ФИЗИОЛОГИЯ РАСТЕНИИ, 2011, том 58, № 3, с. 453-460
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
Fine Mapping of a Quantitative Trait Locus qHD3-1, Controlling the Heading Date to a 29.5-kb DNA Fragment in Rice
© 2011 W. Y. Wang*, X. Liu*, H. F. Ding*, M. S. Jiang**, G. X. Li**, W. Liu*,
C. X. Zhu***, F. Y. Yao*
* High-Tech Research Center, Shandong Academy of Agricultural Science, Jinan, P. R. China ** Shandong Rice Research Institute, Jining, Shandong, P. R. China *** State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, Shandong, P. R. China
Received August 6, 2010
In our previous studies, a single segment substitution line (SSSL) W23-03-8-9-1 with substituted interval of PSM301-PSM306-PSM305-PSM304-RM3894-RM3372-RM569-RM231-RM545 on chromosome 3 has been found to comprise a gene for extremely early heading date. To map this gene, the SSSL W23-03-8-9-1 was crossed with the recipient Huajingxian (HJX74) to develop an F2 segregating population. The distribution of early and late heading plants in this population fitted a segregation ratio of 3 : 1, indicating that early heading was controlled by a dominant gene. Using a random sample of 520 individuals from the F2 segregation population, the qHD3-1 locus was mapped between two SSR markers, RM3894 and RM3372, with genetic distances of 1.2 and 1.1 cM, respectively. For fine mapping of qHD3-1, a large F2:3 segregating population was developed, with 6000 individuals from the F2 plants heterozygous in the RM3894 and RM3372 regions. The analysis of recombinants in the qHD3-1 region put the gene locus into an interval of 29.5 kb flanked by the left marker 3HD8 and the right marker 3HD9. Sequence analysis of this fragment predicted eight open reading frames. One of them, ORF8, with its molecular function predicted to encode ribonuclease III activity and RNA binding, is considered the most interesting candidate gene.
Keywords: Oryza sativa — heading date — single segment substitution line — physical mapping — sequence information
INTRODUCTION
Heading date (HD) plays a principal role in the regional adaptability of rice (Oryza sativa L.) and is a key factor in attaining the desired yield level. Control of HD is one of the leading objectives in rice breeding. Growth period from sowing to heading consists of the vegetative growth phase and reproductive phase. The latter does not differ considerably across cultivars, while the former, which is subdivided into the basic vegetative phase (BVP) and photoperiod-sensitive phase (PSP), is the main determinant for HD in rice [1, 2].
In rice, earliness is a key target in improving economical efficiency of rice production. Some genetic studies have been conducted on extremely early heading in rice [3—5]. It has been reported that two genes, qDTH-7-1 and qDTH-7-2, both on chromosome 7 related to extremely early heading, are involved in HD
Abbreviations: BVP — basic vegetative phase; HD — heading date; ORF — open reading frame; PSP — photoperiod-sensitive phase; QTL — quantitative trait locus; SSSL — single segment substitution line.
Corresponding author: Fang-Yin Yao. High-Tech Research Center, Shandong Academy of Agricultural Science, Jinan 250100, P. R. China. Fax: 86-531-8317-9440; e-mail: yaofy@163.com
variation among cultivars adapted to the northern limit of rice cultivation, such as Hokkaido and Europe [6]. Although QTLs for extremely early heading have been identified during the last two decades, little is known about the corresponding genes.
Recently, more and more QTLs for HD have been fine-mapped and cloned. Using a large segregating population derived from an advanced backcross progeny between ajaponica variety, Nipponbare, and an indica variety, Kasalath, the Hd3 locus was genetically dissected into two loci, Hd3a and Hd3b [6]. Using a QTL-NIL population, qSPP7, a major QTL for HD, was fine-mapped to a 0.2 cM region [7], renamed Ghd7, and finally cloned [8]. At present, seven QTLs for heading date (Hd1, Hd6, Hd3a, Ehdl, Ehd2, RFT1, and Ghd7) have been cloned using map-based strategy [9—15], and the underlying rice genes were shown to share a high degree of similarity with those from Arabidopsis thaliana L., although Arabidopsis and rice are model long- and short-day plants, respectively, for understanding the genetic mechanisms of floral control.
With completely sequenced rice genome, practically unlimited numbers of molecular markers can be developed using the sequence information available in
public databases. Chromosome-walking will be greatly reduced or even avoided in the progress of map-based gene isolation, and the Rice Genome Automated Annotation system (http://RiceGAAS.dna.affrc.go.jp), combined with high-resolution of gene fine mapping, is helpful in identifying the candidate genes. Thousands of QTL have been mapped in the latter two decades, and the focus ofQTL research in rice is being gradually transferred from fine mapping to QTL isolation.
In our previous studies, a QTL qHD3-1 affecting HDwas mapped to the substituted interval of PSM301— PSM306-PSM305-PSM304-RM569-RM231-RM489— RM545 on rice chromosome 3 flanked by SSR markers PSM306 and RM489 [16]. Presently we aimed at fine-mapping this QTL as a single Mendelian factor based on a large secondary segregation population.
MATERIALS AND METHODS
Mapping population. Based on previous research of our group, the SSSL W23-03-8-9-1 with substituted interval of PSM301-PSM306-PSM305-PSM304-RM3894-RM3372-RM569-RM231—RM545 on chromosome 3 was found having a gene for extremely early HD and manifested stable and early heading in various environments of Shandong, Guangdong, and Hainan provinces.
In order to fine-map the gene, the SSSL W23-03-8-9-1 was crossed with the recipient HJX74 to develop an F2 segregating population. The resultant F1 was selfed to produce F2 seeds. The gene for HD was mapped on the short arm of chromosome 3 between two SSR markers, RM3894 and RM3372, by using a random sample of 520 individuals from the F2 segregating population. The F2 plants, with the heterozygous region around the target gene locus, were used to develop an F3 segregating population containing 6000 individuals for high-resolution linkage mapping of the qHD3-1 locus. Meanwhile, extremely early heading and weak or no PSP were identified in W23-03-8-9-1.
Field experiment and HD investigation. The segregating population and the corresponding parents were planted in April—October 2008 and 2009 on the farm of the High-Tech Research Center, Shandong Academy of Agriculture Science, Jinan, P. R. China. Each entry was planted at a spacing of16.6 cm from plant to plant and 25 cm between rows. Days to heading of each plant were scored when the first panicle (1-cm-long) was emerged. The HD phenotypes in the segregating population revealed a large variation.
DNA extraction and PCR amplification. Rice ge-nomic DNA was extracted from fresh leaves harvested from each plant using a microisolation method described by Zheng et al. [17] with a minor modification.
The PCR was performed in 20 |l reaction mixtures containing 30 ng of template DNA, 0.15 |l of 10 mM dNTPs, 1.5 units of Taq DNA polymerase, 2 |l of10X PCR buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3),
1.5 mM MgCl2, and 0.01% gelatin), and 1.5 |l of 2 |M forward and reverse primers. Cycling conditions were 5 min at 94°C followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 5 min. PCR products were subjected to electrophoresis on 6% polyacrylamide gel. After completion of the electrophoresis, the gels were silver-stained.
Marker selection and primer design. SSR markers were used to determine the genotypes of marker loci for each plant. Known SSR markers were adopted from the public database released by the International Rice Microsatellite Initiative (www.gramene.org); other SSR and InDel markers were developed at the Plant Molecular Breeding Research Center, South China Agricultural University, Guangzhou, China. The sequence between RM3894 and RM3872 downloaded from a publicly available rice genome sequence (www.ncbi.nlm.nih.gov) was exploited to design new SSR markers by using SSRIT procedures (www. gramene.org/db/searches/ssrtool) and the primer premier version 5.0. Sequence diversities between indica '93-11' andjaponica 'Nipponbare' were used to develop InDel markers. All primer pairs flanking SSRs or InDels were designed in light of the following parameters: 18—25 nucleotides in length, absence of secondary structure, a GC content around 50%, and a melting temperature around 55°C.
Map construction. The genetic map of the gene locus was developed using MAPMAKER/EXP version 3.0 based on genotypic and phenotypic data for 520 segregating individuals in F2 population. The genetic distance (in centimorgans, cM) was calculated using the Kosambi function.
Fine physical mapping of the gene for HD. An F3
segregating population comprising 6000 individuals was used to fine-map the gene locus. SSR markers RM3894 and RM3372, which flanked the target gene, were first used to detect the recombinants in these plants. Additional PCR-based markers were subsequently developed according to the sequence information of the reference Nipponbare and 93-11. The physical map of the qHD3-1 locus was constructed by using bioinformatics analysis. Molecular markers linked with the qHD3-1 locus were landed on the BAC or PAC clones of the reference Nipponbare and released by IRGSP using the sequence homology search tool BLASTN (www.blast.ncbi.nlm.nih.gov/BLAST.cgi). Sequences of these clones were aligned using the pair-wise BLAST sequence tool (ncbi.nlm.nih.gov/blast. cgi/b12seq/b12.html) for constructing the BAC/PAC contigs spanning the qHD3-1 gene locus. At the same time, the genotype and phenotype of the key recombinants and their progenies were analyzed in the present study.
FINE MAPPING OF A QUANTITATIVE TRAIT LOCUS qHD3-1
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RESULTS
Frequency distribution and inh
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