Development of Chromosomal Segment Substitution Lines from a Backcross Recombinant Inbred Population of Interspecific Rice Cross

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1 Rice Science, 2006, 13(1): Development of Chromosomal Segment Substitution Lines from a Backcross Recombinant Inbred Population of Interspecific Rice Cross CHEN Jie 1, 2, Hafeez Ur Rahman BUGHIO 3, CHEN Da-zhou 4, LIU Guang-jie 1, ZHENG Kang-le 1, ZHUANG Jie-yun 1 ( 1 Chinese National Center for Rice Improvement and State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou , China; 2 Anhui Agricultural University, Hefei , China; 3 Pakistan Atomic Energy Commission Nuclear Institute of Agriculture, Tando Jam Sindh , Pakistan; 4 Rice Research Institute, Jiangxi Academy of Agricultural Sciences, Nanchang , China) Abstract: A backcross recombinant inbred line population consisting of 202 lines was developed from Xieqingzao B // Xieqingzao B / Dongxiang wild rice. The population was assayed with DNA markers and phenotyped on planthopper resistance and yield traits. A linkage map consisting of 119 DNA markers and spanned for 1188 cm over the 12 rice chromosomes was constructed. Thirty-two chromosomal segment substitution lines were selected based on the percentage of Xieqingzao B allele at marker loci. These lines are of great potential for gene mapping and alien gene introgression. Key words: alien introgression; DNA marker; Dongxiang wild rice; chromosomal segment substitution line Genetic variation in cultivated rice has been reduced tremendously during domestication and modern plant breeding. As a consequence, slow progress in rice breeding and genetic vulnerability in rice production has been witnessed since 19s. Wild relatives of cultivated rice remain to be highly diversified and hold various genes conferring resistance to biotic and abiotic stresses, thus providing a valuable gene pool for rice genetic improvement. More and more attentions have been paid to introgression of beneficial alleles from wild rice into elite breeding lines [1-2], and molecular tagging of new resistance genes from wild rice species has been greatly facilitated with advances in DNA marker technology [3-5]. Most of the agronomically important traits are inherited quantitatively. While it is relatively straightforward to identify and transfer major genes from unadapted germplasm into adapted germplasm, identification of useful quantitative trait loci (QTLs) from unadapted germplasm is difficult and requires specialized genetic design. An approach known as the introgression line analysis [6] has been extensively used in rice and other crop species [7-8]. A number of Received: 14 July 2005; Accepted: 9 December 2005 Corresponding author: ZHUANG Jie-yun (jz13@hzcnc.com) studies in rice have reported on the DNA marker-assisted establishment of chromosomal segment substitution lines (CSSLs) in inter-species and inter-subspecies crosses [9-12], and on the application of CSSLs for QTL verification and QTL fine mapping [7, 13-14]. While most studies employed backcrossing for CSSL development, an alternative approach was proposed to select rice lines carrying a few loci from the donor parent in a permanent population that was used to construct a molecular linkage map and displayed skewed segregation in favor of the recipient parent [15]. These lines could be used as intermediate in the development of near isogenic lines (NILs) that carry a single chromosomal segment from the donor parent. Dongxiang, Jiangxi Province, China, is the northernmost natural habitat of common wild rice (Oryza rufipogon). In addition to the utilization of cold tolerance and pest resistance of Dongxiang wild rice, introduction of the cytoplasm of a male sterile accession into indica rice cultivar resulted in the development of dwarf-abortive cytoplasmic male sterile (CMS-DA) line Xieqingzao A in 19. Development of alien introgression lines will facilitate the discovery of valuable genes from Dongxiang wild

2 16 Rice Science, Vol. 13, No. 1, 2006 rice. In this paper, we described the construction of a genetic linkage map using a BC 1 F 5 population of Xieqingzao B // Xieqingzao B / Dongxiang wild rice, and the development of 32 CSSLs from the population. MATERIALS AND METHODS Population development A BC 1 F 1 population of Xieqingzao B// Xieqingzao B/Dwr was developed in 1999 by Rice Research Institute, Jiangxi Academy of Agricultural Sciences, Nanchang, China. The recurrent parent Xieqingzao B (hereafter referred to as XB) is the maintainer line of CMS-DA line Xieqingzao A (O. sativa subsp. indica), and Dwr is an accession of O. rufipogon from Dongxiang, Jiangxi Province, China. The BC 1 F 1 population was grown in 2000 at the China National Rice Research Institute (CNRRI), Hangzhou, China. Seeds were harvested from each true BC 1 F 1 plant, respectively. The population was advanced by single seed descent. A backcross inbred line (BIL) population consisting of 202 lines at BC 1 F 5 generation was obtained in DNA marker analysis The 202 BC 1 F 5 lines and the recurrent parent XB were grown in 2003 at CNRRI. Roughly 10 grams of fresh leaf from a single plant per line were collected for DNA extraction following the method of Lu and Zheng [16]. A total of 95 RFLP probes and 169 SSR primers were selected for the survey of parental polymorphism. As the original Dwr accession is no longer available, two DNA pools, each consisting of 10 randomly-selected BILs, and the DNA from the recurrent parent XB were used. Parental RFLP analysis was conducted in combination with five restriction enzymes (BamHⅠ, DraⅠ, EcoRⅠ, EcoRⅤ and Hind Ⅲ). Polymorphic probe/enzyme combinations were selected for assaying the BILs. Southern blotting was performed according to the standard method [12]. Probe hybridization and signal detection were carried out by using ECL direct nucleic acid labelling and detection systems (Amersham Pharmacia Biotech). SSR primers were amplified in a total volume of 10 µl containing 2 µl of 5 SSR buffer, 0.5 µmol/l of each primer, 0.2 mmol/l dntps, 2.5 mmol/l of MgCl 2, 0.5 unit of Taq DNA polymerase and ng of genomic DNA. The PCR was performed in PTC-200 Thermal Cycler (MJ Research). Samples were pre-denatured at 94 for 2 min, followed by 30 cycles of 45 s at 94, 45 s at 55 and 45 s at 72, and a final extension at 72 for 8 min. PCR products were separated in 6% non-denaturing polyacrylamide gel and visualized by silver staining as described previously [18]. In assaying the BILs using polymorphic SSR primers, PCR products were separated in 2-4% agarose gel, stained with ethidium bromide and visualized by using GDS-7600 (UVP), or separated in 6% non-denaturing polyacrylamide and visualized by silver staining. Data analysis At each marker locus, the DNA fragment detected from XB was used as the reference to score the genotype of BILs: lines showing XB fragment only were recognized as XB homozygotes, showing non-xb fragment only as Dwr homozygotes, and showing both XB and non-xb fragments as heterozygotes. Chi-square test was used for examining the segregation of marker alleles to the expected ratio of 1:3 in BC 1 F 5 population. To alleviate the influence of segregation distortion on linkage test and the estimation of recombination fractions, a four-step analysis was employed for map construction in this study. Firstly, the markers were assigned to each rice chromosome according to the maps of Rice-Cornell-RFLP-1994, Rice-Cornell-RFLP-2001, Rice-Cornell-SSR-2001 and Rice-IRMI-2003 ( Secondly, Mapmaker 3.0b [19] was employed to identify the most probable marker order within a linkage group, using data type of F 2 intercross. Thirdly, QTXb19 [20] which used statistic methods that are not sensitive to the effects of segregation distortion was applied to verify the linkage among markers in the same group and to estimate recombination fraction (R) between adjacent markers, using the segregation pattern of F 2 intercross. Fourthly, the recombination fraction (r) in BIL was calculated using the formula r = 2R / (3-4R) [21] and converted to genetic distance using Kosambi function [22]. Analysis of introgressed segments The percentage of XB alleles over the 122 co-dominant markers was calculated for each BIL, using the formula: XB% = (N N 2 )/N, where N 1 is

3 CHEN Jie, et al. Development of CSSL from a Backcross Recombinant Inbred Population of Interspecific Rice Cross 17 the number of XB homozygotes, N 2 the heterozygotes, and N the total number of informative individuals. Lines showing 90% or higher XB proportions were selected. Additional lines were chosen from those showing 85-90% XB proportions to extent the coverage of introgressed segments. Graphical genotyping of the lines selected were performed with GGT32 [23]. Phenotypic evaluation Evaluation for brown planthopper (BPH) and the white-backed planthopper (WBPH) resistance was conducted in the summer of 2004 at CNRRI. The BILs and the parental line XB were tested with two replications in plastic trays of 60 cm 45 cm 10 cm. In each replication, 20 germinated seeds from each BIL were sown in a row of 20 cm with 3 cm spacing between rows, but only 15 healthy seedlings were retained. Three rows of control varieties were grown at random in each tray. The susceptible control TN1 and resistant control RHT were used in both experiments, and additional resistant controls Mudgo and ASD7 were applied for BPH resistance test. During the second leaf stage, 2nd- to 3rd-instar nymphs of planthopper were released to the trays for infestation at a density of 10 insects per seedling. When the seedlings of the susceptible control TN1 cm RG147 RG1 RG532 RM5359 RM6466 RG173 RM1 RZ744 RM246 RG101 RZ730 RM315 RG222 2 RG555 RM154 RZ742 RM1285 RM555 RM57 RM3294 RM71 RM29 RG157 RZ551 RM250 RM213 RM208 RM138 3 RG348 RG450 RG191 RM218 RZ251 RZ22 RG482 RZ519 RZ575 4 RG449 RM142 RM273 RM303 RG214 RM348 RM131 RM127 RG620 RM2 5 RM153 RG556 RM13 RM267 RM289 RM164 RZ70 RZ225 RM87 RM274 RM334 RG119 6 were completely killed, seedling mortality was measured for each BIL. The insects used for infestation were collected from the fields at CNRRI, among which BPH included biotypes 1 and 2. One row of six plants was planted for each BIL and the parental line XB in the paddy field at CNRRI in Heading date (HD) was recorded as the number of days from sowing to the first panicle emerging. Panicles for each line were harvested at maturity. Six traits, number of panicles per plant (NP), number of filled grains per panicle (NFGP), total number of spikelets per panicle (TNSP), spikelet fertility (SF), 1000-grain weight (TGWT) and grain yield per plant (GYD), were measured. RESULTS Linkage map and marker segregation Of the 95 RFLP and 169 SSR markers used for parental polymorphism survey, 127 markers showing polymorphism were employed to assay the 202 BILs. Linkage analysis of the 127 markers was conducted with Mapmaker 3.0b. A linkage map consisting of 119 marker loci (43 RFLP and 76 SSR) was constructed (Fig. 1). The remaining 8 markers were either unlinked to any of the 119 markers or could not be RM204 RM225 RM314 RM253 RM276 RG64 RG264 RG123 RG172 RM3 7 RG528 RM5752 RM6574 RM125 RM214 RM11 RG678 RM234 RG351 8 RM25 RM126 RZ562 RM72 RG978 RM339 RM42 RM210 RM256 RM308 9 RM316 RM219 RZ698 RM201 RG667 RM215 RG451 RM RM7492 RM6370 RM244 RM311 RM304 RM171 RM6704 RM1125 RM1375 RM19 RM247 RM101 RG RM332 RM202 RG2 RM206 RM254 RG303 RM224 RM1233 RM RG236 Fig. 1. Linkage map constructed by using 202 BC 1F 5 lines of Xieqingzao B// Xieqingzao B/ Dongxiang wild rice. Intervals flanked by markers having genetic distance larger than 35 cm are indicated by //.

4 18 Rice Science, Vol. 13, No. 1, 2006 located properly. Two pairs of co-dominant markers on chromosome 1 were completely segregated, respectively, reducing the number of marker loci included in the map to 117. The linkage map covered all the 12 chromosomes of rice and spanned for 1188 cm. Genetic distances between adjacent marker loci ranged as cm. The marker order and genetic distances among markers were in accordance with the published maps. Overall bias towards the recurrent parent XB was observed in the population, with 81.4% XB alleles on average. Significant distorted segregation from the expected 1:3 segregation was found for 81 of the 117 co-dominant markers mapped. The proportion of XB alleles at four markers on chromosome 6, RM204, RM225, RM314 and RM253, were significantly lower than 75% and higher than 50%. The remaining 77 skewed markers all had XB alleles higher than 75%. Selection of CSSLs As calculated over the 122 co-dominant markers, 21 out of the total 202 BILs were shown to have 90% or higher proportion of XB alleles. The linkage map constructed was applied to graphically genotyped the 21 lines by using software GGT32. It was found that a considerable proportion of the linkage map was not covered by transgressed segments (Fig. 2, top 21 lines). From lines showing 85-90% XB proportion, 11 lines (Fig. 2, bottom 11 lines) were selected to fill the gap of non-transgressed regions. Altogether, 32 lines were selected as CSSLs, in which transgressed segments covered 99.3% of the genome as indicated by the linkage map. Characterization of the CSSLs All of the 202 BC 1 F 5 lines were tested for the resistance to WBPH and BPH, as well as for heading date and six yield traits. In comparison with the original population consisting of 202 lines, the sub-population of the 32 CSSLs were more similar to the recurrent parent XB in terms of the agronomical performances (Table 1). A number of CSSLs were shown to have high resistance to WBPH or BPH. Three lines, line 71, XB homozygotes Dwr homozygotes Heterozygotes Fig. 2. Distribution of introgressed segments in pre-isogenic lines. Numbers at the top refer to the chromosome number, and those at the left refer to the BIL number.

5 CHEN Jie, et al. Development of CSSL from a Backcross Recombinant Inbred Population of Interspecific Rice Cross 19 Table 1. Agronomical performances of the 202 BILs, 32 CSSLs and Xieqingzao B. Lines HD (d) NP NFGP TNSP SF(%) TGWT (g) GYD (g) 202 BILs 72.5± ± ± ± ± ± ± CSSLs 70.7± ± ± ± ± ± ±7.5 Xieqingzao B Trait values of the 202 BILs and 32 CSSLs are indicated as population mean±sd. HD, Heading date; NP, Number of panicles per plant; NFGP, Number of filled grains per panicle; TNSP, Total number of spikelets per panicle; SF, Spikelet fertility; TGWT, 1000-grain weight; GYD, Grain yield per plant. and 152 were resistant to WBPH (Fig. 3). It was noteworthy that line 71 had agronomical performances similar to the recurrent parent XB. Two lines, line 36 and 192, were resistant to BPH, of which line 192 had agronomical performances similar to the recurrent parent XB (Fig. 4). The heading date and 1000-grain weight of the remaining three lines were also similar to those of XB, except that line 91 has a later heading date. DISCUSSION Segregation distortion has been commonly observed in interspecific populations [24-25], presumably ascribed to crossability barriers between species [2] or unintentional selection against plants with low fertility or vigor [24]. In this study, 81 out of the total 117 co-dominant markers mapped showed skewed segregation, among which 77 (95%) were in favor of the adapted parent XB. Since segregation distortion is expected to affect the estimation of recombination fractions, a number of analytical tools have been proposed to reduce the errors [26-27]. Software QTX employed statistic methods that are not sensitive to the effects of segregation distortion and has been applied in the construction of molecular linkage map in populations derived from interspecies crosses [28]. In 120 XB Line 71 Line 91 Line XB% WBPH NP NFGP TNSP SF TGWT GYD HD Fig. 3. Performance of three CSSLs resistant to WBPH. WBPH resistance was measured as seedling mortality. 120 XB Line 36 Line XB% BPH NP NFGP TNSP SF TGWT GYD HD Fig. 4. Performance of two CSSLs resistant to BPH. BPH resistance was measured as seedling mortality.

6 20 Rice Science, Vol. 13, No. 1, 2006 this study, recombination fractions were estimated with QTX19b for an F 2 population and then converted to the value in a BIL population. The map constructed matches well to the rice molecular maps of Rice-Cornell-RFLP-1994, Rice-Cornell-RFLP-2001, Rice-Cornell-SSR-2001 and Rice-IRMI-2003, while a map constructed by using Mapmaker differed greatly from the published maps (data not shown). The development and application of NILs have greatly promoted QTL fine mapping in rice and other crop species [6-8, 29]. Taking the advantage of DNA marker data accumulated in the construction of genetic map using XB//XB/Dwr BIL population, and facilitated by the segregation distortion towards the adapted rice line XB, a set of CSSLs were selected in this study. In these lines, one or a few chromosome segments were introgressed from Dongxiang wild rice into the genetic background of CMS-DA maintainer line Xieqingzao B. As shown in the previous section, a number of lines have displayed high resistance to planthopper. Such lines could be used as NILs for QTL fine mapping or as intermediates for NIL development. They could also be used as intermediate to develop new breeding lines carrying beneficial genes from the wild rice. Further work is underway to determine whether the WBPH or BPH resistance genes are located in the introgressed segments observed, to evaluate the CSSLs for other traits, and to develop NIL sets that have a more complete genomic coverage. ACKNOWLEDGEMENTS The authors would like to thank Dr Tanksley s Laborotory, the Cornell University (Ithaca, New York State, USA) for providing the DNA probes. This work is supported by Hi-Tech Research and Development Program of China (2003AA207030, 2004AA227130) and Key Agricultural Program of Zhejiang Province, China (2003G10028). REFERENCES 1 Multani D S, Jena K K, Brar D S, Reyes B D, Angeles E R, Khush G S. Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin.to cultivated rice O. sativa L. Theor Appl Genet, 1994, 88: Brar D S, Khush G S. Alien introgression in rice. Plant Mol Biol, 1997, 35: Huang Z, He G, Shu L, Li X, Zhang Q. Identification and mapping of two brown planthopper resistance genes in rice. Theor Appl Genet, 2001, 102: Renganayaki K, Fritz A K, Sadasivam S, Pammi S, Harrington S E, McCouch S R, Kumar S M, Reddy A S. Mapping and progress toward map-based cloning of brown planthopper biotype-4 resistance gene introgressed from O. officinalis into cultivated rice, O. sativa. Crop Sci, 2002, 42: Gu K, Tian D, Yang F, Wu L, Sreekala C, Wang D, Wang G L, Yin Z. High-resolution genetic mapping of Xa27 (t). a new bacterial blight resistance gene in rice. Oryza sativa L. Theor Appl Genet, 2004, 108: Eshed Y, Zamir D. An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics, 1995, 141: Li J, Thomson M, McCouch S R. Fine mapping of a grain-weight quantitative trait locus in the pericentromeric region of rice chromosome 3. Genetics, 2004, 168: Monforte A J, Tanksley S D. Development of a set of near isogenic and backcross recombinant inbred lines containing most of the Lycopersicon hirsutum genome in a L.esculentum genetic background: A tool for gene mapping and gene discovery. Genome, 2000, 43: Doi K, Iwata N, Yoshimura Y. The construction of chromosome substitution lines of African rice (Oryza glaberrima Stued.) in the background of japonica rice (O. sativa L.). Rice Genet Newsl, 1997, 14: Aida Y, Tsunematsu H, Doi K, Yoshimura A. Development of a series of introgression lines of japonica in the background of indica rice. Rice Genet Newsl, 1997, 14; Kurakazu T, Sobrizal, Ikeda K, Sanchez P L, Doi K, Angeles E R, Khush G S, Yoshimura A. Oryza meridionalis chromosomal segment introgression lines in cultivated rice, O. sativa L. Rice Genet Newsl, 2001, 18: Li W T, Zeng R Z, Zhang Z M, Zhang G Q. Analysis of introgressed segments in near isogenic lines for F 1 pollen sterility in rice. Chinese J Rice Sci, 2003, 17(2): Jiang L, Cao Y J, Wang C M, Zhai H Q, Wan J M, Yoshimura A. Detection and analysis of QTL for seed dormancy in rice (Oryza sativa L.) using RIL and CSSL population. Acta Genet Sin, 2003, 30(5): Ishimaru K. Identification of a locus increasing rice yield and physiological analysis of its function. Plant Physiol, 133:

7 CHEN Jie, et al. Development of CSSL from a Backcross Recombinant Inbred Population of Interspecific Rice Cross Inukai T, Zeigler R S, Sarkarung S, Bronson M, Dung L V, Kinoshita T, Nelson R J. Development of pre-isogenic lines for rice blast-resistance by marker-aided selection from a recombinant inbred population. Theor Appl Genet, 1996, 93: Lu Y J, Zheng K L. A simple method for isolation of rice DNA. Chinese J Rice Sci, 1992, 6: Sambrook J, Fritch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd edn. New York: Cold Spring Harbor Laboratory Press, Shi Y F, Ying J Z, Wang L, Zhu Z W, Zhuang J Y. Screening SSR markers for rice variety identification. Chinese J Rice Sci, 2005, 19(3): Lander E S, Green P, Abrahamson J, Barow A, Daly M J, Lincoln S E, Newberg L. Mapmaker: an interactive computer program for constructing primary genetic maps of experimental and natural populations. Genomics, 1987, 1: Manly K F, Cudmore R H Jr, Meer J M. Map Manager QTX, cross-platform software for genetic mapping. Mamm Genome, 2001, 12: Lin S Y, Sasaki T, Yano M. Mapping quantitative trait loci controlling seed dormancy and heading date in rice Oryza sativa L., using backcross inbred lines. Theor Appl Genet, 1998, 96: Kosambi D D. The estimation of map distance from recombination values. Ann Eugen, 1944, 12: van Berloo R. GGT: Graphical GenoTyping. Wageningen, Netherlands: Laboratory of Plant Breeding, Wageningen University & Research Centre, Tanksley S D, Nelson J C. Advanced backcross QTL analysis: a method for simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor Appl Genet, 1996, 92: Causse M A, Fulton T M, Cho Y G, Ahn S N, Chunwongse J, Wu K S, Xiao J H, Yu Z H, Ronald P C, Harringtom S E, Second G, McCouch S R, Tanksley S D. Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics, 1994, 138: Aluko G, Martinez C, Tohme J, Castano C, Bergman C, Oard J H. QTL mapping of grain quality traits from the interspecific cross Oryza sativa O. glaberrima. Theor Appl Genet, 2004, 109: Lorierx M, Goffinet B, Perrier X, Gonzalez D, Lanaud C. Maximum-likelihood models for mapping genetic markers showing segregation distortion: Ⅰ. Backcross populations. Theor Appl Genet, 1995, 90: Thomson M J, Tai T H, McClung A M, Lai X H, Hinga M E, Lobos K B, Xu Y, Martinez C P, McCouch S R. Mapping quantitative trait loci for yield, yield components and morphological traits in an advanced backcross population between Oryza rufipogon and the Oryza sativa cultivar Jefferson. Theor Appl Genet, 2003, 107: Yamamoto T, Kuboki Y, Lin S Y, Sasaki T, Yano M. Fine mapping of quantitative trait loci Hd-1, Hd-2 and Hd-3, controlling heading date of rice, as single mendelian factors. Theor Appl Genet, 1998, 97: