Gene Mapping and Expression Analysis of a Novel Mutant reproduce organs absent (roa) in Rice Nan Wang, Xian-Chun Sang, Yun-Feng Li, Ying-Hua Ling, Fang-Ming Zhao, * Zheng-Lin Yang and Guang-Hua He Rice Research Institute, Key Lab of Biotechnology and Crop Quality Improvement of the Agricultural Ministry, Southwest University, Chongqing 400715, P. R. China
Received June 29, 2009; accepted September 24, 2009
ABSTRACT Mutant plays an important role in function analysis in plant. A rice flower mutant reproduce organs absent (roa), showing a stable inheritance during several years of study, was identified in rice (Oryza sativa L. ssp. Indica) cultivar Jinhui10 treated with EMS and used in this study. This mutant showed following: elongated palea and pedicle; absence of inner three whorls of floral organs; multi-whorls glume like organs inside the lemma/palea; spikelet meristem like organ upon the pedicle. These phenotypes suggested that ROA is a key gene in rice spikelet development. Genetic analysis confirmed that the mutant traits were controlled by a single recessive nuclear gene. By gene mapping, ROA was restricted between two SSR markers RM221 and RM1342 on the chromosome 2. It concluded that ROA was a novel gene involving in flower development in rice. Besides, the mutation of ROA influenced the transcription level of floral homeotic genes; the expression of floral homeotic genes decreased in roa panicle compared with wild-type, and it suggested that ROA affected flower development by influencing the expression of floral homeotic genes. Key words: rice (Oryza sativa L.), flower mutant, gene mapping, ABCE model.
INTRODUCTION Flower development, as one of the most interesting studies, have been deduced following the research in Arabidopsis, of which ABCE model is the most noticeable in elucidating flower development from the molecular level in dicot (Coen and Meyerowitz, 1991). In Arabidopsis, sepal / petal / stamen / carpel are
*To whom correspondence should be addressed. Email:
[email protected].
classified as four whorls of floral organs, and A/E class genes specify the first whorl sepal, A / B / E class genes specify the second whorl petal, B / C / E class genes specify the third whorl stamen, C / E class genes specify the forth whorl carpel (Coen and Meyerowitz, 1991; Kater et al., 2006; TheiBen and Saedler, 2001). In monocot, some recent studies indicated that the ABCE model can be referenced in elucidating molecular mechanism of rice flower development. In which, lemma / palea, lodicule, stamen and carpel are comparable with sepal, petal, stamen and carpel
(Yamaguchi and Hirano, 2006). ABCE class genes in Arabidopsis have been cloned as following, A class genes: AP1, AP2; B class genes: AP3, PI; C class gene: AG, and E class genes: SEP1 / 2 / 3 / 4 (Kater et al., 2006). In rice, there are three A class genes OsMADS14, OsMADS15 and OsMADS18 (Jeon et al., 2000b; Pelucchi et al., 2002; Kyozuka et al., 2000; Fornara et al., 2004); three B class genes OsMADS2, OsMADS4 and OsMADS16 (Chung et al., 1995; Kang and An, 1997; Prasad and Vijayraghavan, 2003; Kyozuka et al., 2000; Nagasawa et al., 2003); two C class genes OsMADS3 and OsMADS58 (Yamaguchi et al., 2006); five E class genes OsMADS7, OsMADS8, OsMADS1, OsMADS5 and OsMADS34 (Greco et al., 1997; Pelucchi et al., 2002; Kang and An, 1997; Jeon et al., 2000a; Agrawal et al., 2005; Prasad et al., 2005). Additionally, a non ABCE-class gene Drooping Leaf also plays a key role in flower development in rice (Nagasawa et al., 2003; Yamaguchi et al., 2004). Rice flower mutants play more and more important roles in genetic analysis and function identification in the network of molecular regulation in rice. The study on novel rice flower mutant reproduce organs absent (roa) would be a good foundation to reveal the molecular network in flower development of rice.
MATERIALS AND METHODS Plant materials The roa mutant was derived from Jinhui10 treated with EMS, and Jinhui10 was an indica restorer line bred in our lab. Xinong1A (Oryza sativa L. ssp. indica.) as female was crossed with the roa mutant in Chongqing in 2006, and F1 generation propagated in Hainan in autumn 2006 and F2 population propagated in Chongqing in spring 2007. F2
segregating generation was used for mapping the ROA gene. Microscopic analysis Young panicles were fixed in formaldehyde-acetic acid (FAA) overnight at 4C. They were dehydrated through an ethanol series from 50í100%, and then embedded. Samples were sectioned to 10 μm, stained and observed under a Nikon E600 light microscope. For scanning electron microscopy (SEM), flower tissues developing at different stages were collected and fixed in FAA overnight at 4C, then dehydrated through an ethanol series from 50í100%, critical point drying, mounting and carbon coating, specimens were examined in a Hitachi S-3000 scanning electron microscope. DNA isolation Genomic DNA of individual plants was extracted following the description of Rogers and Bendich (1988). Each bulk contained equal amounts of DNA from ten normal plants and mutant plants in F2 population, respectively. Microsatellite (SSR) analysis SSR markers were downloaded from gramene (http://www.gramene.org/microsat) and synthesized by Shanghai Invitrogen. Based on the Rice Genome Program (RGP) and Beijing Genomics Institute (BGI), SSR markers were newly designed to fine map the ROA gene according to rice genome sequences (markers sequences are listed in 5BCMF ). The protocols for PCR amplification, electrophoresis, and silver-staining were the same as those reported (Luo et al., 2007) with a slight modification. Total volume of PCR solution was 12.6 ⼿, reactions containing 1.25 ⼿ of 10 × stock PCR buffer, 1 ⼿ of 50 ng / ⼿ DNA, 0.75 ⼿ of 25 mM Mg2+, 0.5 ⼿ of 2.5 mM dNTPs, 8 ⼿ ddH2O, 1 ⼿ of 10 mM primers and 0.1 ⼿ of 5 U / ⼿ Taq DNA
5BCMF5IFTFRVFODFTPGNBSLFSTUIBUMJOLBHFEXJUI30" Markers name RM5804 RM3512 RM221 RM1342 RM3212 RM497 RM5460
Forward sequence GCATCGATCTTAATCTGCCC TCTGATCTCACGAGTCACAT ACATGTCAGCATGCCACATC GAAGCAAGAAACCAAAGATG AGACGACAAACACCTGCCTC TCCTCTTCACCTATGGGTGG AAGAGAACAAGCCATGGTGC
Reverse sequence CATGGCCATCCCTAAAACAC AAGATAGGGGTGAACACTTG TGCAAGAATCTGACCCGG CTTTCGGTCTCAAGCAATAT CAAACACAAACGCAGCCTC GCCAGTGCTAGGAGAGTTGG GCCTTTTCTTGCCTTTGGAC
5BCMF5IFTFRVFODFTPGQSJNFSTVTFEJOTFNJRVBOUJUBUJWFBOERVBOUJUBUJWF1$3 Primer name
OsMADS2 OsMADS4 OsMADS16 DL OsMADS3 OsMADS58 OsMADS13 OsMADS1 ACTIN1
Forward sequence CCACCACTGGATTGAATGTCC GATGGACCACTGGAGGATGC CGGCAAGTACCACGAGTTCTG CCCATCTGCTTACAACCGCTT AGAGAACAGGCTGGAGAAAGGC AACAGTGACACCTCCAACGC CAGGAGTCTGCCAAACTGCG TCTTGGTGAGGATTTGGGC TGCTATGTACGTCGCCATCCAG
polymerase. Amplification was carried out on a MyCycler Thermal Cycler (Bio-Rad, Hercules, CA. USA) under the following procedure: 5 min at 94C, 35 cycles of 30 sec at 94C, 30 sec at 55C and 1 min at 72C, and 10 min at 72C. Amplified products containing microsatellite regions were segregated by electrophoresis on 10.0% w / v polyacrylamide gels, and the band patterns were visualized using silver staining as described previously. RNA isolation and RT-PCR Total RNA was isolated from various tissues of the roa and wild-type with Watson RNeasy Plant Mini kit (Watson, China) according to the manufacturer’s instructions. The first strand of cDNA was synthesized from 2 Gof total RNA using oligo (dT) 18 primers in 25 ⼿ reaction volume using Super Script III Reverse Transcriptase kit (Invitrogen, USA). 0.5 ⼿ of the reverse transcribed RNA was used for PCR templates with gene-specific primers (primer sequences are listed in 5BCMF). The PCR conditions were: 5 min at 94C, 25í30 cycles of 30 sec at 94C, 30 sec at 60C and 1 min at 72C, and 10 min at 72C. The PCR products were separated by 2% agarose gels in 1 × TAE buffer. Quantitative PCR Quantitative PCR reactions were carried out using the same RNA samples, which were used for RT-PCR as described earlier. First strand cDNA was synthesized by reverse transcription using 2 Gof total RNA in 25 ⼿ of reaction volume using Super Script III Reverse Transcriptase (Invitrogen, USA). Diluted cDNA samples were used for quantitative PCR analysis with 200 nM of each primer mixed with SYBR Green PCR master as per manufacturer's instructions.
Reverse sequence CAGAATGGCAAGCACAGAGC CTGGAGGTTGGGGTGGCT GAACTCCAGCCCGTCCAGAT GTTGGAGGTGGAAACCGTCG GGCTGCTGTCCCCTCTCATTC TTGGTGATCTGTTGCTTCAGC CCTTTCTCCAGGCGGCTTTC CCTTGCTCTTCAGATCAAACAG AATGAGTAACCACGCTCCGTCA
The reaction was carried out in 96-well optical reaction plates (Applied Biosystems, USA), using ABI Prism 7000 Sequence Detection System and software (PE Applied Biosystems, USA). At least three PCR reactions using the same templates were performed to get average values of expression levels. The PCR conditions were 10 min at 95C, 40 cycles of 30 sec at 95C, 1 min at 60C and 30 sec at 72C, and 10 min at 72C. For roa mRNA expression, the specific primers (primer sequences are listed in 5BCMF) were used. To normalize the variance among samples, ACTIN1 was used as endogenous control. Relative expression values were calculated after normalizing against the maximum expression value.
RESULTS roa affects spikelet development In wild-type spikelet, rudimentary glumes, empty glumes and palea / lemma formed in a 1 / 2 alternate arrangement in the pedicle ('JH", #and*). Inside the palea/lemma, there were two lodicules which formed on the lemma sides, six stamens and one carpel in the orderly whorls ('JH$, %, +, ,, 2 and ). Compared with wild-type, there was no difference at vegetative growth stage in roa ('JH). At spikelet growth stage, the entire four whorls of floral organs of roa were affected except for the lemma: palea and pedicle was elongated ('JH&, 3, / and ); almost all the inner three whorls of floral organs were absent; multi-whorls glume like organs were inside the lemma / palea ('JH', (, ), -, ., 3and ), and a spikelet meristem like organ was upon the pedicle ('JH), 0, 3 and ). Almost all the roa spikelets absented the inner three
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recessive gene (5BCMF ). Gene mapping of ROA
'JHVSF4DIFNBUJDSFQSFTFOUBUJPOPGXJMEUZQF MFGU BOE SPB SJHIU TQJLFMFU HM HMVNF MP MPEJDVMF TU TUBNFO DB DBSQFM HMP HMVNF MJLF PSHBOT TNMP TQJLFMFUNFSJTUFNMJLFPSHBO
whorls of floral organs, however very few normal spikelets were detected in roa inflorescence and the normal spikelets ranged from 1 to 20 in one plant ('JH 1), such plant occupied about 20% in the roa mutant plants. Genetic analysis of roa At heading stage, individual plants from the F1 and F2 population derived from the cross between Xinong1A and mutant, along with their parents, were investigated. All F1 plants exhibited a normal phenotype and the segregation ratio of normal phenotype over mutant phenotype was 3 : 1 in the F2 population, so the mutant traits were controlled by one
To map the ROA, 1040 plants showing mutant phenotype in F2 population derived from cross between Xinong1A and roa were acquired. Four hundreds SSR markers were used to amplify the parents’ and bulks’ genomic DNA simultaneously, and RM5804 and RM5460 on chromosome 2 were polymorphic both in the parents and the gene bulks, suggested the primary linkage with ROA, which was further confirmed by analysis of F2 recessive individuals and the genetic distances were 2.019 cM and 1.635 cM respectively. To restrict ROA to an exercisable region, thirty SSR markers between RM5804 and RM5460 were developed. Five markers exhibited polymorphism between the parents, and then were used to amplify the recombinants of RM5804 and RM5460. Fine linkage analysis showed ROA was located between RM221 and RM1342, genetic distances were both 0.144 cM ('JH ).
roa affects the transcription level of floral homeotic genes To make clear whether the mutant phenotypes were
'JHVSF1IFOPUZQFPGXJMEUZQF MFGU BOESPB SJHIU BUWFHFUBUJWFHSPXUITUBHF 5BCMF(FOFUJDBOBMZTJTPGSPB Cross combination Xinong1A / roa
Total 4065
NO. of normal plants 3025
NO. of mutants 1040
Chi-square( Ȥ20.05, 0.74
1
= 3.84)
'JHVSF5IFHFOFNBQQJOHPG30"PODISPNPTPNF5IFOVNCFSTVOEFSUIFNBSLFSTJOEJDBUFSFDPNCJOBOUT
caused by the change of the expression of floral homeotic genes, we compared the transcription level of OsMADS2, OsMADS4, OsMADS16, DL, OsMADS3, OsMADS58, OsMADS13 and OsMADS1 between Jinhui10 and roa panicle. By semi-quantitative PCR and quantitative PCR results, they showed that the transcription level of all the floral homeotic genes above mentioned decreased distinctly in roa panicle ('JH ).
DISCUSSION The mutation of ROA affected all the four whorls of floral organs, the lodicule, stamen and carpel were replaced by glume like organs in roa. In Arabidopsis thaliana sep1sep2sep3 triple mutant petals, stamens, and carpels were converted into sepals (Pelaz et al., 2000; Honma and Goto, 2001), and all floral organs converted into leaf like organs in sep1sep2sep3sep4 quadruple mutant (Ditta et al., 2004). According to the
mutant traits, it suggested that ROA may be E class gene in rice. So far, only one E class genes OsMADS1 (LHS1) was studied well in rice. In the weak phenotype of the lhs1 mutant, the spikelet consists of leafy palea and lemma, two pairs of palea- and lemmalike structures, fewer stamens, and more carpels. In plants with the strong phenotype, the lhs1 mutation results in generation of new flowers within the spikelet (Jeon et al., 2000a). OsMADS1 knockdown perturbs the differentiation of specific cell types in the lemma and palea, creating glume like features, with severe derangements in lemma differentiation. In many OsMADS1 knockdown florets glume-like organs occupy all the inner whorls (Prasad et al., 2005), those mutant traits were similar to those of roa, and the transcription level of B / C class genes didn’t alter in OsMADS1 knockdown plant (Prasad et al., 2005). In roa, the transcription level of B / C / E class genes OsMADS2, OsMADS4, OsMADS16, DL, OsMADS3, OsMADS58, OsMADS13 and OsMADS1 were
'JHVSF&YQSFTTJPOBOBMZTJTPGGMPSBMIPNFPUJDHFOFTJOSPBQBOJDMF " $PNQBSJTPOPGGMPSBMIPNFPUJD HFOFTUSBOTDSJQUTCFUXFFOXJMEUZQFBOESPBQBOJDMFXJUITFNJRVBOUJUBUJWF351$3"DUJOXBTVTFEGPS OPSNBMJ[BUJPOPGD%/"RVBOUJUZ # $PNQBSJTPOPGGMPSBMIPNFPUJDHFOFTUSBOTDSJQUTCFUXFFOXJMEUZQFBOE SPBQBOJDMFXJUIRVBOUJUBUJWF351$3"DUJOXBTVTFEGPSOPSNBMJ[BUJPOPGD%/"RVBOUJUZ
decreased distinctly. B class genes (OsMADS16 / 4 / 2), C class genes (OsMADS3 / 58), E class gene OsMADS1 and Drooping leaf were involved in the development of all four whorls of floral organs, D class gene OsMADS13 played key role in ovule development in rice (Chung et al., 1995; Kang and An, 1997; Prasad and Vijayraghavan, 2003; Kyozuka et al., 2000; Nagasawa et al., 2003; Yamaguchi et al., 2006; Nagasawa et al., 2003; Yamaguchi et al., 2004; Jeon et al., 2000a; Prasad et al., 2005). It suggested that ROA might be the upstream gene of floral homeotic genes, and it regulated the floral organ development by influencing transcription level of the floral homeotic genes in rice. Gene mapping restricted the ROA between RM221 and RM1342 on chromosome 2, genetic distances were both 0.144 cM. In this region, no genes have been reported with similar roa phenotype or function. It is confirmed that roa is a novel flower mutant, and further studies could promote the elucidatory of rice flower development and mending of ABCE model.
ACKNOWLEDGEMENTS This research was supported by the National Natural Sciences Foundation (30800598), the Excellent Youth Foundation Project of Chongqing (CSTC, 2008BA1033), the Natural Sciences Foundation Project of Chongqing (CSTC, 2008BB1258) and the Fine Animals and Plants Breeding Project of Chongqing (CSTC, 2007AA1019).
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