Breeding of Marker-free Indica Three-lines Restorer with ZmC 4 Ppc by Marker-assisted Selection

Enhancing photosynthetic productivity of rice is always the key goal of researchers by to use exogenous C4 genes. The full-length of intact maize gene of C4-specific PEPCase (ZmC4Ppc) is 6781 bp that makes it difficult for marker-assisted selection (MAS) in rice. The specific marker of intact ZmC4Ppc was designed and termed as MRpc. MRpc was verified that it was a gene marker because ZmC4Ppc could be overexpressed at every developmental stage and it was used for MAS of indica restorer Shuhui881 with ZmC4Ppc and FPM881was gotten, which was tested for genetic background, PEPCase activity, net photosynthetic rate (Pn), general combining ability (GCA) and specific combining ability (SCA). The results indicated that FPM881, which similarity of genetic background had arrived at above 95%, showed a higher PEPCase activity and net photosynthetic rate than those of control. Analysis of yield components showed that some of the progenies carrying ZmC4Ppc had better GCA and SCA than those of the control. Then, six lines of FPM881 were planted in larger population by selfing and gotten selectable-marker free plants with target gene using MRpc and hpt primers. Our results indicated that introduction of ZmC4Ppc via MAS and its stable expression could increase grain yield of hybrid rice.


Introduction
Photosynthesis is main source power of crop production.Most of crops, such as rice and wheat, are classified as C 3 plants because the first product fixed atmospheric CO 2 is 3-PGA.However, up to 50% carbon fixed is lost during photorespiration because of competitive inhibition of O 2 and CO 2 in active site of Rubisco.Because of possessing the C 4 photosynthetic pathway as CO 2 -concentrating mechanisms (CCM), C 4 plants can achieve high photosynthetic performance and high water-and nitrogen-use efficiencies.In terrestrial C 4 plants, CO 2 release from C 4 acids and the resulting elevation of cellular CO 2 levels take place at a site that is physically separated from the site of initial carboxylation.This separation occurs through the bundle sheath and mesophyll cells in typical/classical C 4 plants (Hatch, 1987;Leegood, 2002), and through two distant subcellular compartments in the discovered single-cell C 4 plants (Edwards et al., 2004).By contrast, in some aquatic plants (H.Verticillata) C 4 photosynthesis is accomplished in a single cell without any compartmentation and chloroplast differentiation (Bowes et al., 2002;Leegood, 2002).Therefore, the key of C 4 pathway activation is enzymes relative to CCM.
The key enzymes of C 4 pathway consist of phosphoenolpyruvate carboxylase (PEPCase), pyruvate phosphate dikinase (PPDK), NADP-dependent malic enzyme (NADP-Me) etc.The function of PEPCase, namely, control coefficients (C j ), is the largest (0.35) than the other key enzymes during photosynthesis in C 4 plant (Furbank et al., 1997;Dever et al., 1998;Bailey et al., 2000).PEPCase catalyzes irreversibly PEP to yield OAA and Pi using HCO - 3 as the inorganic carbon substrate, which plays a key role while atmospheric CO 2 is fixed in photosynthesis of C 4 and CAM plants.The genetic structures of key enzymes of C 4 pathway such as C 4 -specific PEPCase etc have been decoded and ZmC 4 Ppc has 10 exons and 9 introns with 6781 bp (Matsuoka et al., 1989).C 4 pathway isn't the privilege of C 4 plants.There are a lot of experimental evidences that C 3 plants have C 4 photosynthetic biochemical characteristics (Hibberd et al., 2002).The pathway with C 4 photosynthetic characteristics in C 3 plants is named as C 4 -like pathway because of the absence of the kranz anatomy.It has been proved that Kranz anatomy is not essential for terrestrial C 4 plant photosynthesis (Voznesenskaya et al., 2001).There is C 4 -like pathway in rice lemma because it has been proved that 14 CO 2 is fixed by PEPCase using 14 CO 2 tracer and LED technology (Imaizumi et al., 1997).
Introducing a C 4 -like pathway into C 3 plants is one of the proposed strategies for the enhancement of photosynthetic productivity (Endo et al., 2008).The intact genes of key enzymes of C 4 pathway, ZmC 4 Ppc, ZmC 4 Pdk etc have been transformed to Kitaake, respectively by Ku et al. (1999) and Fukayama et al. (2001) by using the Agrobacterium-mediated transformation and they were over expressed.Ku et al. (2000) reported that transgenic rice plants expressing the maize PEPCase had higher photosynthetic capacities (up to 30% higher) than untransformed plants.Besides improving photosynthetic capacity and yield, introduction of key enzymes genes in C 4 pathway into rice had a good potential to enhance its tolerance to stress of photoinhibition and photooxidation under field conditions.Jiao et al. (2001) reported that the light saturated photosynthetic rate could be greatly increased (55%) by overproduction of the maize PEPCase in transgenic rice plants and PEPCase activity can be greatly increased up to 20-fold than that of non-transformants.Ku et al. (2001) found that the grain yield was about 10~20% higher in Kitaake with ZmC4Ppc transgenic rice lines under small-scale field trial, as compared with the untransformed plants.Bandyopadhyay et al. (2007) reported that overproduction of the maize PEPCase enhanced photosynthesis and increased crop yield of transgenic rice plants.PEPCase was overexpressed in the same intracellular compartment, cytosol, in C 3 plants as in C 4 plants (Miyao, 2003).It will be an important biotechnology pathway to transfer key enzyme genes of C 4 pathway from transgenic rice materials to elite rice varieties by marker-assisted selection (MAS).However, its band is not clear and repetition is poor if the intact genes are amplified.We designed the specific marker of ZmC4Ppc and applied in the breeding of indica restorer with ZmC4Ppc by MAS.On the other hand, transgenic rice, namely trans-ZmC 4 Ppc Kitaake, has selectable marker hygromycin phosphotransferase gene (hpt), which serves no useful purpose once transgenic plants being regenerated and continues to express its gene products.Furthermore, it results in more and more environmental and consumer concerns.Therefore, breeding of marker-free indica restorers with target gene is crucial.

Plant materials and DNA extraction
Rice varieties, Shuhui 881 was afforded by Rice Research Institute of Sichuan Agricultural University, Kitaake， a extremely early Japonica variety from Hokkaido of Japan by Mr. Fujimoto Kan and Kitaake transformed with ZmC4Ppc by Mr. DM Jiao.Maize varieties，Yayu 10, Yuyu 22 and Linao 4 were afforded by High Technology Agricultural Company of Sichuan Agricultural University.Their DNAs were extracted according to the method of Xiang et al. (2007).(Altschul et al., 1990) BLASTs were carried out between the nucleotide sequences of ZmC 4 Ppc, and indica 9311, respectively.(http： btn.genomics.org.cn：8080/blast.php？name= rice).Meanwhile, BLASTs were also carried out between the nucleotide sequences of ZmC 4 Ppc, and all GenBank＋EMBL＋DDBJ＋PDB rice sequences (but no EST, STS, GSS, or phase 0, 1, or 2 HTGS sequences), respectively (http：// www.ncbi.nlm.nih.gov/blast/Blast.cgi).All the jobs were done at the server of Sichuan Agricultural Biotechnology Center.

Design of specific primers
The software of designing primer -Primer Premier 5.0 was applied.
The synthesis reaction of primers was done by Shanghai Sangon.
The reaction condition of simple sequence repeats (SSR) was carried out according to Chen et al. (1997).The difference of electrophoresis was agarose gel concentration of 3% at 160 V for 3 h as compared with the electrophoresis of PCR production of ZmC 4 Ppc.The aim of SSR reaction was to screen differential primers and confirmed similarity degree of genetic background for FPM881, as compared with Shuhui881.

Determination of PEPCase activity
The assay of PEPCase activity was carried out according to the method of Huang et al. (2002).The developmental stages of determination were initial-tillering, tillering, jointing, initial-heading, full-heading and maturing.Five plants were used at each stage and the determination of each plant was repeated for 3 times.

Measurements of net photosynthetic rate (P n )
The portable photosynthesis system LI-6400 was used.The last second leaves from main stem were used for the measurement of P n at c. 10: 00 AM under controlled condition of a CO 2 concentration of 400 µmol•mol -1 , 30 , ℃ and a light intensity of 1200 µmol•m -2 •s -1 .The dates of P n measurements were the same as those for the determination of PEPCase activity.Five plants with 3 repetitions were used in the assay.

PCR analysis of hpt gene
PCR analysis was carried out using primers specific for the hpt as previously described (Ma et al., 2004), Forward: 5' TAC ACA GCC ATC GGT CCA GA 3', Reverse: 5' TAG GAG GGC GTG GAT ATG TC 3'.

Measurements of general combining ability (GCA) and Special combining ability (SCA) for improved Shuhui 881 with ZmC 4 Ppc (FPM881) gotten by MAS
3×7 incomplete diallel cross design was used (there were 3 male sterility lines and 7 FPM881 restorer lines).All of the F 1 was sowed in experiment farmland of Rice Research Institute of Sichuan Agricultural University.The plantlets contained ZmC 4 Ppc gene, which were analysed by PCR in third leaf stage, were preserved and transplanted to field when seedling-age arrived at 40 days.Random block, 1.43 m 2 of every plot area with three times replication.Standard of transplanting: Row spacing, 0.27 m, socket spacing, 0.16 m, having same management compared with field.At maturity, five plants were harvested for every plot and measuring number of panicles per plant (No. of panicles per plant, panicles), number of spikelets per panicle (No. of spikelets per panicle, grains), seed setting rate (%), thousand-grain weight (1000-grain weight, gram) and grain yield per plant (gram).Their results were analyzed by analysis of variance (ANOVA) and calculating of combining ability.All of the data were operated in DPS software.

The specific primer sequences
By BLAST, the fragments of ZmC 4 Ppc existed only in maize rather than in rice were 1-1330 bp, 2180-3065 bp and 6320-6781 bp.Using software Primer Premier 5.0, the 1-1330 bp of ZmC 4 Ppc was used for primer design.Its primer sequences were as follows and named as MRpc：Forward: 5' AAG CAG GGA AGC GAG ACG 3', Reverse: 5' GAT TGC CGC CAG CAG TAG 3'.The site of amplification by the primers was 970-1280 bp in ZmC 4 Ppc, and the size of the product was 311 bp, Tm=58.0 .Its site of fragment amplified was showed in ℃ Fig. 1.

Verification
The genomic DNAs of the materials Yayu 10, Yuyu 22, Linao 4, Kitaake with ZmC 4 Ppc, Kitaake, Shuhui 881 and FPM 881 were amplified with MRpc by PCR (Fig. 2).The amplified bands could be seen for Yayu 10, Yuyu 22, Linao 4, Kitaake with ZmC 4 Ppc and FPM 881, with the same size of 310 bp as compared with DNA marker (Aim in showing FPM881, the later being bred line, on figure2 was to compared it with maize having same band).
MRpc was used to screen transgenic material, Kitaake with ZmC 4 Ppc and the transgenic plants with specific band were reserved.Then, their PEPCase activities and net photosynthetic rate were determined, together with their wild-type Kitaake in next year (Fig. 3A).The PEPCase activity values of Kitaake with ZmC4Ppc were significantly higher than that of Kitaake at 6 different stages.The PEPCase activity was the highest at elongation stage and the lowest at maturing.The PEPCase activities of Kitaake with ZmC 4 Ppc were 4.47~25.26times as much as Kitaake at 6 different stages.
The net photosynthetic rate (P n ) of Kitaake and Kitaake with ZmC 4 Ppc were shown in Fig. 3B.The P n of Kitaake with ZmC 4 Ppc began to increase gradually from initial-tillering and reached its maximum at initial-heading, then began to decrease gradually.The P n of Kitaake began to increase from tillering and reached its maximum at elongation, then began to decrease.The P n of Kitaake with ZmC 4 Ppc at different stages was higher than that of Kitaake except initial-tillering.The result was similar to the report of Li et al.(2001), who determined P n of Kitaake with ZmC 4 Ppc and Kitaake when the transgenic material were selected by determining PEPCase activity.
In a word, MRpc is a specific marker, which means band could be amplified uniquely from rice variety that has been transformed with ZmC 4 Ppc, and has included the target gene in rice genome.The ZmC 4 Ppc in transgenic materials, which was selected by MRpc, could be over expressed.The fact indicates that MRpc is gene marker of ZmC 4 Ppc.

Performance of improved restorer bred by MAS
MRpc was applied in MAS.Shuhui 881 (recurrent parent) and Kitaake with ZmC 4 Ppc (donor parent) were crossed in Nanjing.The hybrid was backcrossed and subjected to MAS continuously in Wenjiang, Sichuan (summer), and Lingshui, Hainan (spring), and improved Shuhui 881 with ZmC 4 Ppc (FPM 881, BC 6 F 2 ) was bred successfully.The breeding procedure for FPM 881 by MAS was shown in Fig. 4. To analyze its genetic background, the polymorphism was analyzed between Kitaake with ZmC 4 Ppc and Shuhui 881 by using SSR primers of 530 pairs distributed in the rice genome.One hundred and three pairs of them had polymorphism.The F values (Nei et al., 1979) of similarity of 10 plants of FPM 881 were between 95.15%~99.03% as compared with Shuhui 881, the recurrent parent (Table 1).
To detect the photosynthetic characteristics of FPM 881, the plants with F value 99.03% were selected.Their PEPCase activity and P n were measured at same developmental stage above.Their PEPCase activities of FPM 881 was the highest(1264.2µmol/(mg•h))at the elongation stage, and was the smallest (355.0 µmol/(mg•h)) at the initial-tillering stage (Fig. 5A).They were increased significantly, and reached 23.3, 4.4, 4.4, 3.7, 1.3 and 3.7-fold at 6 different stages, respectively.The fact indicated that ZmC 4 Ppc could be over expressed during MAS by using MRpc.
The P n values of FPM 881 at 6 different stages were shown in Fig. 5B.They were higher than that of Shuhui881 at initial-tillering, tillering, elongation, initial-heading, increased by 18.5%, 1.4%, 22.3% and 0.3%, respectively.The value of both PEPCase activity and P n at elongation stage was the highest.It means that a higher PEPCase activity could be transformed to improve P n .The P n of FPM 881 at full-heading and maturing stage did not differ significantly from Shuhui 881, which might be due to the overcast and rainy weather at the time of measurement.

Analysis of GCA and SCA of FPM 881
The GCAs of 6 lines of FPM 881 and Shuhui 881 had different performances in different traits (Table 2).To number of panicles per plant, number of spikelets per panicle, seed setting rate and thousand-grain weight, the highest was line 5, Shuhui 881, line 3 and line 2, respectively.Grain yield per plant of line 1 had the highest GCA, secondly line 6, which had not significant difference, however, compared with line 1.Therefore, line 1 and line 6 could be further examined and utilized for GCA.SCA of each combination was listed in Table 3.The highest combination of number of spikelets per panicle, seed setting rate and thousand-grain weight was 2480A/Shuhui 881, 776A/line 3 and 776A/line 3, respectively.SCA of seed setting rate and thousand-grain weight of 776A/line 3 was the highest, respectively, the reason existing in higher GCA of line 3 in seed setting rate and thousand-grain weight.However, line 3 had lower GCA in grain yield per plant, its reason existing in too low GCA of line 3 in number of spikelets per panicle (Table 2).To grain yield per plant, SCA of 776A/line 6 was the highest, arrived at 18.01.

Identification and elimination of hpt gene
In BC 6 F 4 of MAS, ZmC 4 Ppc and hpt genes were amplified together in plants of above 6 lines, which began to selfing with size of 72 plants for each line, but not backcross again, and the elite plant with ZmC 4 Ppc was reserved.In BC 6 F 6 , there were 3 plants in line 6, which had ZmC 4 Ppc but not hpt (Fig. 6).It indicated that target gene and marker-gene had segregated in this generation and the plants with ZmC 4 Ppc gene but not hpt gene had gained.Therefore, marker-free indica three-line restorer FPM881 has been bred successfully.

Discussion
The existence of the biochemical characteristics of C 4 photosynthesis in the stems and petioles of C 3 plants indicates that the essential biochemical components and regulatory elements control the cell specific gene expression required for C 4 photosynthesis are already present in C 3 plants (Jeanneau et al., 2002).C 3 plants have necessary genetic mechanism of high level expressing C 4 -specific genes.Therefore, to transfer the key enzyme genes in C 4 pathway into C 3 crops will be an efficient way to enhance its biomass production and grain yield.Wang et al. (2004) reported that the light saturated photosynthetic rate of F 1 hybrids of Kitaake with ZmC 4 Ppc and photo-sensitive genetic sterile rice lines were 50% higher than that of the receptor parents.Huang et al. (2002) observed that the carboxylation efficiency of Kitaake transformed with ZmC4Ppc increased by 49.3% and the CO 2 compensation point decreased by 26.2% than that of untransformed rice, Kitaake.Our results also proved that the introduction of ZmC 4 Ppc into rice can enhance its photosynthetic capacity and efficiency.
MRpc is a gene marker of ZmC 4 Ppc because its PCR results showed that MRpc only amplifies specific band from the materials that contain ZmC 4 Ppc gene and its size of product is about 311 bp.The results of PEPCase activity and P n of Kitaake with ZmC 4 Ppc and FPM881 indicated that ZmC 4 Ppc could be overexpressed.The highest PEPCase activity and P n of FPM 881 were at elongation during 6 different developmental stages.PEPCase activity and P n of FPM 881 were increased by 4.4-fold and 22.3% over control, Shuhui 881 at elongation, respectively.It showed that the increase of PEPCase activity could be transformed into elevating of P n .However, the main increase of PEPCase activity was not at elongation, but at initial-tilling.By comparison with Shuhui 881 at same stage, PEPCase activity of FPM 881 increased 23.3-fold.The fact indicated that the expression of ZmC 4 Ppc was constitutive, and highly efficient.
Besides MRpc reported in this study, there is no another report about specific marker of ZmC 4 Ppc gene in rice.Related reports that tried to breed of restorers with ZmC 4 Ppc by MAS are little at present; Wang et al. (2002) studied on 5129 and Wanjing 97 of two-line restorers by using Kitaake with ZmC 4 Ppc and the MAS markers, p 1 GTTCCAACCACGTCTTCAAAGC; p 2 ATTTACCCGCAGGACATATCC.However, there was not any introduction about their origin, its specificity, background screening and combining ability in his report.Therefore, it was not known that similarity degree of genetic background and combining ability of improved restorers compared with recurrent parent.The studies have revealed that C 3 plants have at least two different type of genes; one type encode enzymes with 'housekeeping' function, and the other type are very similar to the C 4 genes of C 4 plants, though expression of the latter is very low or even undetectable in C 3 plants.These C 4 -like enzymes have high homology compared with C 4 -specific enzymes, and C 4 -like enzyme genes are progenitor of C 4 -specific enzyme genes in evolution (Ku et al., 1996;Miyao, 2003).Besides these, there is C 3 -specific PEPCase in rice.Specificity of amplification by marker designed will not be assured if specific fragments are not found by nucleotide BLAST.
Six lines of FPM 881 and their control, Shuhui 881 were analyzed by GCA and SCA, and the better improved restorers could be selected out from offspring lines of MAS, which similarity of genetic background had arrived at 95% and more.Line 1 and line 6 had higher GCA in grain yield per plant and 776A/line 6 had the highest SCA in grain yield per plant of all F 1 , which also had the highest grain yield per plant, and its competing heterosis was 34.7%.Therefore, line 1 and line 6 can be further test-crossed with other sterile lines.These results proved that ZmC 4 Ppc exerts gene effect in rice genetic background.The specific marker MRpc should be applied to breed elite indica restorers, maintainers and male sterile lines with ZmC 4 Ppc, because they likely have higher net photosynthetic efficiency and grain yield.
Along with the continuous increase of commercial cultivation of trans-genetic plants, its safety problem in environment and food has been concerned widely, especially, the presence of marker genes of antibiotics or herbicide resistance in trans-genetic crops.Nowadays, without selectable-marker genes is an important prerequisite of breeding trans-genetic crops.It is a simple method for getting selectable-marker free plants with target gene by sexual crossing (Kai, et al., 2002).Yu et al. (2005) obtained selectable-marker free transgenic rice plants with improved disease resistance from the selfing progeny of the transgenic plants.We have also gained success by this method.

Figure 1 .
Figure 1.Structure of intact ZmC 4 Ppc and the selective antibiotic resistance gene used for rice transformation Marker showed the site of band amplified on the intact ZmC 4 Ppc.

Table 1 .
Result of genetic background analysis of FPM 881

Table 2 .
Relative values of general combining ability of 5 grain yield components for 7 restorer lines

Table 3 .
Relative values of specific combining ability of 5 grain yield components for 21 combinations Male sterile line