Comparative Metabolomics Reveals Two Metabolic Modules Affecting Seed Germination in Rice (Oryza sativa)

The process of seed germination is crucial not only for the completion of the plant life cycle but also for agricultural production and food chemistry; however, the underlying metabolic regulation mechanism involved in this process is still far from being clearly revealed. In this study, one indica variety (Zhenshan 97, with rapid germination) and one japonica variety (Nipponbare, with slow germination) in rice were used for in-depth analysis of the metabolome at different germination stages (0, 3, 6, 9, 12, 24, 36, and 48 h after imbibition, HAI) and exploration of key metabolites/metabolic pathways. In total, 380 annotated metabolites were analyzed by using a high-performance liquid chromatography (HPLC)-based targeted method combined with a nontargeted metabolic profiling method. By using bioinformatics and statistical methods, the dynamic changes in metabolites during germination in the two varieties were compared. Through correlation analysis, coefficient of variation analysis and differential accumulation analysis, 74 candidate metabolites that may be closely related to seed germination were finally screened. Among these candidates, 29 members belong to the ornithine–asparagine–polyamine module and the shikimic acid–tyrosine–tryptamine–phenylalanine–flavonoid module. As the core member of the second module, shikimic acid’s function in the promotion of seed germination was confirmed by exogenous treatment. These results told that nitrogen flow and antioxidation/defense responses are potentially crucial for germinating seeds and seedlings. It deepens our understanding of the metabolic regulation mechanism of seed germination and points out the direction for our future research.

OsPRR37 Alternatively Promotes Heading Date Through Suppressing the Expression of Ghd7 in the Japonica Variety Zhonghua 11 under Natural Long-Day Conditions

Heading date is an important agronomic trait of rice (Oryza sativa L.) and is regulated by numerous genes, some of which exhibit functional divergence in a genetic background-dependent manner. Here, we identified a late heading date 7 (lhd7) mutant that flowered later than wild-type Zhonghua 11 (ZH11) under natural long-day (NLD) conditions. Map-based cloning facilitated by the MutMap strategy revealed that LHD7 was on the same locus as OsPRR37 but exhibited a novel function as a promoter of heading date. A single-nucleotide mutation of G-to-A in the coding region caused a substitution of aspartic acid for glycine at site 159 within the pseudo-receiver (PR) domain of OsPRR37. Transcriptional analysis revealed that OsPRR37 suppressed Ghd7 expression in both ZH11 background under NLD conditions and the Zhenshan 97 background under natural short-day conditions. Consistently, the expression of Ehd1, Hd3a and RFT1 was enhanced by OsPRR37 in the ZH11 background. Genetic analysis indicated that the promotion of heading date and reduction in grain yield by OsPRR37 were partially dependent on Ghd7. Further investigation showed that the alternative function of OsPRR37 required an intact Ghd7-related regulatory pathway involving not only its upstream regulators OsGI and PhyB but also its interacting partner Hd1. Our study revealed the distinct role of OsPRR37 in the ZH11 background, which provides a more comprehensive understanding of OsPRR37 function and enriches the theoretical bases for improvement of rice heading date in the future.

DAYA REGENERASI KALUS DAN TUNAS IN VITRO PADI VARIETAS TARABAS PADA BERBAGAI KONSENTRASI 2,4-D

In Vitro Callus and Plant Regeneration Rate of Tarabas Rice on Several Concentrations of 2,4-D The Agricultural Research and Development Agency and the West Java Provincial Government are developing new superior varieties with Japonica rice standards, namely the Tarabas variety. However, the equivalence of somatic embryogenesis ability of Tarabas rice with original Japonica variety has not been reported. In this study, the frequency of callus regeneration of Tarabas vs Hwayoung rice varieties was compared. Induction of callus from mature embryos with several concentrations of 2,4-D showed the same extent of callus formation in both rice varieties. Callus induced by 1 ppm of 2,4-D showed the higher rate of shoot formation. On the other hand, percentage of callus formation of Tarabas rice was not affected by the increase of 2,4-D concentrations and was able to show 100% regeneration rate at the fourth week in the regeneration medium, although the shoot growth was not as fast as those from medium with 1 ppm 2,4-D. Therefore, these results suggest that Tarabas variety has a somatic embryogenesis capacity equivalent to that of japonica rice and has the potential as research objects in the field of biotechnology. Badan Penelitian dan Pengembangan Pertanian serta Pemerintah Provinsi Jawa Barat mengembangkan varietas unggul baru dengan standar padi Japonica yaitu varietas Tarabas. Namun, kesetaraan varietas Tarabas dengan varietas Japonica asli dalam kemampuan embriogenesis somatik belum dilaporkan. Penelitian ini membandingkan respons kultur jaringan antara beras Tarabas dan padi Japonica varietas Hwayoung. Induksi kalus dari embrio matang dengan beberapa konsentrasi 2,4-D menunjukkan respons pembentukan kalus yang sama pada kedua varietas padi. Kalus yang diinduksi 1 ppm 2,4-D menunjukkan laju pembentukan tunas yang lebih tinggi. Di sisi lain, kalus Hwayoung yang diinduksi konsentrasi 2,4-D yang lebih tinggi menunjukkan penghambatan dalam pembentukan tunas. Di lain pihak, pembentukan kalus padi Tarabas tidak terpengaruh oleh peningkatan konsentrasi 2,4-D dan mampu menunjukkan 100% laju regenerasi tanaman pada minggu keempat di media regenerasi walaupun pertumbuhan tunas tidak secepat pada perlakuan 1 ppm 2,4-D. Karena itu, hasil penelitian ini menunjukkan bahwa varietas Tarabas memiliki kapasitas embriogenesis somatik yang setara dengan padi japonica dan padi Tarabas mempunyai potensi sebagai obyek penelitian di bidang bioteknologi.

Two SNP Mutations Turned off Seed Shattering in Rice

Seed shattering is an important agronomic trait in rice domestication. In this study, using a near-isogenic line (NIL-hs1) from Oryza barthii, we found a hybrid seed shattering phenomenon between the NIL-hs1 and its recurrent parent, a japonica variety Yundao 1. The heterozygotes at hybrid shattering 1 (HS1) exhibited the shattering phenotype, whereas the homozygotes from both parents conferred the non-shattering. The causal HS1 gene for hybrid shattering was located in the region between SSR marker RM17604 and RM8220 on chromosome 4. Sequence verification indicated that HS1 was identical to SH4, and HS1 controlled the hybrid shattering due to harboring the ancestral haplotype, the G allele at G237T site and C allele at C760T site from each parent. Comparative analysis at SH4 showed that all the accessions containing ancestral haplotype, including 78 wild relatives of rice and 8 African cultivated rice, had the shattering phenotype, whereas all the accessions with either of the homozygous domestic haplotypes at one of the two sites, including 17 wild relatives of rice, 111 African cultivated rice and 65 Asian cultivated rice, showed the non-shattering phenotype. Dominant complementation of the G allele at G237T site and the C allele at C760T site in HS1 led to a hybrid shattering phenotype. These results help to shed light on the nature of seed shattering in rice during domestication and improve the moderate shattering varieties adapted to mechanized harvest.

Genome sequence of the model rice variety KitaakeX

Background: The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species. Results: Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics. Conclusions: The high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.

Genome sequence of the model rice variety KitaakeX

Background: The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species. Results: Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics. Conclusions: The high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.

Genome sequence of the model rice variety KitaakeX

Background: The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species. Results: Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics. Conclusions: The high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.

Genetic effects of F1 pollen sterility genes S-b, S-d and S-e in rice (Oryza sativa L.)

An experimental population commonly used in genetic analyses of gene or quantitative trait loci (QTLs) in rice is chromosome segment substitution lines (CSSLs). In the present study, with the typical indica variety Guangluai 4 as a donor and japonica variety Taichung 65 as a recipient, seven CSSLs carrying F₁pollen sterility genes S-b, S-d, S-e, S-b/S-d, S-b/S-e, S-d/S-e, and S-b/S-d/S-e were obtained by specific selection for the target genes, non-specific selection for the genome of the recurrent parents in four backcross populations (BC₁F₂, BC₂F₂, BC₃F₂ and BC₃F₃). We evaluated the genetic effect of the F₁ pollen sterility genes using 35 F₁hybrid individuals in crosses derived from CSSLs and Taichung 65. Pollen fertility of F₁ hybrid plants was observed and the results indicated that the single genes S-b, S-d and S-e can cause 67.7%, 14.6% and 53.2% of pollen sterility, respectively. Multiple genes S-b/S-d, S-b/S-e, S-d/S-e, and S-b/S-d/S-e can cause 76.6%, 85%, 68.7%, and 93% of pollen sterility, respectively.

The Arrangement of Endosperm Cells and Development of Starch Granules Are Associated With the Occurrence of Grain Chalkiness in Japonica Varieties

The endosperm chalkiness in grains is one of the most important traits in rice grain qualities, and grain chalkiness directly determines grain quality and market price. Therefore, the endosperm chalkiness reduction is an important goal of breeding scientist in rice. However, little is known about the relationship of the formation of rice endosperm chalkiness and the morphological structure and development in fragrant japonica and conventional japonica varieties. In this study, we have mainly investigated the chalkiness characters such as degree of endosperm chalkiness (DEC), chalkiness rate (CR) and chalkiness area (CA) in 12 fragrant japonica varieties and 9 conventional japonica varieties. Furthermore, the endosperm structure and starch granules of rice grains were also observed with scanning electron microscope (SEM). The results indicated that the 21 varieties including the different fragrant japonica and conventional japonica varieties has a linear relationship between the DEC and the CR (P < 0.05). Among the fragrant japonica and conventional japonica varieties, the biggest difference is the CR, follow by CA and DEC. In addition, there is a certain correlation between the arrangement of endosperm cells, the distribution of starch granules and the occurrence of chalkiness characters in the different fragrant japonica and conventional japonica varieties. For the same fragrant japonica or conventional japonica variety, there is no significant difference between the starch granules from the transparent parts of chalkiness and non-chalky, while the starch granules of grains between chalky and non-chalky has obvious difference. Our results would provide important references for high quality rice breeding.

Genetic analysis and fine mapping of the RK4 gene for round kernel in rice (Oryza sativa L.)

Grain shape of rice is an important trait for both yield and quality. A rice rk4 (round kernel) mutant was obtained from the japonica variety Zhonghua 11 by radiation of ⁶⁰Co-γ. The grain width of the mutant was increased and the length was decreased. Simultaneously, the 1000-grain weight was slightly reduced, therefore the grain shape of the mutant tended to be small and round. In this study, genetic analysis and gene mapping of the mutant gene were carried out using the F₂ and F₃ populations derived from the mutant and the indica variety Xianhui 8006. The results suggested that the round kernel was controlled by a single recessive allele (rk4) which was located on chromosome 5. The RK4 gene was further mapped between the molecular markers LSTS5-77 and LSTS5-60 with 0.57 and 0.096 cM, respectively. A BAC clone was found to span the RK4 locus, and the RK4 gene was placed in a 46 kb region that contains six annotated genes according to the available sequence annotation database. This result will help us to isolate the RK4 gene and reveal the molecular mechanism of the round kernel in rice.