A new approach for obtaining rapid uniformity in rice (Oryza sativa L.) via a 3x x 2x cross

PLANT GENETICS

SHORT COMMUNICATION

A new approach for obtaining rapid uniformity in rice (Oryza sativa L.) via a 3x x 2x cross

Shaochen Xing^I; Yuhong Cai^II; Kaida Zhou^III

^IBiotechnology Research Center, Jilin Academy of Agricultural Sciences, Changchun, China

^IIResearch Center of Agricultural Environment and Resources, Jilin Academy of Agricultural Sciences, Changchun, China

^IIIRice Research Institute, Sichuan Agricultural University, Chengdu, China

^{Send correspondence to}

ABSTRACT

A triploid (2n = 3x = 36) rice plant was obtained by screening a twin seedling population in which each seed germinated to two or three sprouts that were then crossed with diploid plants. One diploid plant was chosen among the various F₁ progenies and developed into an F₂ population via self-pollination. Compared with the control variety Shanyou 63, this F₂ population had a stable agronomical performance in field trials, as confirmed by the F-test. The stability of the F₂ population was further substantiated by molecular analysis with simple sequence repeat markers. Specifically, of 160 markers assayed, 37 (covering all 12 chromosomes) were polymorphic between the parental lines. Testing the F₁ hybrid individually with these markers showed that each PCR product had only a single band instead of two bands from each parent. The bands were identical to either maternal (23 markers) or paternal (eight markers) bands or distinct from both parents (six markers). The amplified bands of all 60 randomly selected F₂ plants were uniform and identical to those of the F₁ hybrid. These results suggest that the F₁ plant is a non-segregating hybrid and that a stable F₂ population was obtained. This novel system provides an efficient means for shortening the cycle of hybrid rice seed production.

Key words: F-test, polyploidy, rice, SSR marker, stability.

Since the discovery that tetraploid plants can be regenerated from callus tissue on cut stems of diploid Solanum nigrum (Winkler, 1916) polyploidy has been recognized as a common phenomenon in nature and an important factor in the evolution of plant genomes. Polyploidy occurs in many taxa and is particularly widespread in flowering plants. At least half of the known angiosperm species have experienced polyploidy in their evolutionary history (Hieter and Griffiths, 1999; Echardt, 2001; Wu et al., 2001). Polyploidy often results in considerable genomic changes such as chromosomal rearrangements, gene loss and changes in DNA methylation (reviewed by Adams, 2007).

Compared to their diploid and haploid counterparts, polyploid organisms often express specific characteristics such as larger cell and body sizes (Sugiyama, 2005) and a propensity to develop apomixis (Naumova et al., 1999). Studies in rice have identified stable lines in an early generation from the progeny of 3x x 2x or 4x x 2x crosses (Wu et al., 1999; Xing et al., 2000). Wang et al. (1999) also reported that loss of heterozygosity (LOH) from 2x x 2x crosses led to stable panicle rows in F₂ progeny and subsequently proposed a mechanism of "assortment mitosis" (Wang et al., 2001) that was supported by cytological evidence (Wang et al., 2006).

In this study, we screened another triploid x diploid cross that differs from the crosses reported by Wu et al. (1999) and obtained a diploid F₁ plant that generated a stable F₂ population. This system will be helpful in providing new insights into the potential application of polyploidy and should allow the development of an efficient breeding system to greatly shorten the breeding cycle.

Individuals of the triploid plant DB43, originally derived from a twin seedling population, served as the maternal parent. A diploid japonica-type cultivar, ZD2, served as the paternal parent. The 25 F₁ seeds from a DB43/ZD2 cross were obtained by direct hybridization followed by embryo rescue. Five plants among the F₁ seedlings were cytologically confirmed to be diploid (Xing and Zhou, 2000). Self-pollinated F₂ seeds were collected to generate five F₂ populations in the following year. Only one of these five populations appeared to be phenotypically uniform in the field.

To verify the phenotypic uniformity of the F₂ population, five major morphological traits (plant height, panicle length, number of productive tillers, seed-setting rate and 1000-grain weight) were investigated and compared with the very widespread Shanyou 63 as the control variety by using the F-test (Table 1). The F value (sd₁/sd₂) for each trait was < 1.0, indicating that the F₂ population was stable for these agronomical traits under the field conditions used.

Microsatellite markers were used to assess the relationship between parents and the F₁ hybrid and to test the stability of the F₂ population. PCR was done with the following assay mixture in 25 mL: 40 ng of template DNA, 200 mM of each of the four dNTPs, 2.5 mL of 10x buffer, 1 unit of DNA Taq polymerase, 2 mM MgCl₂ and 0.25 mM of each of the two primers. The PCR amplifications were done in a Perkin Elmer 9600 GeneAmp PCR System with the following conditions: 94 °C for 7 min, followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, and a final extension at 72 °C for 10 min. The amplification products were separated by electrophoresis in 3% (w/v) agarose gels followed by staining with ethidium bromide and examination under UV light.

One hundred and sixty simple sequence repeat (SSR) markers were used to screen for polymorphisms in the parental lines: 37 of these markers covering all 12 rice chromosomes were polymorphic (Figure 1). More importantly, when these polymorphic markers were used to amplify the F₁ DNA template individually, each F₁ product showed only a single band instead of the expected two bands that were supposed to be identical to those from the two parents. Comparison of the PCR patterns of the parents with those of the F₁ hybrid plant allowed the polymorphic SSR markers to be classified into three groups: Group 1 included 23 SSR markers for which the size of the band amplified from F₁ was identical to that of the maternal parent (Figure 2A), Group 2 included eight SSR markers for which the size of the band amplified from F₁ was identical to that of the paternal parent (Figure 2B) and Group 3 included six SSR markers for which the size of the F₁ amplified band was completely different from either parent (Figure 2C). The 31 SSR markers in Groups 1 and 2 originated from either the maternal or paternal parent, rather than from both parents, implying that these loci are truly homozygous.

To confirm the uniformity of the F₂ population, 60 DNA samples were randomly selected from the F₂ population, together with DNA from both parents and the F₁ hybrid, and used as templates for PCR amplification. The resulting PCR products from all of the polymorphic SSR markers were compared to each other on the same agarose gel. The resulting pattern indicated that all of the 60 samples were uniform and coincided with the genotype of F₁ plant. Three markers representing each of the different groups and 30 F₂ samples were chosen to illustrate this uniformity (Figure 2D).

Six markers had completely different PCR patterns with F₁ DNA template from those of their parents. This phenomenon has also been observed in wheat (Liu et al., 1998), although the mechanism of allele loss following hybridization remains unclear.

Various studies have shown that polyploidy can lead to immediate, extensive changes at the genic and genomic levels, resulting in differential gene silencing or gene loss (reviewed by Udall and Wendel, 2006). Josefsson et al. (2006) showed that maternal imprinting of PHERES1(PHE1), the gene of type I MADS-box, and paternal imprinting of MEDEA(MEA), the gene encodes a polycomb group (PcG) protein, appeared to be lost in hybrids between tetraploid Arabidopsis thaliana and diploid Arabidopsis arenosa. This phenomenon, known as early generation stability, has previously been reported in rice from apomixis (Chen, 1992), although not all studies have confirmed this (Shi et al., 1996). The results of our experiment cannot be explained by apomixis because the markers tested in non-segregating diploid progeny were of mixed paternal and maternal origins. The most probable explanation in this case was recombination followed by chromosomal elimination in mitotic cells of the F₁ hybrid.

Our results indicate that the F₂ population was non-segregating and should theoretically be stable in subsequent generations. This unusual phenomenon, which differs from the findings previously reported by Wang et al. (1999), should prove useful for breeding restorer lines of hybrid rice (Zhou et al., 2007).

Acknowledgments

We thank Professor Lihuang Zhu of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, for his support during this work.

Received: May 27, 2009; Accepted: December 7, 2009.

Associate Editor: Everaldo Gonçalves de Barros

• Adams KL (2007) Evolution of duplicate gene expression in polyploidy and hybrid plants. J Hered 98:136-141.
• Akagi H, Yokozeki Y, Inagaki A and Fujimura T (1996) Microsatellite DNA markers for rice chromosomes. Theor Appl Genet 93:1071-1077.
• Chen J (1992) A Collection of Papers on Breeding of Apomictic Rice. China Science and Technology Press, Beijing, 118 pp.
• Chen X, Temnykh S, Xu Y, Cho YG and McCouch SR (1997) Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.). Theor Appl Genet 95:553-567.
• Echardt NA (2001) A sense of self: The role of DNA sequence elimination in allopolyploidization. Plant Cell 13:1699-1704.
• Hieter P and Griffiths T (1999) Polyploidy - More is more or less. Science 285:210-211.
• Josefsson C, Dilkes B and Comai L (2006) Parent-dependent loss of gene silencing during interspecies hybridization. Curr Biol 16:1322-1328.
• Liu SB, Jia JZ, Wang HG, Kong LR and Zhou YH (1998) Special chromosome markers for E. Genome and DNA polymorphism between Agropyrom elongatum (2n = 14) and common wheat detected by RAPD markers. Acta Agron Sin 24:687-690 (In Chinese, abstract in English).
• Naumova TN, Hayward MD and Wagenvoort M (1999) Apomixis and sexuality in diploid and tetraploid accessions of Brachiaria decumbens Sex Plant Reprod 12:43-52.
• Shi GC, Ni PC and Song JX (1996) The identification of apomixis in rice variety 84-15. Sci Agric Sin 29:56-61 (In Chinese, abstract in English).
• Sugiyama SI (2005) Polyploidy and cellular mechanisms changing leaf size: Comparison of diploid and autotetraploid populations in two species of Lolium Ann Bot 96:931-938.
• Temnykh S, Park WD, Ayres N, Cartinhour S, Hauck N, Lipovich L, Cho YG, Ishii T and McCouch S (2000) Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.). Theor Appl Genet 100:697-712.
• Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S and McCouch S (2001) Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): Frequency, length variation, transposon associations, and genetic marker potential. Genome Res 11:1441-1452.
• Udall JA and Wendel JF (2006) Polyploidy and crop improvement. Crop Sci 46:S3-S14.
• Wang RRC, Li XM and Chatterton NJ (1999) Loss of heterozygosity and accelerated genotype fixation in rice hybrids. Genome 42:789-796.
• Wang RRC, Li XM and Chatterton NJ (2001) A proposed mechanism for loss of heterozygosity in rice hybrids. Euphytica 118:119-126.
• Wang RRC, Li XM and Chatterton NJ (2006) Cytological evidence for assortment mitosis leading to loss of heterozygosity in rice. Genome 49:556-557.
• Winkler H (1916) Über die experimentelle Erzeugung von Pflanzen mit abweichenden Chromosomenzahlen. Z Bot 8:417-531.
• Wu R, Gallo-Meagher M, Littell RC and Zeng ZB (2001) A general polyploid model for analyzing gene segregation in outcrossing tetraploid species. Genetics 159:869-882.
• Wu XJ, Wang XD, Zhou KD and Zhu LH (1999) A non-segregation F₂ population derived from the cross of triploidxdiploid in rice. Acta Bot Sin 41:1067-1071 (In Chinese, abstract in English).
• Xing SC and Zhou KD (2000) Genetic study on the self-bred progenies of special autopolyploid rice. J Sichuan Agric Univ 18:308-310 (In Chinese, abstract in English).
• Xing SC, Zhou KD and Zhu LH (2000) Identification of one early-generation population from 4nx2n cross using microsatallite markers. J Agric Biotechnol 13:365-367 (In Chinese, abstract in English).
• Zhou LJ, Wang L, Wei Q, Ao GH, Wu XJ and Li SG (2007) Utilizing early generation stability characteristic to breed restorer line of hybrid rice. Chin J Rice Sci 21:265-269 (In Chinese, abstract in English).

Publication Dates

• Publication in this collection
  26 Mar 2010
• Date of issue
  2010

History

• Accepted
  07 Dec 2009
• Received
  27 May 2009