085 with QTLs revealed only under the WD0 treatments. In addition, there were QTLs detected only in one treatment or just the treatment average; near marker G164 on chromosome 3 for WD49(97), at marker C701 for WD49-average and at markers RG449 (on chromosome 4), e18m43.4 (on chromosome 8) and C189 (on chromosome 11) for WD0(98).

Broad-sense heritability ranged from zero to moderately high (Table 2). The results of QTL-mapping of the root mass below 90 cm are presented in Table 5 and Fig. 2. A total of six regions contained QTLs detected with a LOD threshold of 3.2, with only one region representing a positive effect from Bala alleles. All but one of these QTLs were detected only in single experiments. A QTL between markers C86 and C949 on chromosome 1 was detected in WD49(97) and WD49-average. 3.4. Root thickness

Root to shoot ratios where higher for Azucena than for Bala in 1997 and showed considerable variation between years for the WD0 treatment (Table 1). Heritability was moderate to high (Table 2). The result of QTL-mapping root to shoot dry weight ratio are presented in Table 4 and Fig. 2. There were 11 regions identi®ed with QTLs of LOD  3:2 of which seven represent a positive effect of the Azucena allele. The region of chromosome 1 between RM212 and C949 had QTLs of opposing effect for WD0(98) which matched those for plant dry weight. A large proportion of chromosome 9 affected root to shoot ratio in the WD0 treated plants. Two loci relatively close to each other with similar effect were suggested, with the upper QTL appearing to have a larger effect. That was centred on marker G385 and revealed QTLs in WD0(97), WD0(98) WD0-average and overall average while the lower QTL, centred on G1085 was revealed only for WD0-average. On chromosome 8 a strong QTL was identi®ed in WD49(97) (which was also revealed in the WD49average) which was close to, but not overlapping with, a QTL detected in the overall average (which matched a putative QTL for WD0(97)). These probably represent two distinct QTLs on chromosome 8. Five other QTLs were detected only in individual experiments and one for only the WD49-average.

Root thickness, at both the base of the plant and at 90 cm depth in the soil, was higher in the WD49 treatment than the WD0, and in 1997 it was higher in Azucena than Bala (Table 1). The broad-sense heritability was consistently moderate to high (Table 2). The results of QTL-mapping root thickness are presented in Table 6 and Fig. 3. There were 14 regions containing root thickness QTLs above the threshold level, most of which represent positive effects of the Azucena allele. More QTL were detected for root thickness at depth than at the base of the plant. On chromosome 9 there were QTL clusters centred on both markers G385 and G1085. At G385 QTLs were detected only for thickness at depth, and these were revealed for all individual experiments except WD49(97). At marker G1085, QTLs for thickness at depth were revealed for all but the WD49(98) treatment and QTLs were also revealed for thickness at the base for the WD0(98) treatment. Several QTLs with high LOD scores were detected for the overall average root thickness (averaged for all treatments at both depths). LOD scores of 7.2 and 9.7 were obtained at markers G385 and G1085 on chromosome 9, respectively, of 7.7 at marker e12m37.13 on chromosome 12 and of 6.9 at marker RM348 on chromosome 4. On chromosome 6, strong QTLs were detected in individual experiments, for root thickness at 90 cm in WD0(98) at marker R2654 (LOD of 6.2) and for thickness at the base in WD49(97) at marker e12m37.7 (LOD 7.0).

3.3. Root mass below 90 cm

3.5. Maximum root length

Root mass below 90 cm was highly variable but tended to be higher in Azucena than Bala (Table 1).

Maximum root length after 28 and 35 days was slightly higher in the WD0 treatments than the WD49

3.2. Root to shoot ratio

A.H. Price et al. / Field Crops Research 76 (2002) 25±43

treatments, but not greatly different between Azucena and Bala in 1997 (Table 1). Broad-sense heritability was zero to moderate (Table 2). Results for mapping maximum visible root length (MRL, calculated from the normalised average of measurements taken at 28 days and at 35 days) are presented in Table 7 and displayed in Fig. 3. A total of six regions contained QTLs, three of which represented a positive effect from the Bala alleles. On chromosome 9 at marker G1085 there was a cluster of QTLs, for two of the individual experiments and all three of the averages. Three QTLs were only detected for averages; WD0-average and overall average at marker C601 on chromosome 2, and only overall average at marker RG190 on chromosome 4 and marker RG650 on chromosome 7. 3.6. Number of roots past 100 cm at day 42 The number of visible roots past 100 cm at day 42 was much lower in 1998 compared to 1997 but was highly variable in both years (Table 1) and hence had zero to low broad-sense heritabilities (Table 2). Nonetheless, a total of 12 regions contained QTLs (Table 8 and Fig. 4). Only two of these contained QTLs for the overall average; on chromosome 5 between makers RG119 and RG346 a QTL for overall average was associated with QTLs for WD49(97) and WD49average and on chromosome 8 between markers R902 and G1010 a QTL for overall average was associated with a QTL for WD49-average. On chromosome 1 at marker C949, QTLs were detected for WD49(97), WD49(98), WD49-average and WD0average in which Azucena alleles had a positive effect in WD49 treatments but a negative effect in WD0 treatments. 4. Discussion This paper identi®es a large number of QTLs for general plant growth and root morphology. For each trait there were often many QTLs and no major genes, con®rming that the traits are multigenic. Different experiments gave different patterns of QTLs indicative of the phenomenon of QTL by environment interaction. Thus the genetic control of root growth is complex. By comparing the maps presented here together,

39

it is possible to conclude that a total of 24 regions signi®cantly affect aspects of root morphology, either in one or several environments. These regions, or QTL, are summarised in Table 9. Also included in the table is evidence of these regions affecting root morphology in other populations from published studies. Comparative mapping was achieved through the alignment of extensive published maps of Kurata et al. (1994) and Causse et al. (1995) and maps on the web site of Oryzabase (http://shigen.lab.nig.ac.jp/rice/oryzabase/). Comparative mapping alignments can be subject to error due to differences in genetic distance between markers in some genomic regions of different populations. Of the 24 regions, 12 represented a positive effect from Azucena, eight from Bala and three had positive effects from both parents, dependent on either the trait (QTL 8-2) or the environment (QTLs 1-3 and 4-2). Thus for QTL 8-2, Azucena alleles increased the root to shoot ratio but decreased the number of roots past 100 cm. For QTL 1-3, the Azucena allele increased deep root weight, root thickness and number of roots past 100 cm in the WD49 treatment, but decreased root to shoot ratio, root thickness and roots past 100 cm in the WD0 treatment. Likewise, for QTL 4-2, in 1998 the Azucena allele increased root thickness in WD49 but decreased it in WD0. 4.1. The most notable QTLs The results on QTLs presented here were compared with ®ndings published from other populations. A total of seven are considered for further discussion here because the evidence from different studies (with the exception of chromosome 1) suggests that the QTLs could be of potential value in breeding for root traits. These QTL are discussed below. The QTL at the bottom of chromosome 1 (QTL 1-3) affected total plant size, mass of roots at depth and number of roots past 100 cm. Under well-watered conditions (WD49), the Azucena allele had a positive effect. This is a region in which the sd-1 semidwar®ng locus maps (Huang et al., 1996) and which has been repeatedly identi®ed as a QTL (not a major gene) for plant height and plant size in this population, since Bala carries the sd-1 locus. Under the WD0 treatment, the Azucena allele in this region had a negative effect on root morphology. This region (between RG690 and RZ801) was shown to affect total root weight and deep

40

A.H. Price et al. / Field Crops Research 76 (2002) 25±43

root weight in a double haploid population derived from a cross of IR64 with Azcuena (IR64  Az) grown in long tubes containing soil (Yadav et al., 1997). Zhang et al. (1999) showed that the same region (between CDO345 and RZ909) contained QTLs for root pulling force (a ®eld-based method of assessing total root strength) in a cross of CT9993-5-10-1M  IR62266-42-6-2 (CT  IR62:). The QTL near the bottom of chromosome 2 (QTL 2-2) was detected for several traits. It is the location of a large QTL for root penetration ability in this population (Price et al., 2000). This root penetration QTL has also been identi®ed in three of the four other populations in which penetration ability has been studied (between markers RG73 and RG324 in Co39  Moroberekan (Co  Mo), Ray et al. (1996); between markers RG188 and RG95 in IR58821-23-B1-2-1  IR5261-UBN-1-1-2 (IR58:  IR52:), Ali et al. (2000); at marker C1408 in CT  IR62:, Zhang et al. (1999)). Champoux et al. (1995) using the Co  Mo population grown in soil-®lled tubes identi®ed a QTL for maximum root length at marker RG324 which is probably in the same location, while Yadav et al. (1997) also reported a QTL for maximum root length here (between markers Pall and RZ58) in IR64  Az. It is surprising, therefore that this QTL was detected relatively infrequently in this study. Near the bottom of chromosome 4 (QTL 4-4), QTLs for root thickness at the base of the plant were detected in both 1997 experiments and for overall average root thickness. In this location QTLs for root penetration have been detected in Co  Mo near marker RG214 (Ray et al., 1996) and in CT  IR62: at marker RG476 (Zhang et al., 1999). QTLs have also been identi®ed near here for the thickness of penetrated roots in IR64  Az between the markers RG163 and RZ590 (Zheng et al., 2000), root to shoot ratio and root thickness in Co  Mo at marker RG214 (Champoux et al., 1995) and for root pulling force in CT  IR62: at marker RG476 (Zhang et al., 1999). In the middle of chromosome 7 (QTL 7-3) a small number of weak QTLs for total plant mass, number of roots past 100 cm at day 42 and maximum root length were detected. In a similar location, at marker RZ337B, Yadav et al. (1997) reported QTLs for total root weight, deep root weight and maximum root length. Just below marker RG650, Zhang et al. (1999) reported a QTL for rooting depth in the

CT  IR population, although there was no description of how it was assessed. In the middle of chromosome 9 is a region (QTL 9-1) were QTLs for overall average for root thickness and root to shoot ratio were detected, accounting for 9.2 and 10.6% of the phenotypic variation for each, respectively. This region has previously been identi®ed as being associated with QTLs for root morphology. Yadav et al. (1997) identi®ed QTLs associated with total root weight and deep root weight at marker RZ206 which is probably near G385. Likewise Champoux et al. (1995) identi®ed QTLs associated with root thickness, root:shoot ratio and root dry weight per tiller also at RZ206. Below this on chromosome 9 is a region (QTL 9-2) which appeared to have a substantial effect on root growth. It affected maximum root length and root thickness under most conditions. Indeed, this QTL accounted for about 13% of the phenotypic variation for both traits in overall average. This region also appeared to in¯uence whole plant mass and root to shoot ratio, but only under the prolonged drought (WD0) treatment, accounting for 12.5 and 8.8% of the WD0-average for total plant mass and root to shoot ratio, respectively. This is probably the same region already identi®ed as affecting root morphology associated with RFLP marker RZ12 in two other populations. QTLs associated with root thickness, root/shoot ratio and root dry weight per tiller below 30 cm at this marker were identi®ed in Co  Mo (Champoux et al., 1995). At the same marker, Yadav et al. (1997) identi®ed QTLs for deep root weight and maximum root thickness in IR64  Az. Using the IR64  Az population grown in soil-containing tubes in the ®eld, Hemamalini et al. (2000), identi®ed a QTL for root thickness in a similar location (in this case the LOD score was below 2, but R2 was 12.7%). Since this region affects root morphology under most conditions and had a positive effect on shoot biomass under prolonged water-de®cit, it may well represent a very useful region for marker assisted improvement of rice for drought prone environments. Finally, in the lower portion of chromosome 11 (QTL 11-1) is a region which was not strongly detected here but has been shown to affect maximum root length of the F2 of this population grown in hydroponics (Price and Tomos, 1997) and root penetration ability (Price et al., 2000). The contrasting

A.H. Price et al. / Field Crops Research 76 (2002) 25±43

magnitude of the QTL detected in hydroponics by Price and Tomos (1997), where R2 was 29%, and that detected here indicates a substantial degree of QTL  environment interaction. Whether the cause is the root or the shoot environment is not clear as yet, since unpublished data on hydroponic screening under relatively low light intensities (250 mmol m 2 s 1 PAR) has also failed to identify this QTL in the F6 population. However, same region is identi®ed in three other populations. In Co  Mo the marker CDO365 has been associated with root to shoot ratio, maximum root length, root thickness (Champoux et al., 1995) and root penetration ability (Ray et al., 1996). This is the location of a QTL for the length of penetrated roots at marker RG103 in IR58:  IR52 (Ali et al., 2000) and may be the same as a QTL for root pulling force in CT  IR62: just above CDO365 (Zhang et al., 1999). It is interesting to note that a QTL affecting root growth has never been observed here in IR64  Az, which may be due to Azucena and IR64 both having the same allele. 4.2. QTL  environment interaction It is known that root growth responds to environmental conditions (both above and below ground). In the companion paper, Price et al. (2002) demonstrated that the long-term water-de®cit (WD0) treatment used here stimulated the depth of rooting and had a effect on the distribution of roots within these arti®cial soil pro®les. It is debatable whether the ideal root morphological traits required for drought resistance are the ability to grow a deep root system before drought begins, or the ability to grow a deep root system in response to drying soils. The former may be costly in terms of carbon resource if drought should fail to occur. The latter could be compromised in soils prone to hardening when dry, since the rooting front may fail to keep pace with the front of deepening soil hardness. It is not known whether genes which contribute to deep rooting under well-watered conditions also contribute in drier soils, or whether there are genes speci®c to drought induced root growth. The results presented here indicate that some QTLs contribute to rooting at depth under both types of treatment (e.g. QTL 9-2) while others contribute only in one type. For example, at QTL 1-3, Azucena alleles acted to increase root length only under the well-watered

41

conditions, before late water-de®cit began, but under long-term water-de®cit, Bala alleles at this locus actually increased the number of roots past 100 cm. Another example is that of QTL 11-1, where QTLs for root length were active only in the early, long-term water-de®cit. This suggests that constitutive and drought-speci®c genes may both contribute to rooting depth under drought and that there is a considerable amount of QTL  environment interaction. While this is a preliminary investigation into the environmental stability of root morphology QTLs, it clearly demonstrates that some of these QTLs will be expressed only under speci®c environmental conditions. It must be emphasised that, although this experiment is not representative of root growth in the ®eld, it does illustrate the need to investigate further the impact of environmental variables on root growth QTLs. In subsequent investigations it will be important to test other variables that are known to effect root distribution (e.g. nutrient distribution) under controlled conditions more representative of the ®eld environment. 4.3. QTLs valuable in marker assisted selection In this and previous studies several regions of the rice genome have been identi®ed as potentially valuable for the transfer of QTLs for root morphological traits into drought susceptible varieties by marker assisted selection (MAS). Work is currently underway to transfer of four of these QTLs from Azucena into a popular Indian variety, Kalinga III. The regions chosen as targets for transfer by MAS are regions on chromosome 2 (centred on marker C601, i.e. QTL 2-2), chromosome 7 (centred on marker RG650, i.e. QTL 7-3), chromosome 9 (centred on G1085, i.e. QTL 9-2) and chromosome 11 (centred on C189, i.e. QTL 11-1). In this study, QTLs for root morphology have been detected at all these target regions, although the region on chromosome 11 was only revealed under the longterm water-de®cit treatment. These MAS-derived lines, and other near isogenic lines containing noteworthy QTLs, will be screened in laboratory and ®eld experiments. The effect that these QTLs have on yield under diverse environments can then be assessed. It will also be important to assess the effect they have on other traits via linkage drag and pleiotropy. Further analysis of these near isogenic lines may lead to

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A.H. Price et al. / Field Crops Research 76 (2002) 25±43

increased understanding of the physiological function of root morphology QTLs, and could enable the individual genes and/or gene products to be identi®ed. Acknowledgements This document is an output from a project (Plant Sciences Research Programme R6673) funded by the UK Department for International Development (DFID) and administered by the Centre for Arid Zone Studies (CAZS) for the bene®t of developing countries. The views expressed are not necessarily those of DFID. All experiments presented here comply with current UK law. The technical assistance of Gwen Edwards and Alison McIntosh for microsatellite screening is gratefully acknowledged. References Ali, M.L., Pathan, M.S., Zhang, J., Bai, G., Sarkarung, S., Nguyen, H.T., 2000. Mapping QTL for root traits in a recombinant inbred population from two indica ecotypes in rice. Theor. Appl. Genet. 101, 756±766. Causse, M., Fulton, T.M., Cho, Y.G., Ahn, S.N., Chunwongse, J., Wu, K., Xiao, J., Yu, Z., Ronald, P.C., Harrington, S.B., Second, G.A., McCouch, S.R., Tanksley, S.D., 1995. Saturated molecular map of the rice genome based on an inter speci®c backcross population. Genetics 138, 1251±1274. Champoux, M.C., Wang, G., Sarkarung, S., Mackill, D.J., O'Toole, J.C., Huang, N., McCouch, S.R., 1995. Locating genes associated with root morphology and drought avoidance in rice via linkage to molecular markers. Theor. Appl. Genet. 90, 969±981. Chen, X., Temnykh, S., Xu, Y., Cho, Y.G., McCouch, S.R., 1997. Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.). Theor. Appl. Genet. 95, 553±567. Dewar, R.C., 1993. A root shoot partitioning model-based on carbon±nitrogen±water interactions and Munch phloem ¯ow. Funct. Ecol. 7, 356±368. Fukai, S., Cooper, M., 1995. Development of drought-resistant cultivars using physio-morphological traits in rice. Field Crop Res. 40, 67±86. Hemamalini, G.S., Shashidar, H.E., Hittalmani, S., 2000. Molecular marker assisted tagging of morphological and physiological traits under two contrasting moisture regimes at peak vegetative stage in rice (Oryza sativa L.). Euphytica 112, 69±78. Huang, N., Courtois, B., Khush, G.S., Lin, H., Wang, G., Wu, P., Zheng, K., 1996. Association of quantitative trait loci for plant height with major dwar®ng genes in rice. Heredity 77, 130±137.

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