Proper plant height is crucial for lodging resistance to improve the rice crop yield. In this paper, we report the identification of a novel plant height gene ph1 by map based cloning strategy. The ph1 gene was mapped to a region closely linked to semi-dwarf 1 on chromosome 1 with additive effects more than 50 cm on the plant height and 2 days on the heading date in a BC4F2 population and its progeny. ph1 was then fine mapped to BAC contig AP003227. Gene annotation indicated that LOC_OS01g65990 encoding a Chitin-inducible gibberellins-responsive protein (CIGR), which belongs to the GRAS family, might be the right candidate gene. Co-segregation analysis finally confirmed its identity as the candidate gene. A higher expression level of ph1 was detected in the tissues of tall plants compared to those of short plants. ph1 contributed a tremendous genetic effect on plant height, which could be a new resource for breeding semi-dwarf varieties. The evaluation of CIGR as the ph1 candidate will allow us to discover the mechanism underlying plant height determination.
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Keywords: Rice (Oryza Sativa); Plant height; Fine mapping; Gene identification; Map based-cloning.
Rice is the main food crop in many parts of the world, but predominantly in Asia and Africa. The rice yield is determined either directly or indirectly by many traits. Among them, plant height plays a pivotal role, and dwarfism is a valuable trait in crop breeding, because it increases lodging resistance and decreases crop damage due to wind and rain, thereby increasing the crop yield. The second half of the 20th century is very prominent in crop science, because of the famous 'Green Revolution', where the semi-dwarf variety IR8, also known as 'miracle rice', enabled dramatic yield increases and helped to avert predicted food shortages in Asia during that period . The short stature of IR8 is due to a mutation in the plant's sd1 gene, which was later identified as encoding an oxidase enzyme (GA20ox-2) involved in the biosynthesis of gibberellin, a plant growth hormone .
Moreover with the availability of the complete rice sequence and advancement in molecular marker technology, different types of mapping populations such as double-haploids, F2, F3, recombinant inbred lines (RILs), backcross inbred lines (BILs) and introgression lines (ILs) were developed for quantitative trait locus (QTL) identification [3, 4]. A limited number of near-isogenic lines (NILs), BILs, advanced backcross (AB) lines or ILs can be used to precisely identify QTLs instead of a large F2 or RIL population [5, 6]. Since such lines are homozygous, numerous genetically identical plants can be evaluated, thus increasing the accuracy of phenotyping without increasing the efforts of genotyping. These IL series help the fine mapping of QTLs as well as the precise estimation of the QTL effects . Several research groups have reported the plant height genes in rice [8, 9]. Among them, sd1 is the dominant one being widely used in breeding to develop semi-dwarf varieties.
However, the frequent use of single sd1 gene sources may cause genetic vulnerability to pests and diseases . Therefore, it is necessary to develop alternate or new sources of dwarfs for broadening the genetic base of dwarfism. In addition, the functional dissection of more gene-regulated plant height traits will be helpful to further understand the molecular mechanism involved in semi-dwarfism. Identifying useful novel semi-dwarf genes is important for the genetic manipulation of plant architecture in practical rice breeding.
In this study, we identified a major QTL on chromosome 1, ph1, which controls plant height and heading date from introgression lines derived from two contrasting parents in plant height. Further, we report here the fine mapping of ph1 and validation of its candidate gene by co-segregation analysis, comparative sequencing and expression analysis.
Materials and Methods
Plant materials and development of mapping population
A set of introgression lines (BC4F2) was developed from a cross between two contrasting Oryza sativa L. indica varieties in plant height, namely Zhenshan 97, a Chinese native, and Pokkali, an Indian native (Fig 1A); Zhenshan 97 was used as the recurrent parent. One introgression line (BC4F2 family) showed a clear segregation in plant height in 2003. The family, which consisted of 172 plants, and its progeny (BC4F3) were grown in summer 2004 and 2005 in a bird-net-equipped experimental farm of Huazhong Agricultural University, Wuhan, China (29°58'N, 113°41'E). Field experiments were carried out in a randomized complete block design.
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For the progeny test, 24 plants of each BC4F3 family were grown in a two-row plot in Wuhan in 2006. The 20 plants growing in the middle were investigated for plant height and heading date. Field management, including irrigation, fertilizer application and pest control, followed essentially the normal agricultural practices.
Fine mapping population
A large BC4F2 population of 6400 plants was grown for plant height in 2005 in Wuhan. The progenies of twelve recombinants between the gene ph1 and two closely linked flanking markers were sowed in summer 2007 in the same field where the progeny of BC4F2 population grown.
DNA extraction, PCR and marker analysis
DNA was extracted from fresh leaf samples collected at the seedling stage using a modified CTAB protocol . PCR was performed using a hot start Taq polymerase (TaKaRa) under the following conditions: 95°C for 4 min, 35 cycles of 95°C for 40 s, 55-58°C for 40 s and 72°C for 1 min, followed by 72°C for 6 min. SSR markers were used for developing a genetic linkage map. In addition to SSR markers, three newly developed InDel markers for fine mapping are listed in Table S1, according to the publicly available rice genome sequences (http://www.rgp.dna.affrc.go.jp). Markers were designed according to Temnykh et al. . The SSR assay was conducted as described .
Plant height and heading date measurement
Plant height was recorded from the field surface to the top of the highest panicle. At the seed maturation stage, the average panicle length, number of internodes and their lengths were recorded for both Zhenshan 97 and Pokkali. The heading date was recorded as the days from sowing to the first panicle appearance.
Bulk segregant analysis for primary mapping
Five extremely tall individuals with a plant height greater than 200 cm and five extreme short individuals with plant height less than 90 cm were selected from the BC4F2 population for developing two DNA bulks. Equal quantities of leaf tissue from each individual were bulked, and DNA was extracted from the bulk. To identify the polymorphic markers, the two parents Zhenshan 97 and Pokkali were genotyped with 440 SSR markers covering 12 rice chromosomes.
Genetic linkage map and genetic effect analysis
The molecular linkage map was constructed using Mapmaker/EXP 3.0 . The Kosambi function was used to calculate the genetic distance. Interval mapping was conducted using Mapmaker/QTL . The LOD threshold was of 3.0, determined by 1,000 random permutations at a false positive rate of 0.05 for the trait.
Sequencing analysis of sd1 gene and probable candidate gene
Based on the BAC clones AP003561 and AP003227 sequences (http://www.ncbi.nlm.nih.gov), eight and five specific primer pairs were designed to sequence the promoter and coding regions of the sd1 gene and the probable candidate gene, respectively (Table S1). Sequencing was performed by using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed with the ABI Prism 3730 DNA Analyzer (Applied Biosystems). The sequence alignment was performed with the BLAST network service (http://www.ncbi.nlm.nih.gov) and ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html).
RNA extraction and quantitative real time PCR analysis (qRT-PCR)
For gene expression analysis, total RNA was extracted from duplicate biological samples leaf sheath, root and leaf blade tissues of Zhenshan 97 and Pokkali using the TRIzol reagent (Invitrogen) and first-strand cDNAs were synthesized as described previously .
Real time PCR was performed in an optical 96-well plate with an ABI PRISM 7500 real-time PCR system (Applied Biosystems). Gene-specific primers were designed for the sd1 gene and the candidate gene (CIGR) (Table S1). Each reaction contained 12.5 µL of SYBR Premix Ex Taq (TAKARA), 0.5 µL of ROX Reference Dye (TAKARA), 5.0 µL of cDNA samples and 10 µM gene specific primers in a final volume of 25 µL. The rice actin1 gene was used as the endogenous control. All qRT-PCR reactions were carried out in triplicate. The relative expression levels were analyzed as described .
Phenotypic variations of plant height in BC4F2 population and its progeny
The two parents, Zhenshan 97 and Pokkali, showed highly significant differences in plant height (Table 1). In the BC4F2 population, the difference in average plant height between short and tall plants was up to 100 cm (Fig. 1A). The major differences were caused by both the length of internodes and the number of internodes (Fig. 1B). The short plants had an average of four elongated internodes, while tall plants had six (Fig. 1C). In tall plants, the first and second internodes were of similar length and together contributed approximately 60% to the total culm, while in short plants, the first internode alone contributed more than 60% to the total culm (Fig. 1D).
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In BC4F2, plant height varied widely and ranged from 70 to 233 cm. Among the 172 BC4F2 plants, there were 40 short and 132 tall plants, which was in agreement with the expected segregation ratio (1:3) of a single Mendelian gene (χ2=0.22, P=0.639). Further progeny tests (BC4F3) confirmed that 40 and 44 plants were homozygous allele for Zhenshan 97 and Pokkali, while 88 plants are heterozygous at the locus of target plant height gene. The mean height for short, heterozygote and tall plants was 90.2 cm, 177.9 cm and 204.4 cm, respectively (Fig. 2). The frequencies of the three genotypes matched the expected Mendelian ratio (1:2:1) for single locus segregation (χ2=0.28, P=0.869). This analysis suggested that one gene (termed ph1) controlled the variation of plant height in the BC4F2 population.
Primary mapping of ph1
Among the 150 polymorphic simple sequence repeat (SSR) markers, screened for two bulks, no polymorphisms were observed at the loci of 143 markers. While polymorphisms were identified at the loci of seven markers (RM3304, RM3825, RM6439, RM472, RM5382, RM1339 and RM1387) located on chromosome 1 (Fig. S1). Consequently, these seven markers were used to genotype the 172 BC4F2 plants. A local linkage map covering 35.8-cM was constructed (Fig. 3A). The individual BC4F2 genotypes at ph1 were determined by progeny (BC4F3) tests. Then, ph1 treated as a marker was directly localized into the linkage group, located in a 0.6-cM region flanked by markers RM5382 and RM1339 and co-segregated with RM1339 (Fig. 3A). It is closely linked to sd1.
The interval mapping method was conducted to estimate ph1 genetic effects. ph1 explained 90.3% of the phenotypic variance with additive and dominant effects of 57.1 cm and 30.6 cm, respectively. In the progeny test (BC4F3), ph1 explained 92.7% of plant height variation. ph1 acted as partially dominant in both generations; the Pokkali allele increased plant height (Table 2). Variations in heading date were also observed in the BC4F3 population (Fig. S2). Tall plants always flowered 3-4 days earlier than short ones, indicating the pleiotropic effect of ph1 on heading date. One QTL for heading date was also detected in the same interval where ph1 is located. This QTL could explain 31.2% of the total phenotypic variance with LOD scores of 13.7. The additive effect was 1.5 days. Pokkali allele promoted heading date (Table 2).
Fine mapping of ph1
The revolutionary gene sd1 is located on the right side of the marker RM1339, 180 kb from sd1. In order to find the relation of ph1 to sd1, we performed sequencing for Zhenshan 97 and Pokkali with sd1. The sequencing analysis of sd1 showed no sequence difference in the three exons and two introns (Fig. S3). However, one SNP was detected in the promoter region of sd1. To further investigate the role of sd1, we performed expression analysis. The real time PCR results showed no difference in the expression levels of sd1 in root, leaf sheath and leaf blade tissues in both Zhenshan 97 and Pokkali (Fig. S4). Thus, clear molecular level evidence determined that ph1 is distinct from sd1.
It is worth isolating ph1 because it is a novel plant height gene. In total, 1250 extremely short plants which were assumed to be Zhenshan 97 homozygotes at Ph1, were selected from 6400 plants of the BC4F2 population to screen recombinants between Ph1 and markers RM5382 and RM1339. Nine and three recombinants were identified between RM5382 and ph1, and RM1339 and ph1, respectively. The progeny tests of the recombinants between RM5382 and RM1339 showed an identical short plant height, which was highly significantly shorter than the control Pokkali homozygotes (Table 3). This result confirmed the homozygous identity of the twelve Zhenshan 97 recombinants at ph1. To enhance the resolution of the ph1 local linkage map, we used one SSR (RM11960) and four InDel markers (Table S1) to screen the twelve recombinants; seven recombinants were identified between RM11960 and Ph1, six between PHA and Ph1, four between PHB and Ph1 and one between PHC and Ph1 (Fig. 3B). Thus, Ph1 was narrowed down to the region of approximately 92 kb bounded by markers PHB and PHC (Fig. 3B). One Nipponbare BAC (AP003227) spanned the region exactly.
The candidate gene in the 92-kb target region
There are 17 predicted genes in the 92-kb region according to the rice genome automated annotation database (http://rice.plantbiology.msu.edu/) (Fig. 3C, Table S2). Of these, 12 genes have homology with rice full-length cDNAs and other five are of unknown function. Gibberellin-responsive genes were reported to be associated with plant height [18, 19]. Among all the 12 putative genes, LOC_Os01g65900, which encodes a chitin induced gibberellin responsive protein (CIGR), was gibberellin responsive; thus, CIGR was regarded as the ph1candidate.
Sequencing analysis of the 3640-bp CIGR gene showed single-base substitutions in the promoter region and exon 1 of both Zhenshan 97 and Pokkali, and also detected a 4-bp deletion in the intron region of Zhenshan 97 (Fig. 3D). Between the parents, four SNPs were found at 706 bp, 1048 bp, 1151 bp and 1377 bp upstream of ATG, and two SNPs were located in exon 1. CIGR contains three exons; it encodes a protein with 553 amino acids, but modifications in two amino acids were observed between the parents (Fig. S6).
Co-segregation and expression analysis of the candidate
The genotype data showed that all the five recombinants (32, 58, 67, 83 and 121) are homozygous to Zhenashan 97 at gene specific marker (C1) (Fig. S5) and confirmed that CIGR co-segregated with plant height. Thus, CIGR might be the real candidate for ph1. In addition, our qRT-PCR results showed that Pokkali had significantly higher expression levels of CIGR in root, leaf sheath and leaf blade tissues than Zhenshan 97 (Fig. 4).
Comparison of the genetic effects of ph1 to sd1
In this study, it was clearly confirmed that ph1 is distinct from sd1 by fine mapping with a large BC4F2 population. Comparative sequencing between the parents further confirmed that sd1 did not contribute to plant height variation. We detected no difference in the expression level of sd1 between ZS97 and Pokkali, and this was in agreement with the result that ph1 is a novel gene distinct from sd1. Although ph1 was not the allele of sd1, it showed similar genetic characters on plant height but with larger effects. The two homozygotes of ph1 showed a huge difference of 110 cm in plant height.
The variation in the plant height is mainly contributed by the difference in the length of the panicle, the second, third and fourth uppermost stem internodes; in particular, the second and third showed differences of about 25 cm; But the uppermost stem length showed no significant difference (Fig. 1B). While variation in the plant height by sd1 results from differences in stem length of the first (subtending panicle) and second (subtending flag leaf) stem internodes . Meanwhile, the average number of elongated internodes of tall and short lines is different (Fig. 1C). That is to say, ph1 mainly controls the number of internodes and the length of uppermost internodes except the first one. In addition, ph1 has a pleiotropic effect on the heading date. Pokkali alleles increase the plant height and decrease the days to heading, which is in agreement with their negative correlation (r=-0.55, P< 0.01). Taken together, these results confirm that ph1 is a novel gene based on phenotypic variation.
Chitin-inducible gibberellin-responsive gene might be the probable candidate gene
It is a well known fact that gibberellin plays a key role in the plant height. Many important genes like sd1 and d18 are influenced by gibberellins [2, 20]. In this study, the identity of CIGR as candidate gene was validated by co-segregation analysis (Fig. S5). CIGR genes are gibberellin response genes and were reported to play key roles in defense and plant development [21, 22, and 23]. Moreover, the CIGR gene belongs to the GRAS family. Comparative analysis showed important genes like SCARECROW LIKE 1 (SCL1) in Arabidopsis, Dwarf8 in maize and MOC1 (MONOCULM 1) in rice belong to this family and are related to plant growth and development. CIGR is 85% similar to SCL in Aeluropus littoralis and 77% to the SCL in Arabidopsis. It was reported that SCL6 is involved in shoot branching and plant height by testing double and triple mutants of SCL6-III, SCL6-IV and SCL6-V in Arabidopsis .
In addition, it was showed that the induction of CIGR1 and CIGR2 was observed only upon application of phytoactive GA, and the accumulation of mRNAs for CIGR1 and CIGR2 is correlated with the bioactivities of GA in suspension-cultured cells of rice . CIGR1 and CIGR2 are excellent markers of GA signal transduction . In fact, high levels of CIGR1 and CIGR2 mRNAs accompany with a high concentration of phytoactive GA, which promotes plant height. In this study, the expression level of ph1 was significantly higher in root, leaf sheath and leaf blade tissues in tall plants compared with short plants; this finding indicates that tall plants contain a higher concentration of phytoative GA that resulted in increased plant height (Fig. 4). Cloning of this gene is under way in our laboratory which will ultimately reveal its biological functions on plant height.
Plant height gene cluster on chromosome 1
In rice, disease resistant (R) gene clusters contributing significantly to plant defense were observed and studied extensively to elucidate their mechanisms [25, 26]. The characteristic clustering of R genes has been proposed to facilitate the evolution of novel resistance specificities via recombination or gene conversion . Therefore, the organization of functionally related genes in clusters is expected to have an evolutionary advantage to the organism. Besides, many genes controlling the same agronomic traits were located in a very small region, making it difficult to determine whether they are one or two genes in a primary mapping population. For example, qCL1 controlling plant height is located 1.4 cM and 2.6 cM away from two plant height genes, d18 and d2, which are located with 1.2 cM distance on chromosome 1 . QTLph1 and sd1 are linked on chromosome 1 with a 1.7-Mb physical distance [9, 29]. The important plant height genes sd1, QTLph1, d2, d18, qCL1 and ph1 from this study are all located on chromosome 1; many of these genes are located at the distal end, indicating a hot spot region for plant height genes. However, the mechanism or functions of plant growth or development gene cluster genes are not clear. Activating these clusters will uncap a vast resource of novel enzymes, compounds, pathways and diverse chemistries, which can be exploited for a wide range of applications in plant height adaptation.
The authors kindly thank farm technician Mr. J.B. Wang for his excellent field work. This work is partly granted by Natural Science Foundation of China (30830064, 30921091), National Special Program for Research of Transgenic Plant of China (2009ZX08009-103B). Kovi M.R. gratefully acknowledges the China Scholarship Council (CSC) for supporting his Ph.D.