Theor Appl Genet (1996) 93:1211 1217

C. L u 9 L. S h e n

9 Z. Tan

9 Y. X u

9 Springer-Verlag 1996

9 P. He

9 Y . Chert

L. Z h u

Comparative mapping of QTLs for agronomic traits of rice across environments using a doubled haploid population

Received: 10 January 1996 / Accepted: 23 February 1996

We report here the RFLP mapping of quantitative triat loci (QTLs) that affect some important agronomic traits in cultivated rice. An anther culturederived doubled haploid (DH) population was established from a cross between an indica and a japonica rice variety. On the basis of this population a molecular linkage map comprising 137 markers was constructed that covered the rice genome at intervals of 14.8cM on average. Interval mapping of the linkage map was used to locate QTLs for such important agronomic traits as heading date, plant height, number of spikelets per panicle, number of grains per panicle, 1000-grain weight and percentage of seed set. Evidence of genotype-byenvironment interaction was found by comparing QTL maps of the same population grown in three diverse environments. A total of 22 QTLs for six agronomic traits were detected that were significant in at least one environment, but only 7 were significant in all three environments, 7 were significant in two environments and 8 could only be detected in a single environment. However, QTL-by-environment interaction was traitdependent. QTLs for spikelets and grains per panicle were common across environments, while traits like heading date and plant height were more sensitive to environment.

"Abstract

Key words Doubled haploid population 9 Quantitative trait loci (QTLs) 9 Molecular map 9 Rice 9 G x E interaction Communicated by G. Wenzel C. Lu 9 L. Shen 9 P. He 9 Y. Chen 9 L. Zhu ( 9 ) Plant Biotechnology Laboratory, Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, China Z. Tan The Rice Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan 610013, China Y. Xu Department of Agronomy, Zhejiang Agricultural University, Hangzhou 310029, China

Introduction Quantitative genetic studies have been facilitated by the development of molecular markers (Paterson et al. 1988; Stuber 1992). High-density molecular linkage maps permit one to locate quantitative trait loci (QTLs) by linkage analysis using segregating populations (Paterson et al. 1988; Lander and Bostein 1989). Most of the mapping studies have used F 2 or backcross populations, and these studies are difficult to replicate to obtain accurate phenotypic values for precise QTL mapping. The use of recombinant inbred lines (RILs) provides many advantages in QTL studies, but it will take a long time to develop such populations (Burr et al. 1988). Recently, many studies have employed doubled haploid (DH) populations to construct genetic maps and locate QTLs (Heun 1992; Barua et al. 1993; Backers et al. 1995; Lefebvre et al. 1995; Toroser et al. 1995; Uzunova et al. 1995). In contrast to RILs, DH lines derived from anther culture can reach homozygosity through a single generation. Because the lines are genetically homozygous, they can be propagated without further segregation. This characteristic allows for the precise measurement of quantitative traits by repeated trails and for a reduction in the environmental component of the total phenotypic variance. Many studies of QTL mapping have been conducted in a fixed environment to evaluate the phenotype. These studies have thus ignored the genotype-by-environment (G x E) interaction, which is an important component influencing quantitative traits. A few exceptions are the studies conducted by Paterson et al. (1988) and Stuber et al. (1992). Paterson et al. (1991) investigated the predictive value of QTLs across environments in tomato by comparing QTL maps of F 2 populations and their F 3 families, which were grown in different environments. Their results showed that only 4 out of 29 QTLs could be detected in all of the environments tested. Stuber et al. (1992) studied the genotype-by-environmentinteraction for QTLs of maize by field evaluation of back-

1212 Table 1 Means (top row) and variation range (bottom row) of six quantitative traits measured in three locations Location a

Heading date

Plant height (cm)

1000-grain weight Spikelets per (g) panicle

Grains per panicle Seed set percentage (%)

BJ

100.8 66.6-131.6 79.7 67.3-97.5 89.4 67.5-106.2

82.3 48.6-112.0 84.2 44.2-114.8 72.5 50.8-96.6

23.6 16.0 31.7 24.4 15.3-34.3 23.5 16.7-30.9

106.9 15.0-252.8 67.5 2.2-150.7 72.6 2.0-195,7

HZ HN

136.7 34.2-269.2 108.7 34.3-197.3 114.2 43.0-252.0

76.8 8.9-95.5 60.8 4.6-86.6 63.4 1.1-95.0

a BJ, Beijing; HZ, Hangzhou; HN, Hainan c r o s s families in six d i v e r s e e n v i r o n m e n t s , b u t l i m i t e d

evidence of G x E interaction was found. However, the populations they used were F 2 a n d F 3 o r b a c k c r o s s families, so t h e c o m p a r i s o n a c r o s s e n v i r o n m e n t s m i g h t be c o n f o u n d e d b y different g e n e r a t i o n s . I n t h e i n v e s t i g a t i o n p r e s e n t e d here, we h a v e u s e d a p e r m a n e n t D H p o p u l a t i o n to i n v e s t i g a t e t h e G x E i n t e r a c t i o n at i n d i v i d u a l Q T L s b y c o m p a r i n g Q T L m a p s g e n e r a t e d in three diverse environments. Rice is o n e o f t h e m o s t i m p o r t a n t c r o p s in t h e w o r l d . High-density restriction fragment length polymorphism (RFLP) maps have been recently constructed (Causse et al. 1994; K u r a t a et al. 1994), a n d s o m e q u a n t i t a t i v e traits have also been studied using molecular markers ( A h n et al. 1988; W a n g et al. 1994 X i a o et al. 1994; L i et al. 1995a,b). I n this p a p e r r , w e d e s c r i b e a s t u d y u s i n g a D H p o p u l a t i o n a n d a n R F L P m a p to l o c a l i z e Q T L s for a n u m b e r o f a g r o n o m i c c h a r a c t e r s a n d to e x p l o r e t h e possible G x E interaction.

Materials and methods Experimental population and phenotypic evaluation One doubled haploid (DH) population of consisting of 132 DH lines was utilized in this study. This population was developed through anther culture of the F~ between indica rice var. 'Zhai-Ye-Qing 8'(Z) and japonica var 'Jing-Xi 17'(J). The agronomic performance of the DH population was evaluated in field experiments at three locations: Beijing, which is located at 39~ (degree of northern latitude); Hangzhou, which is at 32 ~N and Hainan, which is at 18~N. The DH lines were grown in Beijing and Hangzhou from April to September of 1994, and on Hainan Island from December 1994 to April 1995. All of these locations had previously been used for rice field experiments many times and thus there was adequate insect, disease and weed control. In each of the three environments, the DH lines were planted orderly in doubled rows, with the parents being grown between every tenth DH line as the control. The days from sowing to heading (heading date), plant height, number of productive tillers, panicle length, number of spikelets per panicle, number of grains per panicle, seed set percentage and 1000-grain weight were determined based on the average values of 10 individual plants from each line. Except number of productive tillers and panicle length, all other six traits were significantly different between the parents, and the means and variation range in each of the three environments are listed in Table 1. RFLP assays and map contruction Genomic DNA was extracted from young leaves, then digested with restriction enzymes BamHI, Bg/II, Dral, EcoRI, EcoRV, H indIII, ScaI

and XbaI and hybridized with RFLP markers as described by McCouch et al. (1988). DNA markers were kindly provided by Drs. S.D. Tanksley and S.R. McCouch from Cornell University and RGP from Japan. Segregation data on RFLP markers were obtained from 132 DH lines. Chi-square tests were performed to examine if the observed allelic and genotypic frequencies of the marker loci deviated from the expected ratio (1:1) so that the skewedness of the population could be determined. An RFLP linkage map was then constructed using the software MAPMAKER/EXP ver.3.0 (Lander et al. 1987; Lincoln et al. 1993a). The genetical distances were calculated using the Kosambi function. The LOD threshold was fixed at 3.0, and the error detection was used. The assignment of the genetic maps of Causse et al. (1994) and Kurata et al. (1994) were used in assigning linkage groups or markers to their corresponding chromosomes. On the basis of the molecular map established, the graphical genotypes of each line were established and the percentages of the parent genomes were estimated by the computer program HypeGene (Young and Tanksley 1989).

QTL identification Interval QTL mapping was carried out using the software MAPMAKER/QTL ver. 1.i (Paterson et al. 1988; Lincoln et al. 1993b) on the SUN SPARC-10 workstation. The unconstrained model was used. A LOD score threshold of 2.4 would be needed to test at the P = 0.05 level of significance for the entire rice genome (Lander and Bostein 1989). In this investigation, we used a more stringent threshold of 3.0 for declaring the presence of a QTL, and we considered LOD scores between 2.0 and 3.0 as "suggestive". LOD peaks for each significant QTL were used to position the QTL on the linkage map. In case more than one peak was found on the same chromosome for the same trait, multiple-QTL models were used to determine whether the chromosome possessed single or multiple QTLs. Gene effect (additive effect, free of dominance) and percentage of phenotypic variation attributable to individual QTL were estimated at the peaks.

Results RFLP map A genetic map was constructed based on segregating R F L P d a t a in 132 D H lines. T h i s m a p c o n s i s t s o f 137 R F L P m a r k e r s w i d e l y d i s t r i b u t e d o v e r 12 c h r o m o somes with an average distance of 14.8cM (Kosambi m a p units) b e t w e e n m a r k e r s (Fig. 1). T h e l i n e a r o r d e r of the m a r k e r s w a s f o u n d t o be c o n s i s t e n t w i t h t h a t o f C a u s s e et al. (1994) a n d K u r a t a et al. (1994) e x c e p t for G 3 9 a n d R Z 3 3 7 , w h i c h m a p p e d to different c h r o m o somes, and one inversion on chromosome 2 (RG654, R G 3 2 2 a n d R G 5 2 0 ) . T h e g e n o m e c o v e r a g e w a s es-

1213

1 G39 Cl12 RG536 C225 c94~

- ~ - RG654 (J) 223 I I I -G1234 (j) 21.~

38.~'~

- RG322 e) RG62o ~)

4.6

a37o

-

G275 (j)

-YSOOTa e)

RG541

27.4

RGI01 C49 RG345 RG233

- -C424 (j) 13.9 7.1- - G45 [ " ~ G1314A

RG214

'RGI04

15.1t

7.2.S__G175 .--C74A (z) 15.1 4.6- --RG450 " --RG266 (z)

RG620A 25.2

(z)

6'0tZ -RG369A 3.c

-RG369B

8.7 ~

G62

17.

20.5 -

-

G144

9

RG482

-- - - G243A 17.9

26A - - C746

C513

(i)

18.3 G271 (j) C975

7.5

21.4

RG207

11.6

--

10.3

RG449(j)

11.4 -

40.3

355 1 1 -- RG360

.,22 II

II

1L,

-c63

~r

29.5

-

26.3

R210

5

4

2

11.5 .- RG776B (j)

1~II

22.4

4 0-~

RG13 (J)

10.9 17.2

i~ 12.:

G1314B

RGg08

- G318

~

16.3 CDO456

25.

G294(j) G122 (j)

7.2

.;111-- G81 (j) ~ (j)

11.4

23.0

G329 C236 RG653

--RZ337 RG612

13.9_~_ G1327

29.3

21.7 1~_ G365

7

8

-~-RG351 23.9

II

G379A

I

I

I

I

G278

-- CDO590

23.9

s10~

i~

12 RZ536B

- ~ - Gl106

31.411

74--~-RG181A (z)

2~

4 L . _ ,G, 7

G103 RG885

RG650

31.! - - RZ617 (z) 9.6 G192(z) 11.1 7.4 G1073 "" RG1 8.5 -- G187 24.6

"

ii

12 4-11--RG257

1i

18

18.9 -- C285 10.3 L. G20 15.6 ! C492 (z) 8.3 RG477(z) 4"~ ~ RG511(z) 16 .. RG769(Z) 6.1 RG528(z)

i0

9

"" C400A

13.8

t7.3

9

14.2

-- RZ66

1' LI-I- G29,

C356A 5.~"-- C356B

22.8 --

RG662

12.8 --

10.4

RZ404

11-0,o

19-~0223

I t - aG11O9(z) 2.'H-I--aa~(z)

7"3-[LY12817R (z)

:;':~-PTA818 "-'lPI-- RG2

4"1"143 . 5 - ~ G 1F:ff'397 2 4 1A 9'1'~ - G124-1B(z)

"" G295

15.4 "

"

RG451

"-t'~ G320 9"41~ 0496

16.2-[1--RG574

18

11.6

-

RG136 RG418B

Fig. 1 A rice molecular linkage map with 137 RFLP markers constructed from 132 DH lines derived from the cross of 'Zhaiyeqing 8' x 'Jingxi 17'. Scale in Kosambi centiMorgans (cM) is shown on the left of each chromosome. The markers showing distorted segregation are marked with (z) or (j) indicating their favoring parent genotype Z/Z or J/J, respectively

timated to be approximately 95% on the basis of the two saturated maps mentioned above. The overall proportion of Z/Z genotypes in the DH population was 49.6% as estimated by HyperGene

1 ] -G193

31All "~

RG304

(Young and Tanksley 1989). Out of 137 loci analyzed in the DH population 41(~31%) deviated significantly (P ~