Development and Identification of Introgression Lines from Cross of Oryza sativa and Oryza minuta

Rice Science, 2013, 20(2): 95í102 Copyright © 2013, China National Rice Research Institute Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(13)60111-0

Development and Identification of Introgression Lines from Cross of Oryza sativa and Oryza minuta GUO Si-bin1, 2, WEI Yu1, LI Xiao-qiong1, LIU Kai-qiang1, HUANG Feng-kuan2, 3, CHEN Cai-hong1, 2, GAO Guo-qing1, 2 (1Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China; 2Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Nanning 530007, China; 3Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China)

Abstract: Introgression line population is effectively used in mapping quantitative trait loci (QTLs), identifying favorable genes, discovering hidden genetic variation, evaluating the action or interaction of QTLs in multiple conditions and providing the favorable experimental materials for plant breeding and genetic research. In this study, an advanced backcross and consecutive selfing strategy was used to develop introgression lines (ILs), which derived from an accession of Oryza minuta (accession No. 101133) with BBCC genome, as the donor, and an elite indica cultivar IR24 (O. sativa), as the recipient. Introgression segments from O. minuta were screened using 164 polymorphic simple sequence repeat (SSR) markers in the genome of each IL. Introgressed segments carried by 131 ILs covered the whole O. sativa genome. The average number of homozygous O. minuta segments per introgression line was about 9.99. The average length of introgressed segments was approximate 14.78 cM, and about 79.64% of these segments had sizes less than 20 cM. In the genome of each introgression line, the O. minuta chromosomal segments harbored chromosomal fragments of O. sativa ranging from 1.15% to 27.6%, with an overall average of 8.57%. At each locus, the ratio of substitution of O. minuta alleles had a range of 1.5%í25.2%, with an average of 8.3%. Based on the evaluation of the phenotype of these ILs, a wide range of alterations in morphological and yield-related traits were found. After inoculation, ILs 41, 11 and 7 showed high resistance to bacterial blight, brown planthopper and whitebacked planthopper, respectively. These O. minuta-O. sativa ILs will serve as genetic materials for identifying and using favorable genes from O. minuta. Key words: Oryza sativa; Oryza minuta; introgression line; bacterial blight; brown planthopper; whitebacked planthopper

Rice is one of the most important crops and provides the staple food for more than 50% of the world’s population. Genetic variation in cultivated rice has been reduced tremendously during domestication and modern plant breeding. Currently, the narrow genetic base of breeding programs has resulted in a bottleneck effect in rice variety development (Tanksley and McCouch, 1997). Wild relatives of cultivated rice remain to be highly diversified and hold various genes conferring resistance to biotic and abiotic stress, thus unlocking the tremendous genetic potential from wild rice might break the genetic bottleneck and improve modern rice varieties (Zamir, 2001). Received: 8 January 2012; Accepted: 10 May 2012 Corresponding author: GAO Guo-qing ([email protected])

More and more attention has been paid to introgression of beneficial alleles from wild rice into elite breeding lines (Multani et al, 1994; Brar and Khush, 1997), and molecular tagging of new resistance genes from wild rice species has been greatly facilitated with advances in DNA marker technology (Gu et al, 2004). 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. Exploitation and utilization of the favorable genes from wild rice could overcome the yield plateaus of cultivated rice improvement. An approach known as introgression line (IL) analysis (Eshed and Zamir, 1995) has been extensively used in rice and other crop

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species (Monforte and Tanksley, 2000a; Li et al, 2004). ILs can be obtained through consecutive backcrossing and selfing to introgress small chromosomal segments from the donor into the recurrent parent, with markerassisted selection (MAS) to identify the donor segment number and length. Due to its simple genetic background, ILs have become a useful experimental material for genetic analysis and molecular breeding and could be used to evaluate the action and interaction of genes over multiple years and in multiple site experiments (Monforte and Tanksley, 2000b). Using a set of ILs carrying the whole donor genome, near isogenic lines (NILs) could be constructed rapidly through back-crossing and MAS after a target gene is found. In addition, for fine mapping and positional cloning of certain target genes or QTLs, an enormous secondary F2 population derived from an advanced backcross between selected ILs and the recipient parent could also be rapidly developed (Tian et al, 2006b). Recently, several sets of ILs were constructed in crop species including rice (Li et al, 2005; Tian et al, 2006a; Tan et al, 2007), wheat (Pestova et al, 2001), barley (Matus et al, 2003), tomato (Eshed and Zamir, 1995; Chetelat and Meglic, 2000; Monforte and Tanksley, 2000a), Brassica napus (Howell et al, 1996) and melon (Eduardo et al, 2005). These IL populations would accelerate molecular breeding and improve the traits of agronomic importance. Oryza minuta, a tetraploid wild relative of cultivated rice with BBCC genome, is rich in gene sources, such as resistance to blast blight, bacterial blight (BB), brown planthopper (BPH) and whitebacked planthopper (WBPH). Moreover, a number of resistance genes had been successfully transferred into cultivated rice from O. minuta (Amante-Bordeos et al, 1992; Rahman et al, 2009). In order to identify and use more desired genes from this wild species, it is still very important to construct O. minuta -O. sativa introgression lines. In this study, a set of 131 ILs derived from a backcross between an accession of O. minuta, as the donor, and an elite indica cultivar IR24, as the recurrent parent, were constructed by MAS. The characteristics of introgressed segments, including the introgression ratio in various chromosomal regions, and the length and number of introgressed segments in each individual were assessed. Based on the evaluation of the phenotype of these ILs, BB, BPH and WBPH resistance was also identified.

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MATERIALS AND METHODS Population development An elite indica cultivar IR24 (O. sativa ssp. indica) was used as a recipient parent in a backcross program and crossed with an accession of the tetraploid species O. minuta (accession No. 101133) with BBCC genome, which was kindly provided by the International Rice Germplasm Centre of the International Rice Research Institute in 2004. The F1 plants were backcrossed four times consecutively with IR24 until a BC4 population consisting of 351 plants was obtained. Then BC4F1 plants were self-pollinated for three generations, and 351 BC4F3 families were obtained. On the basis of the results of genotypic analysis, 192 individuals containing specific different introgressed segments were selected from 351 BC4F3 families. The 192 plants of BC4F3 were self-pollinated for three generations consecutively. Finally, 131 ILs containing the chromosomal segments from O. minuta covering the whole O. sativa genome were developed. There was little artificial selection to any traits during the course of BC4F1 development, but there were strong phenotypic selection in selfing process from BC4F2 to BC4F6. DNA extraction and SSR analysis Genomic DNAs were extracted from young rice leaves by using the CTAB method (Murray and Thompson, 1980). Totally, 208 rice SSR primers, which were distributed across the 12 chromosomes of cultivated rice, were retrieved from the publicly available website (http://www.gramene.org). Marker orders for the SSR were identical to the published map (Temnykh et al, 2000). PCR was performed in 25 PL reaction-solution containing 20 ng of genomic DNA, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.8), 0.001% gelatin, 2.5 mmol/L MgCl2, 0.2 mmol/L each of dNTPs, 0.2 mmol/L each primer, and 1 U of Taq polymerase. DNA amplification was carried out in a PTC-100 Programmable Thermal Controller (MJ Research USA), using a program consisting of an initial denaturation for 5 min at 94 °C, followed by 35 cycles of 50 s under 94 °C, 45 s under 55 °C, and 1 min under 72 °C, and a final extension for 5 min under 72 °C. PCR products were separated on 6% polyacrylamide denaturing gels (PAGE), and the allelic similarity and diversity of the SSRs were determined according to the bands in the gel revealed by silver staining (Panaud et al, 1996).

GUO Si-bin, et al. Development and Identification of Introgression Lines

Percentage of O. minuta genome in each introgression line Graphical genotype from the GGT program (van Berloo, 1999) was carried out to determine the percentage of the total genome in each IL that came from each parent. Basically, if two consecutive loci had alleles coming from the same progenitor, the marker interval between them was considered to have the genome of that progenitor. If one locus had alleles from one parent and the consecutive locus had alleles from the other parent, then half of the marker interval between them was considered to have the genome of one progenitor and the other half from the other one (Young and Tanksley, 1989). Field cultivation and trait evaluation One hundred and thirty-one ILs and the recurrent parent IR24 were planted at the experimental farm of Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning City (23° N, 108° E), China, in the spring of 2010, following a complete randomized block design, with two replications, five rows per plot, 10 plants per row, 15 cm between plants within each row and 25 cm between rows. The field management followed essentially the normal agricultural practice. At harvest, eight plants in the middle of each plot were selected and nine yield-related traits including days to heading, plant height, panicle number per plant, panicle length, grain number per panicle, spikelet number per panicle, seed-setting rate, 1000-grain weight and grain yield per plant were evaluated. Evaluation for bacterial blight resistance To evaluate the BB resistance of ILs, one Xanthomonas oryza pv. Oryzae (Xoo) strain (PXO112, race 5) was used. The recurrent parent IR24 and 131 ILs were tested for their reaction to the Xoo strain at the experimental farm of Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China in the spring of 2010. IR24 and 131 ILs plants were clonally propagated by growing auxiliary buds from nodal cuttings. Ten clones of each plant were used for each strain inoculation test. IR24 plants were inoculated approximately 50 d after sowing. All plants were inoculated by the leaf clipping method described by Kauffmann et al (1973). The bacterial inoculums were prepared as described by Guo et al (2009). The bacterial cell suspension was applied to the 10 youngest fully expanded leaves of each tiller by clipping 2í3 cm from the tip of the leaf using a pair of

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scissors dipped in the inoculums. Lesion lengths of the leaves were measured at 14í21 d after inoculation and scoring was done following Machmud (1978). Evaluation for BPH and WBPH resistance Evaluation for BPH and WBPH resistance was conducted in the summer of 2010 at Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China. The 131 ILs and the parental line IR24 were tested with two replications in plastic trays of 60 cm × 45 cm × 10 cm. In each replication, 20 germinated seeds from each IL 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 rice varieties were grown at random in each tray. The susceptible control TN1 and the resistant control RH (Rathu Heenati) were used in the both experiments. During the 2-leaf stage, 2nd- to 3rd-larva 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 were completely died, seedling mortality was measured for each IL. The reaction against the WBPH and BPH was scored following the guidelines of Standard Evaluation Systems for Rice (IRRI, 1988): 0, No damage; 1, Very slight damage; 3, The first and second leaves of most plants partially yellowing; 5, Pronounced yellowing and stunting or about 10% to 25% of the plants wilting; 7, More than half of the plants wilting or dead and remaining plants severely stunted or dying; 9, All plants dead. The insects used for infestation were collected from the fields at Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China.

RESULTS Polymorphism between two parents detected by SSR markers Among the 208 SSR markers distributed throughout 12 chromosomes of rice, 193 (92.8%) could detect polymorphism between IR24 and O. minuta. The ratio was similar to the SSR polymorphism between O. minuta and IR24 in the previous studies (Guo et al, 2009). Finally, 164 polymorphic SSR markers equably distributed throughout 12 rice chromosomes were used to screen the genotypes of ILs. Development of ILs To construct the ILs, the F1 plants, derived from a

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cross between O. minuta and IR24, were backcrossed three times consecutively with IR24 until BC3 generation (186 BC3F1 individuals) was obtained. The genotypes of 186 BC3F1 individuals were surveyed by 150 polymorphic SSR markers in the previous studies (Guo et al, 2009), and all the plants were backcrossed one time consecutively to produce 351 BC4F1 individuals. According to the results of phenotypic evaluation and genotypic analysis, 192 BC4F3 individuals were selected to develop primary ILs. During the course of self pollination for three generations, the segregations of partial traits occurred in a few lines. Finally, 131 unique ILs were obtained, and each of the alleles from O. minuta was found to be in at least one introgression line via genotypic analysis with 164 SSR markers. Based on the genotypes of the 131 ILs, 89 ILs were further selected as the core set of ILs, which substituted segments that covered 85.1% of the whole O. sativa genome (Supplemental Fig. 1).

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terminal position, but it was similar with the results obtained by Tan et al (2007). This may have resulted from selecting different target traits during the course of developing the introgression lines. Phenotypic evaluation of introgression lines As shown in Table 1, there were a wide range of phenotypic variation in nine traits including days to heading, plant height, and seven yield-related traits in the introgression line population. Among these nine investigated traits, the variation range of grain yield per plant was the largest in ILs, while that of days to

Average number and length of introgressed segments Genotypes of 131 ILs were determined by using 164 polymorphic SSR markers distributed evenly across all 12 rice chromosomes, with an average distance of 10.6 cM between neighbor markers. A total of 1 309 substituted segments were detected in the set of ILs, and 1 081 (82.6%) introgressed segments were homozygous and 228 (17.4%) were heterozygous. The number of introgressed segments in each individual ranged from 2 to 19, with an average of 9.99 (Fig. 1). The number of introgressed segments in 68.53% ILs was less than eight. The length of introgressed segments ranged from 1.05 to 70.4 cM, with an average of 14.78 cM. The length of 79.64% introgressed segments was less than 20 cM (Fig. 2).

Fig. 1. Frequency distribution of the number of donor segments in 131 introgression lines.

Ratio of introgression in each individual and each locus

Fig. 2. Frequency of introgressed chromosomal segments (homozygous, heterozygous) according to genetic length.

The O. minuta chromosomal segments in each introgression line harbored chromosomal fragments of O. sativa ranging from 1.15% to 27.6%, with an overall average of 8.57% (Fig. 3). In 86 ILs (65.65%), each contained less than 10.0%. The ratio of substitution of each O. minuta allele was presented in 131 ILs with ranges of 1.5%–25.2%, and an average of 8.3% (Fig. 4). The results did not coincide with other studies by Chetelat and Meglic (2000) and Tian et al (2006a) that concluded the distribution of the introgressed segments along the chromosomes was not random and the majority of introgressed segments were often at the

Fig. 3. Frequency distribution of donor segment length in 131 introgression lines.

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Fig. 4. Ratio of substitution of Oryza minuta alleles in 131 introgression lines.

Table 1. Statistics of nine traits of IR24 and introgression line population in the spring of 2010 at Nanning, China. Trait Days to heading (d) Plant height (cm) Panicle number per plant Panicle length (cm) 1000-grain weight (g) Grain number per panicle Spikelet number per panicle Seed-setting rate (%) Grain yield per plant (g)

IR24 Mean ± SD 102.0 ± 6.3 81.3 ± 4.2 8.6 ± 1.5 22.5 ± 2.1 24.5 ± 1.8 116.5 ± 15.2 128.6 ± 13.6 91.2 ± 1.3 21.3 ± 2.9

heading was the smallest. BB resistance of ILs After inoculation with the Xoo strains, IR24 was susceptible to PXO112 (lesion lengths of more than 15.0 cm), while amongst the 131 ILs, different reaction to PXO112 was observed (Fig. 5). Of the 131 ILs, 41 introgression lines showed high resistance to Xoo (lesion lengths of less than 3.0 cm).

Mean ± SD 100.8 ± 10.5 87.5 ± 19.2 9.2 ± 4.5 23.4 ± 6.4 23.2 ± 3.1 118. 8 ± 30.4 131.9 ± 26.7 82.6 ± 16.1 20.1 ± 11.6

Introgression line CV (%) 6.7 11.8 13.4 10.9 7.8 25.4 16.5 15.3 27.2

Range 91.0í117.0 78.5í118.2 6.8í14.5 18.2í29.7 16.1í29.2 76.5í175.2 93.7í188.3 65.7í94.7 9.4í41.5

BPH and WBPH resistance of ILs All of the 131 ILs and IR24 were tested for the resistance to WBPH and BPH. A number of ILs showed high resistance to WBPH or BPH. Seven lines, lines 21, 41, 71, 114, 117, 171 and 172 were resistant to WBPH (Fig. 6). It was noteworthy that lines 21, 41 and 114 had agronomical performances similar to the recurrent parent IR24. Eleven lines, lines 41, 46, 71, 78, 107, 114, 125, 136, 137, 171 and 172, were resistant to BPH (Fig. 6), of which lines 41, 78, 114 and 136 had agronomical performances similar to the recurrent parent IR24.

DISCUSSION Development of ILs

Fig. 5. Distribution of lesion length after PXO112 inoculation in 131 introgression lines (ILs).

More and more evidence suggests that ILs are useful genetic materials for the identification of new genes (Eshed and Zamir, 1995; Chetelat and Meglic, 2000; Kubo et al, 2002), for distinguishing pleiotropy versus linkage as well as pseudo-overdominance versus true-

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Fig. 6. Resistance scales of IR24, RH, TN1 and 12 introgression lines tested for the resistance to whitebacked planthopper (WBPH) and brown planthopper (BPH).

dominance (Monfore and Tanksley, 2000a), and for the map-based cloning of QTLs (Alpert and Tanksley, 1996). However, three key factors, the donor genome, the range of phenotypic variation in the ILs population and the coverage ratio of the donor genome, should be considered during the course of developing ILs. In fact, all of the rice ILs reported in previous studies had AA genome species as a donor, and a large part of the ILs only had a low coverage of donor genomes. For example, Tian et al (2006a) developed a set of 159 ILs carrying only 67.5% of the genome of O. rufipogon (AA), and a fraction of chromosomal regions were not covered by the ILs. Similar results were also reported by Kubo et al (2002) in a chromosome substitution series derived from a japonica (AA) and indica (AA) cross. A low coverage of the donor genome might result from the hybrid sterility, gametophyte genes, heading date genes, or less efficient use of MAS during backcrossing. Introgressions from distantly related genomes other than AA genomes are usually difficult to obtain because of low crossability and abnormality of chromosome pairing and recombination. O. minuta is non-AA genome wild rice. The interspecific hybrid and backcross progenies from the cross of O. sativa and O. minuta were obtained by embryo rescue and subsequent backcrosses (Guo et al, 2009). In the present study, we developed a set of 131 ILs, and each of the alleles from O. minuta (BBCC) were found to be in at least one introgression line via genotypic analysis with 164 SSR markers. Based on the genotypes of the 131 ILs, 89 ILs were further selected as the core set of ILs, which substituted segments that had covered 85.1% of the whole O. sativa genome (Supplemental Fig. 1) Reasons for our successful construction of ILs that cover the whole genome of recipient parents may

include: (i) the donor O. minuta, which is distinguishable from the recurrent parent, an indica cultivar in the morphological, ecological characteristics and DNA markers (Guo et al, 2009); (ii) the F1 plants, derived from the cross between O. minuta and IR24, were backcrossed four times consecutively with the recurrent parent; and (iii) phenotypic and genotypic selection was performed from the BC4F3 generation until the 131 ILs were developed (Supplemental Fig. 1). We believe that a four-time backcross was reasonable to obtain a higher representation of the donor genome in ILs, and phenotypic selection in advanced backcross and selfing generation were necessary to ensure a wide range combined consecutive selfing after consecutive backcrossing and trait-performance at multiple environments was used during the development of O. minutaO. sativa ILs. Favorable alleles in O. minuta Based on the evaluation of the phenotypes of the 131 ILs, a wide range of alterations in morphological and yield-related traits were also found. These results indicated the value of wild germplasm in rice improvement. Similar results were obtained in wide crosses between O. sativa and O. officinalis (Jena and Khush, 1990), O. sativa and O. australiensis (Multani et al, 1994), and O. sativa and O. latifolia (Multani et al, 2003). More importantly, BB, BPH and WBPH resistance was also identified in the ILs. After inoculation, three lines, lines 41, 11 and 7 showed high resistance to BB, WBPH and BPH, respectively. Five lines, lines 41, 71, 114, 171 and 172 were resistant to WBPH and BPH. Potential of application of ILs Due to its simple genetic background, ILs could be used to construct a new platform for genetic and

GUO Si-bin, et al. Development and Identification of Introgression Lines

functional genomic analysis. Li et al (2005) explained the theory and practice and illustrated their potential for genetic analysis behind ILs construction. Notably, the process of construction of ILs was also the process of construction of QTL-NILs, which effectively assesses the location and effect of QTLs (Tanksley and Nelson, 1996). In addition, QTL fine mapping may be rapidly finished by using the secondary F2 population derived from a cross between ILs and the recipient. Tian et al (2006b) reported that one QTL (gpa7) for number of grains per panicle was fine mapped in a 35-kb region on chromosome 7 using a secondary F2 population derived from a cross between Guichao 2 and its introgression line SIL040, showing significantly less grains per panicle than Guichao 2. He et al (2006) fine mapped qGY2-1 for grain yield per plant from wild rice by developing a set of NILs of the target QTL using an introgression line (BIL19). These results confirmed that ILs were ideal genetic materials for fine mapping QTLs. In our study, some significant differences, including morphological, physiological and yield-related traits, were found in the ILs compared to the recipient. Using these specific phenotypic ILs, the genes/QTLs controlling resistance to BB, BPH, WBPH, yield and yield components, will be mapped in latter study. Further isolation and assessment of these genes/QTLs would provide ideal chances to understand the molecular mechanism for rice domestication and to make use of the favor genetic resource in a modern rice breeding program. Some specific ILs with other important traits including resistance to blast blight, drought-tolerance, grain quality improvement and so on, were identified in this ILs population (unpublished data). By means of the new analysis platform, combining with development of ILs, QTL mapping and DNA array technology, possible candidate genes in the QTL regions can be rapidly identified.

CONCLUSIONS We developed a set of 131 ILs derived from a backcross between an accession of O. minuta as the donor, and an elite indica cultivar IR24 as the recurrent parent, constructed by MAS. Each of the alleles from O. minuta was found to be in at least one IL via genotypic analysis with 164 SSR markers. Based on the evaluation of the phenotype of these ILs, lines 41, 11 and 7 showed high resistance to BB, BPH and WBPH, respectively. These O. minuta and O. sativa

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introgression lines will serve as genetic materials for identifying and using favorable genes from O. minuta.

ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (Grant No. 31160277), the Ministry of Science and Technology of China (Grant No. 2010AA101803), the Ministry of Agriculture of China (Grant No. 2011ZX08001-001), the Guangxi Science and Technology Department, China (Grant No. 10100005-8, 2012GXNSFAA 053056), and the Guangxi Academy of Agricultural Sciences, China (Grant No. 2011JZ02 2011JM02, 12-071-09).

SUPPLEMENTAL DATA The following material is available in the online version of this article at http://www.sciencedirect.com/science/ journal/16726308; http://www.ricescience. org. Supplemental Fig. 1. Graphical genotypes of the 89 core ILs of O. minuta in the study.

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