Experimental and genomic evidence for the indica-type cytoplasmic effect in Oryza sativa L. ssp. japonica

Journal of Integrative Agriculture 2016, 15(10): 2183–2191 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Experimental and genomic evidence for the indica-type cytoplasmic effect in Oryza sativa L. ssp. japonica LIU You-hong*, TANG Liang*, XU Quan*, MA Dian-rong, ZHAO Ming-hui, SUN Jian, CHEN Wen-fu Rice Research Institute, Shenyang Agricultural University/Key Laboratory of Northern japonica Rice Genetics and Breeding, Ministry of Education and Liaoning Province/Key Laboratory of Northeast Rice Biology and Genetics and Breeding, Ministry of Agriculture/Collaborative Innovation Center of japonica Rice Genetic Improvement and Production in Northeast China, Shenyang 110866, P.R.China

Abstract Cytoplasmic effects are important agronomical phenomena that have generated widespread interest in both theory and application. In the present study, five high yield rice cultivars (Oryza sativa L. ssp. japonica) in large-scale cultivation in northeast China were determined to possess Oryza sativa L. ssp. indica-type cytoplasm using cytoplasmic subspecies-specific molecular markers. This was confirmed by cytoplasmic genome-wide single nucleotide polymorphisms (SNPs) and functional gene sequencing. Two of these five japonica cultivars were core breeding parents with high yield and the other three were super-high-yield varieties registered by the Ministry of Agriculture of China. We constructed nuclear substitution lines to further demonstrate whether and how this indica-type cytoplasm contributed to yield improvement by comparing yield components. The results showed that under the same japonica nuclear background, the lines with indica-type cytoplasm had a significant decrease in tillers in exchange for increased grain number per panicle compared with their recurrent parents. Our results implied that botanical basis of this cytoplasmic effect was to reduce the plant’s branching differentiation to produce more floral organs under the constant nutrition. Our findings open another door for the utilization of inter-subspecific hybridization for the improvement of rice cultivar. Keywords: cytoplasmic effects, cytoplasmic genome-wide SNPs, super high yield rice, nuclear substitution lines

1. Introduction

Received 7 September, 2015 Accepted 28 September, 2015 LIU You-hong, E-mail: [email protected]; Correspondence SUN Jian, E-mail: [email protected]; CHEN Wen-fu, Tel/Fax: +86-24-88487184, E-mail: [email protected] * These authors contributed equally to this study. © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61190-X

The nuclear genome plays a predominant role in the inheritance of most plant traits; however, cytoplasmic and cytoplasmic-nuclear interactions also influence specific physiological activities in plants. Majority of proteins and components involved in organelle biogenesis, gene expression, and genome organization in chloroplasts and mitochondria are produced by the nuclear genome (Leon et al. 1998; Ramana et al. 2002; Strand 2004). Conversely, organelles are able to send signals to the nucleus that in turn control nuclear gene expression, a process called retrograde

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signaling (Nott et al. 2006). The chloroplast genome is a relic of an endosymbiont that was integrated into the cell and utilized as an organelle; it typically contains 100–200 genes and encodes proteins involved in photosynthesis and other chloroplast functions (Howe 2012). Chloroplast-to-nucleus retrograde signaling is triggered through pathways such as the Mg-ProtoIX dependent pathway and the plastid gene expression-dependent pathway (Nott et al. 2006). Complete plant mitochondrial genome sequence information is available for seven angiosperm species, namely, sugar beet, tobacco, Arabidopsis, rapeseed, rice, maize, and wheat. The size and organization of their mitochondrial genomes are highly variable, but these encode almost the same proteins (Kubo and Newton 2008; Li et al. 2009). Approximately 90% of the total sequence of the mitochondrial genome comprises introns and repeated elements, and the variation types include gain/loss/transfer/duplication of sequences and genome rearrangements (Kitazaki and Kubo 2010; Galtier 2011). Besides respiration, the mitochondrial genome also plays an important role in other biological processes such as DNA repair (Christensen 2013). Some studies have reported that nuclear-cytoplasmic interactions play essential roles during the development of certain agronomic traits in maize (Zea mays L.), wheat (Triticum aestivum L.), onion (Allium cepa L.), and sunflower (Helianthus annuus L.) (Rao and Fleming 1978; Galloway and Fenster 1999; Murai et al. 2002; Mizumoto et al. 2004; Gökçe and Havey 2006; Atienza et al. 2007; Chandra-Shekara et al. 2007). The application of nuclear-cytoplasmic interaction in rice (Oryza sativa L.) and research on its effects has mostly concentrated on cytoplasmic male sterility (Wang et al. 2006; Hu et al. 2012; Luo et al. 2013). A few reports have described cytoplasmic effects on yield, leaf traits and abiotic stress in both rice subspecies, O. sativa L. ssp. japonica and O. sativa L. ssp. indica (Tao et al. 2004; Tao et al. 2011). Some proteins located in the cytoplasm also affects plant development, seed germination and longevity (Huang et al. 2014; Shi et al. 2014). Although some genetic and molecular studies have identified several genes and pathways involved in cytoplasmic-nuclear interaction, it is very difficult to understand the associated factors and mechanisms from a holistic perspective. A previous study involving screening five japonica cultivars utilized in large-scale cultivation in northeast China has identified indica-type in subspecies differentiation site. Interestingly, two of these japonica cultivars were core breeding parents with high yield and the other three were super-high-yield rice varieties registered by the Ministry of Agriculture of China. These findings were most likely the result of breeding selection through an inter-subspecific hybridization strategy (Sun et al. 2012; Xu et al. 2014). Based

on these reports and driven by curiosity, we were prompted to design experiments to determine the cytoplasmic types of the five target accessions by cytoplasmic genome-wide sequence analysis and to evaluate the effect of the cytoplasm on the rice yield components.

2. Materials and methods 2.1. Plant materials To assess cytoplasm types, we sampled 36 varieties utilized in large-scale cultivation in Northeast China. Simultaneously, we collected samples representing five typical categories of O. sativa as controls, including 10 indica, 9 aus, 8 tropical japonica, 5 Japanese temperate japonica, and 2 aromatic varieties. The 36 tested varieties were sampled from the rice germplasm pool of Shenyang Agricultural University, China. Seeds of the control samples were obtained from the China National Rice Research Institute in Hangzhou, China. Information on these collections and tested varieties are listed in Appendix A.

2.2. DNA extraction To avoid the influence of the nuclear genome, we extracted the DNA from mitochondria and chloroplasts of young leaves by using a Plant Mito DNA (PS+PHENOL) Isolation Kit (GenMed Scientifics Inc., Arlington, MA, USA) and a PS/PHENOL Chloroplast DNA Isolation Kit (GenMed Scientifics), respectively. The extractions were conducted according to the manufacturer’s protocols. For specific-locus amplified fragment sequencing (SLAFseq) of each accession, total genomic DNA from individual plants was extracted from young leaves using the cetyl trimethylammonium bromide (CTAB) method.

2.3. Subspecies-specific cytoplasmic molecular marker analysis Using chloroplast DNA as templates, a subspecies-specific chloroplast marker, ORF100, was used to screen the subspecies types of 70 accessions (Nakamura et al. 1998). Primers used for amplification of this region were as follows: 5´-TTGTATTTCCTTAGACTT-3´ and 5´-ATCTATGGGGT CACAGCC-3´.

2.4. Cytoplasmic genome-wide SNP genotyping SLAF-seq is an efficient and high-resolution strategy for large-scale genotyping (Liu et al. 2014). This technology was used in the present study to identify cytoplasmic genome-wide single nucleotide polymorphisms (SNPs) in rice.

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We constructed a specific-locus amplified fragment (SLAF) library for high-throughput sequencing according to Liu et al. (2014). Purified DNA was digested into DNA fragments of approximately 300 bp in length using an appropriate restriction enzyme combination, in this case, EcoRI, NlaIII and MseI. The fragment ends were repaired, ligated with index paired-end adapters, and then adapter-modified ends were obtained. The target fragments were size selected on agarose gel and then PCR amplified. Then, we performed Illumina sequencing of the pooled library using the Illumina HiSeqTM 2000 paired-end sequencing protocol and defined the genotype of each SNP locus (Illumina, Inc.; San Diego, CA, USA) at Biomarker Technologies Corporation in Beijing, China (http://Biomarker.com.cn/). The cytoplasmic genome sequences of O. sativa ssp. japonica cv. Nipponbare were used as references to align the read sequences of the 70 accessions with the SOAP software. The chloroplast and mitochondrial genomes are available at http://rapdblegacy. dna.affrc.go.jp

2.5. Phylogenetic and haplotype network analysis SNPs that had a >0.05 minor allele frequency (MAF) while meeting the genotype calls of more than 60% of the accessions were used for phylogenetic analysis. After obtaining SNPs from the SLAF alignment of 70 samples, a neighbor-joining tree was constructed based on Nei’s genetic distances using the PowerMarker 3.25 and MEGA 4.0 software (Evanno et al. 2005; Liu and Muse 2005; Tamura et al. 2007). The chloroplast and mitochondrial SNPs of each accession were designated as haplotypes because the evolution of the cytoplasmic genome is conservative in rice. To avoid confusing unsuccessful genotyping with deletion-type SNPs, and thus overestimating the diversity level of cytoplasmic haplotypes, chloroplast and mitochondrial SNPs with integrities of >90% were filtered out for the build of the haplotype networks. The haplotype networks were constructed based on the median-joining method by using the Haplotype Viewer software (http://www.cibiv.at/~%20greg/haploviewer).

2.6. Sequences of 14 functional genes in mitochondria and chloroplasts To detect whether novel mutants occurred in the target accessions, we sequenced 10 and 4 important functional genes or gene segments in chloroplasts and mitochondria, respectively, which are involved in the biological processes of photosynthesis and respiration (Webber and Malkin 1990; Tang et al. 2004; Tian et al. 2006; Wang et al. 2006). The sequencing primers and PCR reaction conditions used in the present study were adopted from the above mentioned

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reports. The PCR products were collected for direct sequencing, which was performed by Beijing Genomics Institute (BGI) Corporation, Beijing, China. The information on the 14 genes and their corresponding primer sequences are listed in Appendix B.

2.7. Construction of isonuclear alloplasmic lines Three of the five target japonica varieties with a japonica-type nucleus and indica-type cytoplasm, namely, Shennong 265, Shennong 9741 and Liaogeng 454, were used as female parents in the crosses with two typical japonica varieties that comprised a japonica-type nucleus and cytoplasm, namely, Toyonishiki and Liaogeng 5. We aimed to substitute the nuclear genomes of the four target japonica varieties with japonica-type nuclear genomes from Toyonishiki and Liaogeng 5. Seven backcrosses using the male parents as recurrent parents were conducted. After selfing for purifying selection, six isonuclear alloplasmic lines (BC7F3) were constructed.

2.8. Cultivation and yield investigation The six isonuclear alloplasmic populations and their parents were grown in the paddy rice field at the experimental farm of Shenyang Agricultural University (Shenyang, China, 41°80´N, 123°38´E) during the summer of 2014. Seeds were sown in the seedling nursery on 18 April, and transplanted with one seedling per hill on 18 May. Each plot was 4.8 m2 and included 120 plants planted at 30 cm×13.3 cm intervals. The plots were arranged in a randomized block design with three replications. Fertilizer was applied with a basal dressing amount of 75 kg N, 150 kg P and 75 kg K ha–1. At the mature stage, the height of the plants was measured, and the aboveground parts of the plants per square meter were harvested. After counting the number of tillers and the number of panicles in each plants, average-sized panicles were collected to determine the number of grains and grain weight. The setting rate was determined by the ratio of non-fertilized and partially filled grains to total grains. ANOVA was performed with SPSS 17.0 software (SPSS, Inc., Chicago, USA) to compare yield components.

3. Results 3.1. Screening for indica-type cytoplasm in northeast Chinese japonica varieties To screen indica-type cytoplasmic samples, we used the specific primer ORF100, which was polymorphic between indica- and japonica-type cytoplasm (Kanno et al. 1993; Garris et al. 2005; Sun et al. 2013). The PCR products of

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Sequence alignment with the reference genome identified, valid single-end reads in each sample, with lengths of approximately 80 bp, which were generated through SLAF library construction and high-throughput sequencing. A distribution diagram of the SLAF tags for each accession was drawn on both the chloroplast and mitochondrial genomes (Appendixes C and D). We then extracted three sets of SNPs for subsequent analysis.

consistent with the subspecies differentiation (Fig. 1). Most japonica background accessions, namely, temperate japonica (blue), tropical japonica (cyan), and aromatic (green), were clustered together in one portion of the tree, whereas most indica background accessions, which included indica (red) and aus (magenta), were clustered in another. The five northern Chinese japonica varieties, namely, Shennong 265, Shennong 9741, Shennong 9816, Liaogeng 454, and Liaogeng 326, were also observed in the indica background cluster. After >90% integrity filtration, 218 chloroplast SNPs and 164 mitochondrial SNPs were used for haplotype analysis. 21 chloroplast and 43 mitochondrial haplotypes were detected in 70 accessions. Fig. 2 shows that the size of each circle was proportional to the number of accessions sharing that haplotype. The colors represent the five typical categories of O. sativa, which were also used to describe the branches of the neighbor-joining tree. Subspecies differentiation was also reflected in the two haplotype networks. The five target japonica varieties shared the same or genetically similar haplotypes as the indica varieties in both the chloroplast and mitochondrial networks. The neighbor-joining tree and haplotype analysis confirmed that the five target varieties had an indica cytoplasmic genetic background.

3.3. Phylogenetic and haplotype network analysis

3.4. Sequencing of 14 cytoplasmic functional genes

The first set of cytoplasmic genome-wide SNPs, which included 1 096 loci, was used to construct a neighbor-joining tree. Two main clusters in the neighbor-joining tree

In the analysis of subspecies-specific markers and cytoplasmic genome-wide SNPs, we discovered indica cytoplasmic attributes in the five target varieties. To further reveal the

indica-type and japonica-type ORF100 were 317 and 385 bp in length, respectively. We then tested a core collection of 36 japonica rice varieties from northern China, with seven typical japonica varieties and seven typical indica varieties as controls. Five samples (Shennong 265, Shennong 9741, Shennong 9816, Liaogeng 454, Liaogeng 326) produced PCR fragments of 317 bp in length representing the indica-type cytoplasm, whereas the other samples produced fragments of 385 bp representing japonica-type cytoplasm. It is worth mentioning that the five samples possessed typical japonica nuclear genomes as revealed by morphological indices, subspecies-specific markers and phylogenetic analysis (Sun et al. 2012).

3.2. Analysis of SLAF-seq data

Fig. 1 Neighbor-joining tree of 70 accessions constructed based on Nei’s (1972) genetic distances calculated from a set of 1 096 cytoplasmic genome-wide single nucleotide polymorphisms (SNPs). The branch color of each accession is defined based on its taxon: temperate japonica (blue), tropical japonica (cyan), aromatic (green), indica (red) or aus (magenta). The same as below. Five varieties, Shennong 265, Shennong 9741, Shennong 9816, Liaogeng 454, and Liaogeng 326 ordered from left to right were marked with +.

LIU You-hong et al. Journal of Integrative Agriculture 2016, 15(10): 2183–2191

types of functional genes and whether new mutations occurred in the chloroplast and mitochondrial genomes, we selected 14 important functional genes from the chloroplast and mitochondrial genomes, respectively, for sequence comparison. Besides the five target varieties, typical indica varieties 93-11 and four typical japonica varieties were sequenced as genotype controls. The results of sequencing and alignment indicated that the five target varieties possessed the same genotype combinations as the typical indica varieties 93-11 in all 14 cytoplasmic functional genes segments. Whereas, the four control japonica varieties possessed the same genotypes as the typical japonica (Tables 1 and 2). The results of gene sequencing and phylogenetic and haplotype network analysis, implied that the indica-type cytoplasm interacting with the japonica-type nucleus might have contributed to the higher yield potential of the five target varieties rather than novel mutations. To confirm this hypothesis, we constructed isonuclear alloplasmic lines for further experiments.

3.5. Effect of indica-type cytoplasm on yield components in nuclear substitution lines The Japanese introduced variety Toyonishiki and the local variety Liaogeng 5 are two typical japonica rice that possess japonica type cytoplasm and nucleus, both of which are

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core materials for large-scale cultivation in northeast China. The nuclear genomes of Shennong 265, Shennong 9741 and Liaogeng 454 were substituted with Toyonishiki and Liaogeng 5 by 7 backcross and 3 selfing events. Then, we investigated the yield components of these nuclear substitution lines. Tables 3 and 4 show that all three indica-type cytoplasms showed a significant effect on tiller number per m2, number of panicle per plant and grain number per panicle under the nuclear genome backgrounds of Toyonishiki and Liaogeng 5. Fig. 3 shows that under the Toyonishiki nuclear genome background, the substitution lines with indica-type cytoplasms showed a dramatic decrease in panicle number via tillering inhibition and an increase in grain number per panicle, except for the Shennong 265/Toyonishiki line. Similar trends were also observed with Liaogeng 5 nuclear genome background. The indica cytoplasm influenced a decrease in tiller number and an increase in grain number per panicle.

4. Discussion Extensive genetic studies have been conducted on cytoplasmic effects and its related breeding applications in crops (Rao and Fleming 1978; Galloway and Fenster 1999; Murai et al. 2002; Mizumoto et al. 2004; Gökçe and Havey 2006; Atienza et al. 2007; Chandra-Shekara et al. 2007).

Fig. 2 Haplotype networks of 70 accessions based on the variation in chloroplast and mitochondrial genomes, above network for chloroplast and below for mitochondria. Circle size is proportional to the number of accessions (numbers in each circle) for a given haplotype. Lines between haplotypes imply genetic similarity between haplotypes.

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Table 1 Alignment of polymorphic loci of chloroplast genes between five target varieties and typical japonica/indica varieties1) Varieties2) 93-11 Nipponbare Yanfeng 47 Liaogeng 5 Toyonishiki Shennong 9816 Liaogeng 326 Shennong 265 Shennong 9741 Liaogeng 454 Varieties 93-11 Nipponbare Yanfeng 47 Liaogeng 5 Toyonishiki Shennong 9816 Liaogeng 326 Shennong 265 Shennong 9741 Liaogeng 454

Gene loci and positions (bp) trnG-trnfM 5 907 6 376 12 174 12 198 12 219 12 347 T 7 GG T – C G – TA C 4 T G – TA C 4 T G – TA C 4 T G – TA C 4 T T 7 GG T – C T 7 GG T – C T 7 GG T – C T 7 GG T – C T 7  GG T – C Gene loci and positions (bp) atpA-2 trnT-trnL trnT-trnL-2 psbB-1 34 968 45 582 45 993 46 357 46 388 68 866 68 940 69 603 T T – – – A C TC C G 6 A 5 G A CT C G 6 A 5 G A CT C G 6 A 5 G A CT C G 6 A 5 G A CT T T – – – A C TC T T – – – A C TC T T – – – A C TC T T – – – A C TC T T – – – A C TC psbA 447 G A A A A G G G G G

rps16 6 086 C T T T T C C C C C

trnC-ycf6 rPoc2-1 rPoc2-2 atpA-1 17 899 17 962 28 052 29 073 34 654 32 TT A G C – CC C A – – CC C A – – CC C A – – CC C A – 32 TT A G C 32 TT A G C 32 TT A G C 32 TT A G C 32 TT A G C psbB-2 ccsA ndhH 69 619 69 630 69 633 105 740 113 042 GAC A T AAGC A ACG T C GCTT C ACG T C GCTT C ACG T C GCTT C ACG T C GCTT C GAC A T AAGC A GAC A T AAGC A GAC A T AAGC A GAC A T AAGC A GAC A T AAGC A

1)

Letters indicate the base. Number and – indicate the number of base insertions and deletions. The five target varieties are shown in bold. The same as below.

2)

Table 2 Alignment of polymorphic loci of mitochondrial genes between five target varieties and typical japonica/indica varieties Gene loci and positions (bp) nhd4

Varieties

112 925

113 001

113 190

207 680

207 690

207 697

207 714

207 719

207 816

207 820

207 841

207 847

207 850

207 860

207 862

207 866

cox3

93-11 Nipponbare Yanfeng 47 Liaogeng 5 Toyonishiki Shennong 9816 Liaogeng 326 Shennong 265 Shennong 9741 Liaogeng 454

T C C C C T T T T T

T C C C C T T T T T

T C C C C T T T T T

G C C C C G G G G G

CTG TCT TCT TCT TCT CTG CTG CTG CTG CTG

– 16 16 16 16 – – – – –

T C C C C T T T T T

– 95 95 95 95 – – – – –

A G G G G A A A A A

– 19 19 19 19 – – – – –

C T T T T C C C C C

AT GG GG GG GG AT AT AT AT AT

10 – – – – 10 10 10 10 10

G A A A A G G G G G

CG AA AA AA AA CG CG CG CG CG

CC AA AA AA AA CC CC CC CC CC

Table 3 Yield components of isonuclear alloplasmic populations and their parents Yield improvements Plant height (cm) Tillers number m–2 Number of panicle plant–1 Number of grain panicle–1 Setting rate 1 000-grain weight (g) *

Toyonishiki 106.24±3.49 411.68±28.93 15.87±0.76 100.37±9.20 0.92±0.05 23.08±0.40

Shennong 265/ Toyonishiki (BC7F3) 112.03±5.73 353.50±84.63 13.8±3.02* 118.84±13.35* 0.90±0.05 24.49±1.53

Shennong 9741/ Toyonishiki (BC7F3) 109.19±2.73 344.25±45.13* 13.00±1.35* 121.15±19.33* 0.91±0.09 23.33±1.26

Liaogeng 454/ Toyonishiki (BC7F3) 111.58±4.53 341.75±29.53** 13.49±1.19* 121.91±13.26* 0.93±0.04 24.78±0.91

and ** indicate a significant difference at the 5 and 1% levels based on variance analysis (ANOVA), respectively. ±SD indicate standard deviation. The same as below.

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Table 4 Yield components of isonuclear alloplasmic populations and their parents Yield improvements

Liaogeng 5

Plant height (cm) Tillers number m–2 Number of panicle plant–1 Number of grain panicle–1 Setting rate 1 000-grain weight (g)

103.88±1.30 392.5±27.68 14.80±0.28 126.45±17.47 0.92±0.10 23.99±1.26

Shennong 265/ Liaogeng 5 (BC7F3) 102.26±2.63 320.00±47.55** 12.16±1.83** 161.72±16.20** 0.94±0.04 25.1±0.77

Liaogeng 5

Shennong 265/Toyonishiki (BC7F3)

Shennong 265/Liaogeng 5 (BC7F3)

Shennong 9741/Toyonishiki (BC7F3)

Shennong 9741/Liaogeng 5 (BC7F3)

Liaogeng 454/Toyonishiki (BC7F3)

Liaogeng 454/Liaogeng 5 (BC7F3) 500

450

450 *

400

**

350

350

300

300

250

250

200

*

*

*

*

100

50

50 Grain numbers panicle–1

**

*

*

150

100

Tiller numbers m–2

*

**

200

150

0

Liaogeng 454/ Liaogeng 5 (BC7F3) 109.02±4.73 345.00±52.15* 12.63±2.25* 154.12±16.28* 0.93±0.01 25.49±0.48*

Toyonishiki

500 400

Shennong 9741/ Liaogeng 5 (BC7F3) 111.63±5.18 336.50±56.18* 12.50±1.71* 158.02±26.45* 0.9±0.08 25.88±1.71

0

Tiller numbers m–2

Grain numbers panicle–1

Fig. 3 Comparisons of tiller numbers m–2 and grain numbers per panicle among two isonuclear alloplasmic populations and their parents. * and ** indicate a significant difference at the 5 and 1% levels based on variance analysis (ANOVA), respectively. Bars indicate standard deviation.

However, reports on the agronomic traits other than male sterility affected by nuclear-cytoplasmic interaction in rice are limited. Two previous studies conducted by Tao et al. (2004, 2011) have demonstrated that nuclear-cytoplasmic interaction had an effect on plant height, flag leaf width, low temperature tolerance, and 1 000-grain weight. The main difference between the present study and previous studies was that we focused on the cytoplasmic effects span the two subspecies. Phenotypic variation from hybrid vigor might be even more significant because of the larger divergence level of the genetic backgrounds derived from indica and japonica. The growing number of cloned quantitative trait loci, genome variations, and haplotype blocks related to agronomically important traits in rice have provided a solid foundation for direct selection and molecular breeding, and a number of genes have been successfully introgressed into mega varieties of rice (Rao et al. 2014). In this present study, we were surprised to find that the japonica accessions with confirmed indica-type cytoplasm were officially recognized as super-high-yield rice. This finding implies

that as a result of breeding selection, the accessions with indica-type cytoplasm and a japonica nucleus were not only fully compatible but also possessed a positive advantage. Furthermore, this advantage in yield components might have been derived from shaping the plant type. In this context, besides directly affecting photosynthesis and respiration, the indica-type cytoplasm could also directly or indirectly influence the the formation of organs and tissues under the japonica background. Although we conducted purity selection, heterozygosity in the nuclear genome could have also promoted, to some level, the advantage of yield components as earlier described. We will thoroughly evaluate the effect of genetic and environmental effects on the stable generation. The genetic information of plants is stored in the genomes of three organelles, the nucleus, plastid and mitochondrion. Photosynthesis and respiration are regulated by enzymes encoded by the cytoplasmic and nuclear genomes. Nuclear genes regulate cytoplasmic gene expression and affect cytoplasmic genome organization (Leon et al. 1998; Ra-

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mana et al. 2002; Strand 2004). Simultaneously, plastids and mitochondria send signals to the nucleus to regulate nuclear gene expression, also known as retrograde signaling (Nott et al. 2006). Therefore, we also have reason to believe that both cytoplasmic and nuclear genes were active and played important roles in the advantage of yield components observed in the present study. Regarding the molecular mechanisms of nuclear-cytoplasmic interaction, the most thorough research has been on cytoplasmic male sterility. In BT-CMS and HL-CMS lines, the expression of orf79 causes gametophytic male sterility, which can be restored by the nuclear genes, RF1 and Rf5, respectively (Wang et al. 2006; Hu et al. 2012). In CMS-WA lines, which can be restored by either of two dominant Rf genes, Rf3 or Rf4, the product of the mitochondrial gene WA352 preferentially accumulates in the anther tapetum and triggers premature tapetal programmed cell death and consequent pollen abortion (Luo et al. 2013). Compared to sterility and restoration, the genetic advantage of yield components involving photosynthesis, respiration and plant morphology is even more complex. Thus, this genetic basis is probably involved in various pathways based on the interaction between entire genomes of the japonica-type nucleus and indica-type cytoplasm. Researchers and breeders noticed early on the cytoplasmic effect of rice, however, except for male sterility, the contribution of cytoplasmic genes and nuclear-cytoplasmic interactions to improve agronomic traits has not received enough attention in rice research and breeding. The effect of indica-type cytoplasm described in the present study was selected during breeding practice and thus were accommodated by the locally cultivated ecoregions without drastic adverse interactions, which in turn could be used as potential sources of cytoplasm in future japonica breeding activities.

5. Conclusion Five high-yield japonica rice utilized in large-scale cultivation in northeast China possess indica-type cytoplasm, which was confirmed by subspecies-specific molecular marker, cytoplasmic genome-wide SNPs, and functional gene sequencing. Compared to recurrent parents, the nuclear substitution lines with indica-type cytoplasm had a significant decrease in tillers in exchange for increased grain number per panicle. Branching reduction to win more floral organs might be the botanical basis of this cytoplasmic effect.

Acknowledgements This study was supported by the National Natural Science Foundation of China (31371587 and 31430062), the Cultivation Plan for Youth Agricultural Science and Technology

Innovative Talents of Liaoning Province (2014046), the China Postdoctoral Science Foundation Grant (2014M560221) and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT), China. Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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