Background

Cytoplasmic male sterility (CMS) is a valuable genetic material for the use of advantage of heterosis in rice and other self-pollinated crops. In the triple crossing rice system, maintainer line is the pollen donator of the sterile line reproduction, and has the relationship of isonuclear alloplasmic with sterile lines (Hu et al., 2008). Cytoplasmic source of Dian-type CMS which founded by Professor Li Zhengyou of Yunnan Agricultural University is an important CMS to hybridize Japonica rice in China. Based on the Dian-type sterile line, China has cultivated a batch of good Japonica Hybrid Rice for production, such as Dian-type Japonica hybrid rice, Yongyou, Chunyou and so on. In China, the research of the hybrid Japonica rice and the hybrid indica rice starts in the same period, and realized three lines combination in the same year, but the application of hybrid Japonica rice is quite limited. One of the main reasons is that the Japonica type parents have less genetic difference, and it is difficult to choose hybridized combinations which have large genetic differences (Tan, 1988). The breeding of hybrid Japonica rice parents which with genetic background of indica rice is an effective way to enlarge genetic diversity of parent of Japonica Hybrid Rice. Rice Research Institute of Yunnan Agricultural University selected and bred maintainer line/sterile line and restoring lines of Dian-type Japonica Hybrid Rice which had Indica-Japonica genetic component by the hybridization between Indica and Japonica, for expanding the genetic differences between the sterile lines and the restorer lines. A batch of sterile line/maintainer line and restoring lines of Dian-type Japonica hybrid rice with genetic background of Indica rice have now been bred, such as sterile line Yumi15A and restoring lines Nan34. The combination of the sterile line Yumi15A and the restoring lines Nan34 which named Dian hybrid 31 was planted in 2004 of 10.57 hm² and brought harvest yield up to 12.808,5 X 10³ kg/hm² (Tan, 2006). The breed showed admirable resistance and increase production in Guizhou cold area. The increase in yield is above 10%, and up to 100% (Xu, 2012; Zhang et al., 2014).

However, because the parents of Dian-type Japonica hybrid rice is a complex, multi-generation of selection, it is difficult to make sure the actual Indica genetic component ratio only through the genealogical relationship of parents. The development of molecular biology technology, especially the sequencing of whole genome of rice (Goff et al., 2002; Yu et al., 2002), lays a good foundation for the study of molecular biology in Rice, and provides a solid basis for the development of molecular markers, also for the study of rice parents’ Indica-Japonica component and genetic difference (Zhang et al., 2009; Yan et al., 2010). In order to research the Indica-Japonica genetic components and genetic diversity of materials, researchers explored the InDel (insertion/deletion) maker by the whole genome sequence comparison of Cultivar 9311 (Japonica rice) and Nipponbare (Indica rice) (Shen et al., 2004; Fan, 2015). InDel markers and SSR markers were used to study the polymorphism of rice. The InDel markers had the advantages of clear and stable PCR amplification bands (Feng et al., 2005). Lu et al. (2009) used 44 Indica and Japonica rice varieties that had known Indica and Japonica properties, including Cultivar 9311 and Nipponbare, and developed 34 InDel markers which can effectively identify the Indica-Japonica genetic components.

In this study, 23 polymorphic markers were screened from 34 InDel markers which were developed by Lu et al. (2009). These markers were used to analyze the Indica genetic components and genetic diversity of 231 Dian-type Japonica hybrid rice which conserved by the Institute of rice research of Yunnan Agricultural University.

1 Results and Analysis

1.1 Model of InDel marking for test materials

In this study, two Indica rice varieties (Cultivar 9311 and Haomuxi which is local old varieties of Yunnan Indica rice) and two Japonica rice varieties (Nipponbare and Pantiangemanaogu which grown in Yunnan at the altitude of 2,600 m in Japonica Rice Landraces) were selected as the standard samples, and all 23 InDel marker loci detected had amplification product, displayed distinctly different band type. Two Indica rice standard samples showed the same band type-Indica specific band (II) on all loci detected. The two selected Japonica rice standard samples also showed a consistent band type-Japonica specific band (JJ). At every InDel loci (Figure 1), Most of the maintainer line materials had 1 JJ, part of them had 1 II, and 32.47% (75) of materials had heterozygous type (IJ), make it clear: 1 Indica type band with 1 Japonica type band. These heterozygous type (IJ) showed at 9 loci, among them, the R12M10 markers on chromosome 12 revealed that up to 61 materials with heterozygous loci. One material had up to 2 heterozygous loci. 7 materials, such as Y-60, have 2 heterozygous loci. No heterozygous loci were detected at 67.53% of the InDel loci.

1.2 Indica genetic component ratios of Dian-type Japonica hybrid rice maintainer line materials

Most of Dian-type Japonica hybrid rice maintainer line materials had a certain proportion of Indica genotypes, only 16 (6.93%) materials didn’t showed Indica genotypes at all 23 loci. The Indica genetic component ratios of all materials were Fi ≤ 0.25. The percentage of 0 < Fi ≤ 0.05, 0.05 < Fi ≤ 0.1, 0.1 < Fi ≤ 0.15, and 0.15< Fi ≤ 0.2 were 22.94%, 33.33%, 26.41%, and 7.36%, respectively. The Indica type loci of another 3.03% maintainer line materials reached 0.2 < Fi ≤ 0.25 (Figure 2). 5 materials detected 5 Indica genotype loci and Anfei paddy had the highest Indica genetic component ratios, up to 0.25. The results showed that most of the materials had Indica genetic components, although genetic backgrounds of all the materials were dominated by Japonica ones, and only a few materials were not detected the Indica genotypes loci.

Indica genotype frequency of maintainer line materials used differed with the different sources. The frequency variation of the Indica genotype loci of seven different sources materials was 0.106~0.062. The Indica genotype frequency from foreign materials was the highest, F_i=0.106; the materials from the East China was the lowest, Fi=0.062 (Figure 1).

1.3 Genetic diversity of experimental maintainer line materials

The overall Nei’s genetic diversity index (H) of maintainer line materials was not high, just 0.143,2. However, Nei’s genetic diversity indexes (H) were different among materials from different sources, and the range of variation was from 0.104,4~0.177,5. The highest genetic diversity index was from Northeast China (H=0.177,5) and the lowest was from Yunnan Academy of Agricultural Sciences (H=0.104,4). Genetic diversity of the materials from various sources was ranged as: Northeast China > RRI-YAU > East China > Overseas > other places of Yunnan > CAAS > YAAS (Table 1).

2 Discussion

The identification of rice Indica-Japonica genotypes with InDel molecular markers is a relatively mature technique (Feng et al., 2005; Lu et al, 2009; Lin et al., 2013). However, in the identification of materials’ Indica-Japonica genotype loci with InDel markers, it is necessary to consider how to ensure the reliability of the identification. Representative varieties of Indica rice (9311) and Japonica rice (Nipponbare) were used as control samples in this study, and also used Haomuxi (local old varieties of Yunnan Indica rice) as the control standard of Indica rice and Pantiangemanaogu (grown in Yunnan at the altitude of 2,600 m in Japonica Rice Landraces) as the control standard of Japonica rice. These two local old varieties as control standards can eliminate the interference of the materials’ genetic components of Indica and Japonica in the selection of Indica and Japonica genotypes. In this study, two Indica or Japonica rice varieties as the standard samples were not found to have inconsistent bands type on a certain marker. The method of double variety standard sample ensures the accuracy of identifying the genotypes of the Indica and Japonica loci.

This research found that most of Dian-type Japonica Hybrid Rice Maintainer Line had Indica gene locus in different degrees which provide a genetic basis for the utilization of the Heterosis between Indica and Japonica. In this study, maintainer line materials used were obtained by more than 10 generations self-breeding, however, on the loci of identification of Indica and Japonica genotypes, a few loci on a few materials were found to be heterozygous. This might be because these loci contribute to the adaptability of the material, and it is also possible that these loci haven’t led to segregation of performance traits during the 10 generations self-breeding of our materials selected, so heterozygous sites were not eliminated. The results also explained from the perspective of genetic locus that the characters of some sterile lines were not separated during the maintenance and reproduction of generations in the same environmental condition in the past few years. However, when they were planted under another environment, they showed variation. The results showed the importance of shuttle breeding (exotic breeding) for pure line varieties. As many as 61 (26.41%) materials are heterozygous for the R12M10 marker loci on the twelfth chromosome, which is worthy of a further research.

Genetic diversity analysis showed that source range of Dian-type Japonica rice maintainer Line is wide, and our research selected the materials from Progeny of Indica-Japonica hybrid. However, compared with the analysis results of Chinese local Japonica rice by the use of simple sequence repeat (SSR) markers (Zhang et al., 2012), the genetic diversity index of materials of our research which based on InDel markers was lower. The reason may be that the index of biological genetic diversity revealed by different molecular markers is different because of the different types of markers. Studies have shown that InDel markers are highly polymorphic among Indica and Japonica subspecies, but low polymorphism in subspecies. The level of average polymorphism is significantly lower than that of SSR molecular markers (Feng et al., 2005). Because an InDel marker can only identify two alleles, and a SSR marker can identify more alleles. On the other hand, the materials used of this study were not limited to the materials of Japonica varieties, but also only one of the genetic types-maintainer line, or even part of it-Dian-type Japonica rice maintainer line. Due to the highly directional selection of these materials during their cultivation, the characters and the genetic background that were not consistent with the standard of Dian-type Japonica rice maintainer line were eliminated. Therefore, the genetic background differences of these materials have no possible to be very large.

In this research, the Indica genetic component ratios and the genetic diversity between the maintainer lines in the maintainer materials were basically clear. The result could provide references for exploring how to make use of heterosis between Indica and Japonica subspecies and how to improve the efficiency of excellent hybrid combination selection in Dian-type Japonica hybrid rice.

3 Materials and Methods

3.1 Experimental materials

The research materials: 231 Dian-type Japonica hybrid rice, Nipponbare (Representative variety of Japonica Rice), Cultivar 9311, Haomuxi (local old varieties of Yunnan Indica rice), and Pantiangemanaogu (grown in Yunnan at the altitude of 2,600 m in Japonica Rice Landraces) were provided by Institute of rice research of Yunnan Agricultural University.

3.2 DNA extraction

The total DNA of fresh rice leaves was extracted by CTAB improved (Rogers and Bendich, 1985).

3.3 Primer information

We used Haomuxi (Dian-type Indica Rice), Pantiangemanaogu (Dian-type Japonica) and maintainer line Hexi 42B to screen 23 polymorphic markers (Table 2) from 34 InDel markers which were developed by Lu et al. (2009). The primers were synthesized by Beijing Genomics Institute.

3.4 PCR (Polymerase Chain Reaction) amplification

PCR reaction system: 10 µL Taq PCR MasterMix, 1 µL primer F (100 pmol), 1 µL primer R (100 pmol), 1 µL template DNA, 7 µL ddH₂O.

Reaction conditions: pre-denaturation temperature was 94°C, time was 5 min; denaturation temperature during the circulation time was 94°C, time was 30 seconds; annealing temperature was 55°C, time was 30 s; chain elongation temperature was 72°C, 35 cycles; final elongation temperature was 72°C, time was 7 minutes.

3.5 Electrophoresis detection

The amplification product was electrophoresis on 3% agarose gel. The electrophoretic results were photographed under the ultraviolet light. Three times of PCR amplification and electrophoresis were repeated for the DNA samples which haven’t showed strip. Samples, which didn’t appear electrophoresis strips after three times’ repeat, were treated as a non-electrophoretic strip.

3.6 Genetic component ratio of Indica and Japonica

The results of DNA PCR electrophoresis of representative Indica rice varieties (9311, Haomuxi) and representative Japonica rice varieties (Nipponbare, Pantiangemanaogu) were used as contrast. On the InDel loci, the samples which DNA had the same location of electrophoresis band with the Indica rice (9311 and Haomuxi) were marked as homozygous Indica genotypes (II), which DNA had the same location of electrophoresis band with the Japonica rice (Nipponbare and Pantiangemanaogu) were marked as homozygous Japonica genotypes (JJ), and which DNA had the same location of electrophoresis band with the Indica rice (9311 and Haomuxi) and the Japonica rice (Nipponbare and Pantiangemanaogu) were marked as Indica-Japonica heterozygous genotype (JI). Based on the genotype data of each InDel loci of each rice sample, the gene ratio of Indica or Japonica to the samples was calculated by function of Lu et al. (2009).

3.7 Genetic diversity analysis

On the basis of the genotype data identified by the lnDel loci of the samples, the Nei (1973) gene diversity index (H=1-(∑ P_i²)/N) of the material was calculated by PopGenever1.32 software.

Authors’ contributions

LYF and TXL were researchers of this study; TXL was the architect and person in charge of the project who guided the design of the experiment, the data analysis, the writing and revision of the paper; LYF was the executive of the experimental research, who completed the analysis of the data, the writing and modification of the first draft of the paper; LJ, LZ, JZZ, LDD and ZJL helped to complete the revision and proofreading of the paper; WJC, JSL, YCQ and PSH participated in the planting and management of experimental materials. All the authors read and approved the final text.

Acknowledgments

This study is funded by the National key R & D project (2016YFD0101101).

References

Feng F.J., Luo L.J., Li Y., Zhou L.G., Xu X.Y., Wu J.H., Chen H.W., Chen L., and Mei H.W., 2005, Comparative Analysis of Polymorphism of InDel and SSR Markers in Rice, Molecular Plant Breeding, Vol.3, No.5: 725-730

Goff S.A., Ricke D., Lan T.H., Presting G., Wang R.L., Dunn M., Glazebrook J., Sessions A., Oeller P., Varma H., Hadley D., Hutchison D., Martin C., Katagiri F., Lange B.M., Moughamer T., Budworth P., Zhong J.P., Miguel T., Paszkowski U., Zhang S.P., Colbert M., Sun W.L., Chen L.L., Cooper B., Park S., Wood T.C., Mao L., Quai P., Wing R., Dean R., Yu Y., Zharkikh A., Shen R., Sahasrabudhe S., Thomas A., Cannings R., Gutin A., Pruss D., Reid J., Tavtigian S., Mitchel J., Eldredge G., Scholl T., Miller R.M., Bhatnagar S., Adey N., Rubano T., Tusneem N., Robinson R., Feldhaus J., Macalma T., Oliphant A., and Briggs S., 2002, A draft sequence of the rice genome (Oryza sativa L. ssp. Japonica), Science, 296: 92-100

https://doi.org/10.1126/science.1068275

PMid:11935018

Hu H.Y., Zhang S.H., and Tan X.L., 2008, Study on Relationship between Maintainer Line sand Heterosis of Dian-typeⅠHybridRice (Oryza sativa L.), Send, Vol.27, No.51: 85-87

https://doi.org/10.1016/j.pnsc.2009.01.011

Lu B.R., Cai X.X., and Jin X., 2009, Efficient Indica and Japonica rice identification based on the InDel molecular method: Its implication in rice breeding and evolutionary research, Natural Science, 19: 1241-1252

Nei M., 1973, Analysis of gene diversity in subdivided populations, Proc Natl AcadSci USA, Vol.70, No.12: 3321-3323

https://doi.org/10.1073/pnas.70.12.3321

PMid:4519626

Rogers S.O., and Bendich A.J., 1985, Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues, Plant Mol. Biol., 5(2): 69-76

https://doi.org/10.1007/BF00020088

PMid:24306565

Shen Y.J., Jiang H., Jin J.P., Zhang Z.B., Xi B., He Y.Y., Wang G., Wang C., Qian L., Li X., Yu Q.B., Liu H.J., Chen D.H., Gao J.H., Huang H., Shi T.L., and Yang Z.N., 2004, Development of genome-wide DNA polymorphism data base for map-based cloning of rice genes, Plant Physiology, 35: 1198-1205

https://doi.org/10.1007/BF00020088

PMid:24306565

Tan X.L., 1988, A Study on Geneitc Diversity of Dian Type Hybrid Rice, Journal of Yunnan Agricultural University, Vol.3, No.1: 45-52

Tan X.L., Rong R.K., Jin S.L., Huang D.J., Zhang Z.L., Chen L.J., Wang X., Tan Y.L., Wang S.H., Wen J.C., Zhang S.L., Shi C.J., and Li Z.Y., 2006, Development of Japonica Hybrid Rice Basedon CMS System of Dian Typel, Journal of Yunnan Agricultural University, 21: 33-37

Wang L.Y., Zhang L.X., Gou X.X., Qi Y.B., Jin Q.S., and Wang J.J., 2013, Identification of -Japonica Attribute for Breeding Materials Using InDel Markers in Rice, Journal of Nuclear Agricultural Sciences, Vol.27, No.7: 0913-0921

Xu X.H., 2012, Yunnan miscellaneous 31 change liangshan district stripper face-guizhou rice cold liangshan area miscellaneous 31 yunnan supporting tissue culture technology demonstration and analysis of promotion effect, Culture with Planting, (5): 44-45

Yu J, Hu S.N, Wang J, Wong G.S., Li S.G., Liu B., Deng Y.J., Dai L., Zhou Y., Zhang X.Q., Cao M.L., Liu J., Sun J.D., Tang J.B., Chen Y.J., Huang X.B., Lin W., Tong W., Cong L.J., Geng J.N., Han Y.J., Li L., Li W., Hu G.Q., Huang X.G., Li W.J., Li J., Liu Z.W., Li L., Liu J.P., Qi Q.H., Liu J.S., Li L., Li T., Wang X.G., Lu H., Wu T.T., Zhu M., Ni P.X., Han H., Dong W., Ren X.Y., Feng X.L., Cui P., Li X.R., Wang H., Xu X., Zhai W.X., Xu Z., Zhang J.S., He S.J., Zhang J.G., Xu J.C., Zhang K.L., Zheng X.W., Dong J.H., Zeng W.Y., Tao L., Ye J., Tan J., Chen X.W., He J., Liu D.F., Tian W., Tian C.G., Xia H.G., Bao Q.Y., Li G., Gao H., Gao T., Wang J., Zhao W.M., Li P., Chen W., Wang X.D., Zhang Y., Hu J.F., Wang J., Liu S., Yang J., Zhang G.Y., Xiong Y.Q., Li Z.J., Mao L., Zhou C.S., Zhu Z., Chen R.S., Hao B.L., Zheng W.M., Chen S.Y., Guo W., Li G.J., Liu S.Q., Tao M., Wang J., Zhu L.H., Yuan L.P., and Yang H.M., 2002, A draft sequence of the rice genome (Oryza sativa L ssp. indica), Science, 296: 79-92

https://doi.org/10.1126/science.1068037

PMid:11935017

Yan S., Huang F.D., Li C.S., Tao Y.Z., and Zou G.Z., 2010, Correlation between genetic distance and heterosis based on yield-relate molecular markers in indica rice, Acta Agriculture Zhejiang ensis, Vol.22, No.5: 545-551

Zhang T., Ni X.L., Jiang K.F., Yang Q.H., Yang L., Wan X.Q., Cao Y.J., and Zheng J.K., 2009, Correlation Between Genetic Distance Based on Molecular Markers of Functional Genes and Heterosis in Rice, Chin J Rice Sci, Vol.23, No.6: 567-572

Zhang L.N., Cao G.Q., and Han L.Z., 2012, Analysis of Genetic Diversity of Japonica Rice Landracein China with Microsatellite Marker, Scientia AgriculturaSinica, Vol.45, No.3: 405-413

Zhang G.L., Wang M., and Xu X.H., 2014, Shallow thick soft technology to make fat Japonica hybrid rice Dian miscellaneous 31 reason to increase production, Serves of Agricultural Technology, Vol.31, No.7: 112-113