1Toyama Agricultural Research Center, www.agri.pref.toyama.jp, email firstname.lastname@example.org
2National Institute of Agrobiological Sciences, www.nias.affrc.go.jp/index_e.html, email email@example.com
3National Institute of Crop Science, nics.naro.affrc.go.jp/index-e.html, email firstname.lastname@example.org
4Society for Techno-innovation of Agriculture, Forestry and Fisheries, www.staff.or.jp , email email@example.com
We report a novel breeding approach using DNA-marker-assisted selection introduced based on the development of introgression lines (ILs) developed with a chromosome segment of Kasalath, an indica rice variety, in the genetic background of Koshihikari, an elite japonica variety. ILs are a powerful tool for detecting quantitative trait loci (QTLs) whose genetic contribution is difficult to evaluate. Once a QTL of agricultural interest is detected on a particular chromosome, near-isogenic lines for QTLs (QTL-NILs) can be developed in 1 or 2 additional generations from advanced back-crossed populations. We demonstrate the usefulness of these ILs in the genetic analysis and breeding of spikelet numbers per panicle and culm length.
A novel series of introgression lines with chromosome segments of an indica variety in a genetic background of an elite japonica rice variety were developed to accelerate genetic analysis and breeding.
introgression line, DNA marker, quantitative trait locus, near-isogenic line, rice
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.
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.
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.
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.
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%.
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.
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.
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.
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