Abstract

Media summary

Keywords

introgression line, DNA marker, quantitative trait locus, near-isogenic line, rice

Introduction

Recent advances in rice genome research have provided powerful tools, such as whole genome sequences and DNA markers, for the genetic analysis of rice (Sasaki 2003, Harushima et al. 1998, McCouch. et al. 2002). It is now possible to make markers at arbitrary regions in rice chromosomes. DNA markers enable the genetic analysis of quantitative traits in conjunction with the development of plant materials (Yano et al. 1997, Lin et al. 1998, Xiao et al. 1996). Near-isogenic lines (NILs) are vital to analyzing quantitative trait loci (QTLs), such as positional cloning and marker-assisted breeding, in detail. The long time required to develop these materials has prevented many breeders from using DNA markers in their breeding programs. To solve this problem, a series of chromosome substitution lines was developed in tomato (Eshed et al. 1995), Brassica (Howell et al. 1996), rice (Kubo et al. 2002), and effectively used as plant material for QTL mapping and breeding. We developed a series of introgression lines (ILs) of the elite Japanese rice variety Koshihikari to facilitate genetic analysis and breeding.

This paper details QTL analysis for spikelet numbers per panicle (SNP) and culm length (CL) using the ILs we developed and the development of near-isogenic lines of QTLs (QTL-NIL) for each trait. The advantages of a novel breeding approach developing ILs in rice as potential genetic resources are discussed.

Materials and Methods

IL Development

The rice variety Koshihikari was crossed with Kasalath and the resultant F₁ plant crossed with Koshihikari to produce BC₁F₁. Single-seed descent was used to develop a BC₁F₃ generation. A randomly selected female parent BC₁F₃ of each BC₁F₃ line was crossed with a male parent Koshihikari to produce secondary F₁ (SF₁). Based on the genotype information of 116 RFLP markers in BC₁F₃ plants (Yamamoto et al. 2001), we selected 49 BC₁F₃ plants based on the following selection criteria: First, a relatively large, single chromosome segment is substituted for Kasalath to maintain the higher level of homozygosity of the Koshihikari allele in a nontarget chromosomal region. Second, target chromosome segments were partially overlapped in selected lines to cover 12 rice chromosomes. BC₁F₃ lines were selected for starting materials for the second round of back-crossing. Two or 3 SF₁ plants derived from 49 BC₁F₃ lines were crossed with Koshihikari to produce SBC₁F₁. Two plants whose target chromosome segment is heterozygous were selected from each SBC₁F₁ line based on the genotype of cleaved amplified polymorphic sequence (CAPS) markers and recrossed with Koshihikari to produce SBC₂F₁. Approximately 10 SBC₂F₁ plants were selected based on the genotypes of target chromosome segments. At least 2 plants whose target chromosome segments were heterozygous were crossed with Koshihikari to produce SBC₃F₁.

Plants with desired genotypes that possess no more than 4 heterozygous chromosome segments at nontarget regions and maintain the target chromosome segment as heterozygous were selected from BC₃F₁, BC₄F₁, and BC₅F₁ lines. Finally, 39 populations (BC₃F₂, BC₄F₂, and BC₅F₂) derived from self-pollinated progeny of selected plants were cultivated and 39 candidate plants carrying homozygous chromosome segments of Kasalath at the target regions selected.

QTL phenotyping and detection for SNP and CL

Ten plants per ILs were cultivated in an experimental field in Toyama, Japan. SNP and CL of main stems were scored for each plant and means calculated. QTLs were detected using the t test for the difference between means of the IL line and Koshihikari. A probability of 0.01 was used as the threshold for detecting a putative QTL.

QTL-NIL Development

To finely map QTLs of agriculturally interest, such as a Kasalath allele with increased SNP or decreased CL at QTLs, we cultivated 2 mapping F₂ populations derived from each F₁ plant whose target chromosome segment was heterozygous. These populations consisted of 144 and 94 plants. As a result of linkage analysis, both QTLs were mapped as 1 gene at chromosome 7 for SNP and at chromosome 11 for CL. QTL-NILs were developed by selecting recombinant plants using flanking DNA markers and by selecting plants with homozygosity at the target allele using DNA markers nearest to the QTL.

Results

IL Characterization

Graphical genotypes of 39 ILs were determined using 118 RFLP markers, which were distributed on 12 rice chromosomes (Fig. 1). Although the small chromosomal region at the distal end of the long arm of chromosome 12 and the proximal region of chromosome 2 were not substituted for the Kasalath chromosome, substituted chromosome segments in ILs covered most regions of the 12 chromosomes. Basically, in each IL, a single chromosome segment was substituted for Kasalath in the genetic background of Koshihikari. However, in some lines, small chromosomal segments of Kasalath were not substituted for Koshihikari for the nontarget region in ILs. In addition, 1 small region of chromosome 1 could not be fixed as homozygous of Kasalath and remained heterozygous due to segregation distortion of unknown origin. Whole chromosomes 6, 7, and 10 were likely to be substituted in 3 lines. Based on the genetic distance in the linkage map described by Harushima et al (1998), the percentage of substituted segment in each IL was estimated at from 1% to 9%.

QTL mappings for SNP and CL

SNP of Koshihikari was 144.8±10.4 and the variation of SNP in 39 ILs ranged from 80 to 191. A significant difference in SNP was observed between 8 ILs and Koshihikari. In lines 207, 208, 211, 214, 223, and 232, SNP was decreased compared to that of Koshihikari but increased in lines 201 and 222, suggesting that at least 7 chromosomal regions contained putative QTLs for SNP.

CL of Koshihikari was 93.7±2.0 and the variation of SNP in 39 ILs ranged from 80.5 to 114.9. A significant difference in CL was observed between 10 ILs and Koshihikari. Of 10 lines, 217, 232, 234, and 235 had CL decreased compared to that of Koshihikari but increased in the remaining lines, suggesting that at least 7 chromosomal regions contained putative QTL for CL.

QTL-NIL Traits

For SNP, QTL-NIL was substituted for Kasalath at the small chromosome segment at the near end of the long arm of chromosome 7. SNP was more numerous than that of Koshihikari by 30%, so the number of spikelets per plant of QTL-NIL increased by 20% compared to that of Koshihikari.

For CL, QTL-NIL was substituted for Kasalath at the small chromosome segment of chromosome 11. CL was 10 cm shorter than that of Koshihikari, although the panicle length of QTL-NIL for CL and Koshihikari were equal.

Conclusions

In rice, 3 series of ILs with a japonica background were been developed, i.e., indica (Aida et al. 1997), Oryza glumaepatula (Sobrizal et al. 1999), and Oryza glaberrima Steud (Doi et al. 1997). The genetic background of these ILs is not, however, of an elite variety. To facilitate breeding after detecting QTLs, ILs must be developed with an elite genetic background. We developed 39 ILs in which substituted Kasalath chromosome segments covered most of the 12 chromosomes in the genetic background of Koshihikari.

Since the genetic backgrounds of the ILs are Koshihikari, as described by Tanksley (1996), once we determine that Kasalath possesses economically valuable positive alleles at QTLs, we can quickly and easily develop QTL-NILs using specific IL as parental lines for additional back-crossing. Time required from QTL discovery to construction and testing of improved QTL-NILs is reduced using such materials.

We found desirable alleles in Kasalath at QTLs for SNP and CL, even though Kasalath itself did not appear to be good material for breeding. This suggests that ILs are also useful for mining new alleles for trait improvement.

References

Aida, Y., H. Tsunematsu, K. Doi, and A. Yoshimura (1997) Development of a series of introgression lines of Japonica in the background of Indica rice. Rice Genet. Newsl. 14: 41-43.

Doi, K., N. Iwata, and A. Yoshimura (1997) The construction of chromosome substitution lines of African rice (Oryza glaberrima Steud.) in the background of japonica rice (O. sativa L.). Rice Genet. Newsl. 14: 39-41.

Eshed Y. and D. Zamir (1995) An introgression line population of Lycopersion pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics 141: 1147-1162.

Howell, P.M., D.F. Marshall, and D.J. Lydiate (1996) Towards developing inter-varietal substitution lines in Brassica napus using marker-assisted selection. Genome 39: 348-358.

Harushima, Y., M. Yano, A. Shomura, M. Sato, T. Shimano, Y. Kuboki, T. Yamamoto, S.Y. Lin, B.A. Antonio, A. Parco, H. Kajiya, N. Huang, K. Yamamoto, Y. Nagamura, N. Kurata, G.S. Khush, and T. Sasaki (1998) A high-density genetic linkage map with 2275 markers using a single F2 population. Genetics 148: 479-494.

Kubo, T., Y. Aida, K. Nakamura, H. Tsunematsu, K. Doi, and A. Yoshimura (2002) Reciprocal chromosome segment substitution series derived from Japonica and Indica cross of rice (Oryza sativa L.). Breed. Sci. 52: 319-325.

Lin, S.Y., T. Sasaki, and M. Yano (1998) Mapping quantitative trait loci controlling seed dormancy and heading date in rice, Oryza sativa L., using backcross inbred lines. Theor. Appl. Genet. 96: 997-1003.

McCouch, S.R., L. Teytelman, Y. Xu, K.B. Lobos, K. Clare, M. Walton, B. Fu, R. Maghirang, Z. Li, Y. Xing, Q. Zhang, I. Kono, M. Yano, R. Fjellstrom, G. DeClerrck, D. Scneider, A. Cartinhour, D. Wate, and L. Stein (2002) Development and Mapping of 2240 New SSR Markers for Rice (Oryza sativa L.) DNA Research 9: 199-207.

Sasaki, T. (2003) Rice Genome Analysis: Understanding the Genetic Secrets of the Rice Plant. Breeding Science 53: 281-289

Sobrizal, K. Ikeda, P.L. Sanchez, K. Doi, E.R.Angeles, G.S. Khush and A. Yoshimura (1999) Development of Oryza glumaepatula introgression lines in rice, O. sativa L. Rice Genet. Newsl. 16: 107-108.

Tanksley, S.D. and J.C. Nelson (1996) Advanced backcross QTL analysis: a method for the simultaneously discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor. Appl. Genet. 92: 191-203.

Xiao, J., J. Li, L. Yuan, and S.D. Tanksley (1996) Identification of QTLs affecting traits of agronomic importance in a recombinant inbred population derived from a subspecific rice cross. Theor. Appl. Genet. 92: 230-244.

Yamamoto, T., F. Taguchi-Shiobara, Y. Ukai, T. Sasaki, and M. Yano (2001) Mapping quantitative trait loci for days-to-heading, and culm, panicle, and internode lengths in a BC1F3 population using an elite rice variety, Koshihikari, as the recurrent parent. Breed. Sci. 51: 63-71.

Yano, M., Y. Harushima, Y. Nagamura, N. Kurata, Y. Minobe, and T. Sasaki (1997) Identification of quantitative trait loci controlling heading date in rice using a high-density linkage map. Theor. Appl. Genet. 95: 1025-1032.