Proper works tallness is important for lodging opposition against air current and rain to better the harvest output.
Among many works tallness cistrons, semi-dwarf 1 is a dominant one and led to the green revolution in 20th century, by preponderantly increasing rice output. However, the frequent use of individual sd1 cistron beginnings may do familial exposure to plagues and diseases. Therefore, it is necessary to develop an surrogate or new beginning of midget for broadening the familial base of nanism. Identifying utile fresh semi-dwarf cistrons is of import for the familial use of works architecture in practical rice genteelness.
Introgression lines derived from two contrasting parents in works tallness, Zhenshan 97 and Pokkali were employed to execute the primary QTL function by majority segregant analysis method. A major cistron Ph1 was systematically detected on chromosome 1 in BC4F2 and BC4F3 coevalss explicating more than 90 % of works height fluctuation.
Further by developing a big BC4F2 population, Ph1 was all right mapped to 92 kilobits to a BAC contig AP003227. Sequence and look analysis by existent clip PCR were performed to clarify the function of sd1 and the campaigner cistron.
No sequence difference was observed in the sd1 coding part of Zhenshan 97 and Pokkali and farther no look degree alterations was detected among all tested tissues between parents corroborating that sd1 has no part to the fluctuation of works tallness in this population, therefore claiming Ph1 is a fresh works height cistron. Among the 17 unfastened reading frames in 92 kilobit, LOC_OS01g65990 coding Chitin inducible gibberellin antiphonal protein belongs to GRAS household might be the right campaigner cistron. Sequence analysis of campaigner cistron showed two amino acid alterations and important high look degrees was observed in Pokkali tissues which might act upon the works tallness.IntroductionRice is the chief eating harvest in many parts of universe, preponderantly in Asia and Africa.
Rice output was determined either straight or indirectly by many traits. Among them works tallness plays a polar function and nanism is a valuable trait in harvest genteelness, as it increases lodging opposition and decreases the harvest amendss due to weave and rain, there by increasing the harvest output. Second half of the twentieth century is really outstanding in harvest scientific discipline by the celebrated ‘green revolution ‘ , where IR8 besides known as ‘miracle rice ‘ , a semi-dwarf assortment, enabled dramatic output additions and helped to debar predicted nutrient deficits in Asia during that period [ 1 ] .
At the same period in wheat, another dominant semi-dwarf cultivar, Rht, facilitated a immense addition in productiveness and led to the wheat ‘green revolution ‘ [ 2 ] . The short stature of IR8 is due to a mutant in the works ‘s sd1 cistron, and subsequently identified this cistron encoding an oxidase enzyme ( GA20ox-2 ) involved in the biogenesis of gibberellin, a works growing endocrine [ 3 ] – [ 5 ] . Gibberellin is besides implicated in green-revolution assortments of wheat, but the decreased tallness of those harvests is conferred by defects in the endocrine ‘s signalling pathway [ 6 ] .Furthermore with complete rice sequence and promotion in molecular marker engineering, conventional genteelness was about replaced by molecular genteelness [ 7 ] . 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 by molecular marker engineering for quantitative trait venue ( QTL ) designation [ 8 ] – [ 11 ] . In order to place QTLs, a limited size of near-isogenic lines ( NILs ) , backcross inbred lines ( BILs ) , advanced backcross ( AB ) lines or introgression lines ( ILs ) can be used alternatively of a big F2 or recombinant inbred line ( RIL ) population [ 12 ] – [ 14 ] . Since such lines are homozygous, legion genetically indistinguishable workss can be evaluated, which will increase the truth of phenotyping without increasing the attempts of genotyping. More over the ILs are effectual as a tool for familial analysis of QTLs.
Any differences between ILs and their parents must be due to a QTL located in the introgressed part. These ILs series aid ticket function of QTLs every bit good as precise appraisal of the consequence of each QTL [ 15 ] . Several research groups have reported QTLs that control works tallness in rice [ 16 ] – [ 19 ] . Among all the cistrons identified, sd1 is the dominant one widely used in engendering to develop semi-dwarf assortments.However, the frequent use of individual sd1 cistron beginnings may do familial exposure to plagues and diseases [ 20 ] – [ 22 ] . Therefore, it is necessary to develop an surrogate or new beginning of midget for broadening the familial base of nanism. Undoubtedly, designation of more works tallness related genes/QTLs will supply us with more chances to engender diverse semi midget assortments which can defy to lodging.
In add-on, functional dissection of more gene-regulated works tallness traits will be helpful to further understand the molecular mechanism involved in semi nanism. Identifying utile fresh semi-dwarf cistrons is of import for the familial use of works architecture in practical rice genteelness.In this survey, we identified a major QTL on chromosome 1, ph1, which controls works tallness from introgression lines derived from two contrasting parents in works tallness Zhenshan 97 and Pokkali. Further we report here the all right function of Ph1 and proof of its campaigner cistron by comparative sequencing and look analysis between parents.
Phenotypic fluctuations of works tallness in BC4F2 population and its offspring
The two parents, Zhenshan 97 and Pokkali showed extremely important differences in works tallness. Zhenshan 97 was of short stature with a tallness of 88 centimeters ranged from 83.0-89.5 centimeter, while Pokkali was tall with an norm of 196 centimeters ranged from 195.
0-218 centimeter ( Table 1 ) . In the BC4F2 population, the difference in averaged works tallness between short and tall workss ( carried Zhenshan 97 allelomorphs and Pokkali allelomorphs at targeted cistron severally ) was big, reached up to more than 100 centimeter ( Fig 1A ) . The major differences were caused by both the length of internodes and the figure of internodes ( Fig 1B ) . Short works had an norm of 4 extended internodes, while tall works had 5. The lengths of all the other internodes except the first of tall works were longer than the opposite numbers of short works. Meanwhile, tall works had a longer panicle as compared with short works ( Fig 1C ) .
In tall works, the first and 2nd internodes with a similar length together contributed about 68 % to the entire culm, while in short works, the first internode entirely contributed 60 % to the entire culm ( Fig 1D ) .In BC4F2, the works tallness was ranged from 70-233 centimeter ( I do non believe it is a transgressive segregation, experimental mistake! ) . Among the 172 workss of BC4F2, there were 40 short and 132 tall workss which was in understanding with the expected segregation ratio ( 1:3 ) of individual Mendelian cistron ( ?2=0.22, P=0.639 ) . Further progeny trial ( BC4F3 ) had confirmed that 40 and 44 workss expressed indistinguishable short ( Zhenshan 97 type ) and tall workss ( Pokkali type ) , severally, whereas 88 workss showed varied works height bespeaking they are heterozygous at the venue of mark works height cistron. The average tallness for Zhenshan 97 type, heterozygote and Pokkali type was 90.2 centimeter, 177.
9 centimeter and 204.4 centimeter ( Fig 2 ) . Frequencies of the three genotypes was fitted to the expected Mendelian ratio ( 1:2:1 ) for individual venue segregation ( ?2=0.
28, P=0.869 ) . This analysis suggested that one cistron ( termed Ph1 ) controlled the fluctuation of works tallness in BC4F2 population
Primary function of ph1
Bulk segregation analysis was used to observe the major works height cistron. A sum of 150 polymorphous simple sequence repetition ( SSR ) markers distributed equally on all the 12 chromosomes between the parents were selected and screened for the two majorities, tall majority and short majority. Among the 150 SSR markers, there is no polymorphism between the two majorities at the venue of 143 markers, of which the majorities are indistinguishable to Zhenshan 97 genotype at 135 marker venue, while the majorities are heterozygotes at 8 markers. Polymorphism was identified between the tall and short majorities at the venue of 7 markers RM3304, RM3825, RM6439, RM472, RM5382, RM1339 and RM1387, which are located on chromosome 1 ( Fig 3 ) .
Consequently, these 7 markers were regarded to be linked to the works height cistron ( termed ph1 ) and were used to genotype the 172 BC4F2 workss. A local linkage map covering 35.8-cM was constructed ( Fig 4A ) . The single BC4F2 genotypes at ph1 were determined by offspring ( BC4F3 ) trial. Then ph1 treated as a marker was straight located into the linkage group, where it is located in a 0.6-cM part flanked by markers RM5382 and RM1339, co-segregated with RM1339 ( Figure 4A ) .
It is closely linked to sd1.Interval mapping method was conducted to gauge its familial effects by utilizing the phenotypic informations from BC4F2 and its offspring. A major QTL ( ph1 ) was detected in the 0.6-cM part between the markers RM5382 and RM1339, ( Fig 4A ) .
ph1 explained 90.3 % of phenotype discrepancy with linear and dominant effects of 57.1 centimeters and 30.6 centimeter.
In the progeny trial ( BC4F3 ) , it explained 92.7 % of works tallness fluctuation, but the linear consequence is less than that of the F2 population. ph1 acted as partial laterality in both coevalss, Pokkali allelomorph increased the works tallness ( Table 2 ) . Variation of heading day of the month was observed in the population. Tall works flowered 3-4 yearss earlier than short 1. QTL consequence please continue……
ph1 is distinguishable from sd1
The radical cistron sd1 is located on the right side of the marker RM1339. RM1339 is more than 180 kilobit from sd1.
In order to supply strong grounds that ph1 with big consequence on works tallness is non the allelism of sd1, we have performed sequencing analysis for Zhenshan 97 and Pokkali with sd1. The sequencing analysis of sd1 showed no sequence difference in the 3 coding DNAs and 2 noncoding DNAs ( Fig 5 ) . However, we have identified one SNP in the booster part of sd1 ( Fig 5 ) . For farther uncluttering the uncertainty on the function of sd1, we have performed the look analysis. The qRT-PCR consequences showed that there was no difference in the look degrees of sd1 in root, leaf sheath and foliage blade in both Zhenshan 97 and Pokkali ( Fig 6 ) .
Therefore clear molecular degree grounds determined that Ph1 is distinguishable from sd1.
Fine function of ph1
It is deserving insulating ph1 as it is a fresh works height cistron. 1250 highly short workss with works tallness of less than 100 centimeter, which were assumed to be Zhenshan 97 homozygotes at Ph1, were selected from 6400 workss of BC4F2 population to test recombinants between Ph1 and markers RM5382 and RM1339. Nine and three recombinants were identified between RM5382 and ph1, and RM1339 and ph1, severally. In instance the trait measuring yielded any false, Zhenshan 97 homozygous workss, progeny trials of the 12 recombinants between RM5382 and RM1339 were conducted. Each recombinant offspring showed an indistinguishable short works tallness, which was extremely significantly shorter than the control Pokkali homozygotes, but showed no difference with the control Zhenshan 97 homozygotes ( Table 3 ) .
This consequence confirmed the Zhenshan 97 homozygous individuality of the 12 recombinants at ph1. To heighten the declaration of the ph1 local linkage map, we used two SSR ( RM11960, RM11961 ) and three InDel markers ( Supp. Table ) to test the 12 recombinants: seven recombinants were identified between RM11960 and Ph1, six between PHA and Ph1, five between RM11961 and Ph1, four between PHB and Ph1 and one between PHC and Ph1 ( Fig 4B ) . Therefore, Ph1 was narrowed down to the part of about 92 kilobits bounded by markers PHB and PHC ( Fig. 4B ) . One Nipponbare BAC contig ( AP003227 ) precisely covered the part.
Putative cistrons in the 92-kb mark part
There are 17 predicted cistrons in the 92-kb part harmonizing to rice genome automated note database ( hypertext transfer protocol: //rice.plantbiology.
msu.edu/ ) ( Fig 4C ; Table 4 ) . Of these, 12 cistrons have homology with rice full-length complementary DNA. Among these 17 cistrons, 5 are of unknown map, and the functional notes of staying 12 cistrons were given in table 4. Gibberellin-responsive cistrons was reported to be associated with works tallness ( Murakami, 1972 ; Kobayashi et al.
, 1989 ; Itoh et al. , 2001 ; Sasaki et al. , 2002a ; Sakamoto et al. , 2004 ) . Among all the 12 putative cistrons, LOC_Os01g65900 which encode chitin induced gibberellin antiphonal protein ( CIGR ) was gibberellin antiphonal, therefore, LOC_Os01g65900 ( CIGRP ) was regarded as the campaigner of ph1.Sequencing analysis of the CIGR cistron which is of 3640 bp showed single-base permutations in booster part and exon 1 of Zhenshan 97 and Pokkali, while four base brace omission in the intron part of Zhenshan 97 ( Fig. 8A ) .
Between the parents, four SNPs were found at 706 bp, 1048 bp, 1151 bp and 1377 bp upstrem of ATG and two SNPs in exon 1. CIGRP contained 3 coding DNAs, it encodes protein with 553 amino acids, with alterations in two aminic acids between parents ( Fig 8B ) .
Co-segregation of the campaigner
In order to corroborate whether the campaigner cistron ( CIGR ) is co-segregated with works tallness, we have developed a cistron based marker ( give a name Y ) covering the SNP part ( detailed ) and screened all the five recombinants ( 32, 58, 67, 83 and 121 ) . The genotype information showed that all the five recombinants are homozygous to ZS97 at Y ( Fig. 7 ) . Namely, CIGR is co-segregated with works tallness.
Thus CIGRP might be the existent campaigner for ph1. In add-on, qRT-PCR showed important difference of CIGRP look degrees in root and leaf sheath compared to flick blade in Zhenshan 97 and Pokkali ( Fig 9 ) .
Comparison of the familial effects of ph1 to sd1
In this studyi??it was clearly confirmed that ph1 is distinguishable from sd1 by all right function with a big BC4F2 population. Comparative sequencing between parents farther confirmed sd1 did non lend to the works tallness fluctuation. No difference in look degree of sd1 between ZS97 and Pokkali was in understanding with the consequence that ph1 is a fresh cistron distinct from sd1. Although ph1 was non the allelomorph of sd1, it showed similar familial characters on works tallness but with larger effects in this survey. You should compare the linear and laterality effects.
( twofold biomass possible application for Biofuel ) ph1 has effects on heading day of the monthIn this survey, the fluctuation of concluding works tallness between tall and short workss is chiefly contributed by the difference in the length of the 2nd, 3rd and 4th topmost root internodes, particularly the 2nd and 3rd have big difference of more than 20 centimeter. But the topmost root length has no difference ( Fig 1B ) . Is it similar to some cistrons or different from other cistrons, particularly sd1? Cited [ 46 ] . Meanwhile, the averaged figure of extended internodes of tall and short lines is different ( Fig 1C ) . that is to state, ph1 controls figure of internodes and length of topmost internodes except the first 1. Take together, ph1 is a new one based on phenotype fluctuation.
Chitin-inducible gibberellin-responsive cistron might be the Probable campaigner cistron
In this survey, foremost Ph1 was all right mapped to a BAC ringer AP003227 ( Fig 4C ) incorporating 12 putative known functional cistrons. Then with the aid of bioinformatics analysis, we inferred the CIGR cistron ( LOC_OS01g65900 ) encoding chitin induced gibberellin antiphonal protein as the campaigner of ph1.
Finally, the individuality of the campaigner cistron was validated by co-segregation analysis.It is a good known fact that gibberellin plays a cardinal function in the works tallness. Many of import cistrons like sd1 and d18 are influenced by gibberellin. More over, the CIGR cistron belongs to GRAS household and comparative analysis had showed of import cistrons like SCARECROW LIKE 1 ( SCL1 ) in Arabidopsis, Dwarf8 in corn, MOC1 ( MONOCULM 1 ) , DELLA protein, gibberellin response modulator protein belongs to this household and are related to works growing and development ( need more elaborate information! ) [ 60 ] – [ 64 ] .
Necessitate one sentence to link the following 1.In add-on, it is reported that initiation of CIGR1 and CIGR2 was observed merely upon application of phytoactive GA, and the accretion of messenger RNA for CIGR1 and CIGR2 is correlated with the bioactivities of GA in suspension-cultured cells of rice ( Day et al. 2004 ) . CIGR1 and CIGR2 are first-class markers of GA signal transduction ( Day et al. 2004 ) . This indicated high degree of messenger RNA for CIGR1 and CIGR2 accompany with high concentration of phytoactive GA, which promotes works tallness.
In this survey, look degree of ph1 is significantly higher in root, leaf sheath and foliage blade in tall works compared with short works ; bespeaking that tall works contained higher concentration of phytoative GA ( is possible to mensurate GA? ) resulted more works tallness. Transformation of the campaigner cistron will finally detect its biological maps in works tallness.
Disease opposition cistrons ( members in cistron household? ) were often clustered in some hot spots [ X ] . Two rice bacterial blight opposition cistrons, Xa4 and Xa26, are tightly linked to each other in the long arm of chromosome 11 [ 48 ] , [ 49 ] .
Besides, many cistrons ( non from same cistron household? ) commanding same agronomic traits were located in a really little part which it is hard to find one or two cistrons at that place in a primary function population. For illustration, it is regarded that one cistron, Gn1, commanding rice grains per panicle in the interval between X and Y, but, it was dissect into two closely linked cistrons Gn1a and Gn1b together commanding the trait in advanced populaiton [ 51 and ] . Later, SPP1 commanding spines per panicle was besides detected in the nearby of the same part [ 50 ] . Two of import heading day of the month commanding cistrons ehd1 and ehd2 are closely linked on chromosome 10 in rice [ 52 ] .
For cistrons commanding works tallness, the similar instance is besides observed. qCL1 is located 1.4 centimeter and 2.6 centimeters off from two works tallness cistrons, d18 and d2, which are located with 1.2 centimeters distance on chromosome 1 [ 47 ] . QTLph1 and sd1 are linked on chromosome 1 with a 1.7-Mb physical distance [ 45 ] , [ 19 ] . In this survey, Ph1 is 182 kilobit from sd1.
Interestingly, most of the of import works tallness cistrons sd1, QTLph1, d2, d18, qCL1 including Ph1 in this survey are all located on the chromosome 1, many in the distal terminal, bespeaking a hot topographic point part for works tallness cistrons. ( We need points on the bunch cistrons instead than the phenomenon )
Materials and Methods
Plant stuffs and development of mapping population
A set of introgression lines ( BC4F2 ) was developed from a cross between two contrasting Oryza sativa L. indica assortments in works tallness, Zhenshan 97 a Chinese native and Pokkali an Indian indigen ( Fig 1A ) , in which Zhenshan 97 was used as the recurrent parent. One introgression line ( BC4F2 household ) showed a clear segregation in works tallness in 2003.
The household dwelling of 172 workss ant its offspring ( BC4F3 ) were grown in summer 2004 and 2005 in a bird-net-equipped experimental farm of Huazhong Agricultural University, Wuhan, China ( 29 & A ; deg ; 58 ‘ N, 113 & A ; deg ; 41’E ) . Field experiments were carried out following the randomised complete block design.For progeny trial, Twenty four workss each BC4F3 household was grown in a two-row secret plan in Wuhan in 2006. 20 workss in the center were investigated for works tallness and header day of the month. Field direction, including irrigation, fertiliser application and pest control, followed basically the normal agricultural pattern.
Fine function population
A big BC4F2 population of 6400 workss was grown for works tallness in 2005 in Wuhan.
The offsprings of 12 recombinantsbetween the cistron Ph1 and two closely linked flanking markers were sowed in summer 2007 in the same field where the offspring of BC4F2 population grown.
Deoxyribonucleic acid extraction and marker choice
Deoxyribonucleic acid was extracted from fresh foliage samples collected at seedling phase utilizing a modified CTAB protocol [ 65 ] . PCR was done utilizing a hot start Taq polymerase ( TaKaRa ) with the undermentioned conditions: 95 & A ; deg ; C for 4 min, 35 rhythms of 95 & A ; deg ; C for 40 s, 55-58 & A ; deg ; C for 40 s, and 72 & A ; deg ; C for 1 min, followed by 72 & A ; deg ; C for 6 min. SSR markers were used for developing familial linkage map.
In add-on to SSR markers, three freshly developed InDel markers for all right function are listed in Supp.Table on the footing of the available public rice genome sequences ( hypertext transfer protocol: //www.rgp.dna.affrc.go.
jp ) . Markers of the Monsanto Rice Genome ( MRG ) series were designed harmonizing to the rice genome sequences of the Monsanto Company [ 66 ] and those of the Rice Microsatellite ( RM ) series harmonizing to Temnykh et Al. [ 67 ] , [ 68 ] . The SSR check was conducted by polyacrylamide gel cataphoresis, as described by [ 69 ] , with minor alterations.
Plant height measuring
Plant tallness was recorded from field surface to the top of the highest panicle of each works for BC4F2 and its offspring BC4F3. At seed ripening phase, mean panicle length, figure of internodes and their lengths were recorded for both Zhenshan 97 and Pokkali.
Heading day of the month record!
Bulk segregant analysis for primary function
Five utmost tall persons with works height more than 200 centimeter and five utmost short persons with works tallness less than 90 centimeters were selected from BC4F2 population for developing two Deoxyribonucleic acid majorities. Equal measures of leaf tissue from each person were bulked and DNA was extracted from the majority. The two parents Zhenshan 97 and Pokkali were genotyped with 440 SSR markers covering 12 rice chromosomes for placing the polymorphous markers. Markers demoing polymorphism in the signifier of a clearly seeable difference in set strength between the high and low tail in these experiments were selected for genotyping these two tail majorities.
Familial linkage map and familial consequence analysis
The molecular linkage map was constructed by utilizing Mapmaker/EXP 3.0 [ 70 ] . Kosambi map was used to cipher familial distance. Interval function was conducted by utilizing Mapmaker/QTL [ 71 ] .
The LOD threshold was of 3.0, determined by 1,000 random substitutions at a false positive rate of 0.05 for the trait.
Sequencing analysis of sd1 cistron and likely campaigner cistron
Based on the BAC ringers AP003561 and AP003227 sequences ( hypertext transfer protocol: //www.ncbi.nlm.nih.
gov ) eight and five specific primer braces were designed to sequence booster and coding parts of sd1 cistron and likely campaigner cistron ( Suppl. Table ) . The two cistrons were amplified utilizing LA Taq ( Takara ) from genomic Deoxyribonucleic acid of Zhenshan 97 and Pokkali, and the PCR merchandises were purified. These purified PCR fragments were sequenced utilizing the Big Dye Terminator v3.1 Cycle Sequencing Kit ( Applied Biosystems, XX, USA ) . Data were collected utilizing the ABI Prism 3730 DNA Analyzer ( Applied Biosystems, XX, USA ) and interpreted utilizing SEQUENCHER 4.
6 ( Gene Codes Corporation, XX, USA ) . Sequence alliance was performed with the BLAST web service ( Website National Center for Biotechnology Information, NCBI ) and ClustalW ( European Bioinformatics Institute, EBI ) .
RNA extraction and quantitative existent clip PCR analysis ( qRT-PCR )
Entire RNA was extracted from leaf sheath, root and leaf blade of Zhenshan 97 and Pokkali utilizing the TRIzol reagent ( Invitrogen, XX, USA ) harmonizing to the maker ‘s process. The quality of the RNA samples was evaluated by optical density measurings utilizing the Nanodrop Spectrophotometer ND-1000 ( Nanodrop Technologies, USA ) . All the RNA samples used in the qRT-PCR reactions showed a 260/280 nm optical density ratio of 1.9 – 2.2.
Prior to qRT-PCR, entire RNA samples were pretreated with RNase-free DNase I to extinguish any polluting genomic Deoxyribonucleic acid.The first-strand complementary DNA were synthesized from DNaseI-treated entire RNA utilizing Superscript contrary RNA polymerase ( Invitrogen, XX, USA ) in a reaction volume of 40 ?l harmonizing to the maker ‘s instructions. qRT-PCR was performed in an optical 96-well home base with an ABI PRISM 7500 real-time PCR system ( Applied Biosystems, XX, USA ) . Gene-specific primers were designed for sd1 cistron and the campaigner cistron ( CIGR ) ( supp. Table Ten ) . Each reaction contained 12.
5 µL of SYBR Premix Ex Taq ( TAKARA ) , 0.5 µL of ROX Reference Dye ( TAKARA ) , 5.0 µL of complementary DNA samples, and 10 µM cistron specific primers in a concluding volume of 25 µL. Rice Actin1 cistron was used as endogenous control with the with primers 5?-TGGCATCTCTCAGCACATTCC-3? and 5?-TGCACAATGGATGGGTCAGA-3? . The thermic rhythm used was as follows: 95 & A ; deg ; C of 10 s, 45 rhythms of 95 & A ; deg ; C for 5 s, and 60 & A ; deg ; C for 35 s. All qRT-PCR reactions were carried out in biological extras, each of which was used for RNA extraction followed by qRT-PCR in triplicate. The comparative look degrees were analyzed as described by Livak and Schmittgen [ 72 ] .
The writers kindly thank farm technician Mr. J.B. Wang for his first-class field work.
This work is partially granted by Natural Science Foundation of China ( 30830064, 30921091 ) , National Special Program for Research of Transgenic Plant of China ( 2009ZX08009-103B ) . Kovi M.R. appreciatively acknowledges the China Scholarship Council ( CSC ) for back uping his Ph.D.