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10 September 2016, Volume 30 Issue 5 Previous Issue    Next Issue
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Phenotyping and Gene Cloning of a Floury Endosperm Mutant ws in Rice Peng-yi PAN, Jian-ping ZHU, Yun-long WANG, Yuan-yuan HAO, Yue CAI, Wen-wei ZHANG, Ling JIANG, Yi-hua WANG, Jian-min WAN 2016, 30(5): 447-457.  DOI: 10.16819/j.1001-7216.2016.6048 Abstract ( )   HTML ( )   PDF (2322KB) ( )   In this study, we obtained a stably inherited floury endosperm mutant ws from japonica variety cv. Dianjingyou 1 (DJY), with its 1000-grain weight, grain size, total starch content, and amylose content in mature seeds all decreased compared with wild-type, as well as the swelling power of starch. By observing the structure of mature and developing endosperm, we found that the starch granules in ws mutant were smaller and loosely packed. The WS locus was first mapped to a region near the centromere in chromosome 8 with 92 recessive individuals, and then was narrowed down to a 95 kb region by using 2025 recessive individuals. Sequence analysis revealed that the coding region of the small subunit of adenosine diphosphate glucose pyrophosphorylase (AGPS2) had a single nucleotide substitution, resulting in a change in the amino acid sequence. RT-PCR analysis showed no significant difference in the expression levels of genes encoding the AGPase subunits in the mutant endosperm, while immunoblot analysis revealed a reduced protein content of the AGPS2b. Meanwhile, the enzyme activity of AGPase in the mutant was decreased to half of the wild-type. These results showed that the mutation of OsAGPS2 caused the decreased activity of AGPase, thus affecting the starch biosynthesis in rice endosperm.

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Global Genome Expression Analysis of Root Genes under Drought Stress in Weedy Rice and Up-land Rice Guo-hua DING, Jian SUN, Guang YANG, Feng-ming ZHANG, Liang-ming BAI, Shi-chen SUN, Shu-kun JIANG, Tong-tong WANG, Hong-liang ZHENG, Tian-shu XIA, Xi-hong SHEN, Dian-rong MA, Wen-fu CHEN 2016, 30(5): 458-468.  DOI: 10.16819/j.1001-7216.2016.5180 Abstract ( )   HTML ( )   PDF (2274KB) ( )   The expression changes of root genes in drought-resistance weedy rice HEB07-2 (Oryza sativa f. spontanea) and up-land rice IAPAR9 (Oryza sativa) were analyzed under polyethylene glycol(PEG)-simulated with drought stress condition with Affymetrix GeneChip rice genome array. The results indicated that the extent and direction of transcriptome response of HEB07-2 and IAPAR9 to drought differed greatly. For HEB07-2, among 6878 expressed genes, 4693 were up-regulated and 2185 were down-regulated under drought condition. For IAPAR9, among 2923 expressed genes,983 were up-regulated and 1940 were down-regulated. Analysis of differentially expressed genes in HEB07-2 and IAPAR9 showed that the weedy rice HEB07-2 had a higher changing fold than the up-land rice IAPAR9. Gene ontology analysis revealed that genes of HEB07-2 in potassium ion transporting(GO: 0006813), secondary metabolite(GO: 0019748), cell growth(GO: 0016049), glucose metabolism(GO:0006006), transmembrane ion transporter activity(GO:0015075), ferroheme coalition(GO:0020037), oxidordeuctase activity(GO:0016491)were significantly up-regulated in comparing with IAPAR9. These were consistent with the physiological and phenotypic data.

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Knock-out Efficiency Analysis of Pi21 Gene Using CRISPR/Cas9 in Rice Fang-quan WANG, Fang-jun FAN, Wen-qi LI, Jin-yan ZHU, Jun WANG, Wei-gong ZHONG, Jie YANG 2016, 30(5): 469-478.  DOI: 10.16819/j.1001-7216.2016.6009 Abstract ( )   HTML ( )   PDF (6345KB) ( )   A CRISPR/Cas9 vector, which contains two targets (Target 1 and Target 2) of Pi21gene, was constructed and transformed to rice variety Nanjing 9108. Twenty-eight T0 transgenic lines were obtained and confirmed by T-DNA specific PCR. Restriction digestion analysis revealed 78.57% mutant ratio in Target 1 site, 92.86% mutant ratio in Target 2 site, 78.57% ratio of lines containing both Target 1 and Target 2 mutant in transgenic lines. Through sequencing analysis, we found the mutant types of targets, such as deletion of bases, insertion of bases, insertion behinds deletion of bases, and long fragment deletion of DNA, etc. Moreover, except amino acid deletion, most transgenic lines displayed frameshift mutation,which caused function loss of Pi21. In this way, a series of Pi21-knock-out rice lines were obtained, and further, could be used for functional analysis of Pi21 and rice blast broad-spectrum resistant breeding.

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Genetic Analysis and Gene Mapping of a Dwarf and High-tillering Mutant mz3 in Rice (Oryza sativa L.) Xiao-mei SU, Yu-xing FANG, Yu-long LIU, Feng LIU, Suo-bing ZHANG, Yun-hui ZHANG, Yi-qun BAO 2016, 30(5): 479-486.  DOI: 10.16819/j.1001-7216.2016.6037 Abstract ( )   HTML ( )   PDF (3806KB) ( )   A dwarf and high-tillering mutant, temporarily termed as mz3 was obtained from Zhonghua 11, a japonica rice variety by EMS mutagenesis. Genetic analysis indicated that the mutant phenotype was controlled by a recessive gene. An F2 population was developed by crossing mz3 with Nanjing 11, an indica rice variety for mapping of the mutant gene, which was ultimately restricted within a physical distance of about 747 kb between two SSR markers RM19353 and RM510 on the long arm of rice chromosome 6. Gene prediction revealed that D3, a gene conferring rice plant height and tiller number was located in this region. Sequence analysis identified a single G to A nuclear acid substitution at the position 636 from the ATG start codon, forming a premature translational termination codon. Sequencing of 10 randomly selected recessive plants from mz3/Nanjing 11 mapping population confirmed the same mutation site carried by the 10 individuals. Subcelluar location indicated that D3 protein of the mutant was located in the nuclei as wild type. BiFC analysis showed that D3 protein of the mutant could not interact with D14 protein for lacking of essential amino acids that bind with D14, blocking signal transduction of strigolactones. So we speculated that the mutant phenotype of mz3 was probably caused by mutation in D3 gene.

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Development of Functional Markers and Identification of Haplotypes for Rice Grain Width Gene GS5 Chuan-deng YI, De-rong WANG, Wei JIANG, Wei LI, Xiao-jun CHENG, Ying WANG, Yong ZHOU, Guo-hua LIANG, Ming-hong GU 2016, 30(5): 487-492.  DOI: 10.16819/j.1001-7216.2016.6038 Abstract ( )   HTML ( )   PDF (436KB) ( )   As a major component of rice grain shape, the grain width is an important trait which is correlated with the rice yield and quality. Based on the alignment of gene GS5 genomic DNA sequence, two functional markers were developed based on two missense polymorphisms (ACC/CTA in the exon2, A/C in the exon9, respectively). Subsequently, the markers were used to identify the genotypes of gene GS5 in the 294 accessions of a rice mini-core collection and 65 japonica varieties released in Jiangsu Province from 2007 to 2013. We found that the allelic variations at the two target loci had significant or extremely significant differences in the grain shape traits(grain length, grain width and ratio of length to width). Based on the two variation sites of gene GS5, four haplotypes (combinations) were found in the rice mini-core collection, and had extremely significant effect on grain width, grain thickness and ratio of length to width in the indica group, and on grain width and ratio of length to width in the japonica group, respectively. While in japonica varieties released by Jiangsu, only two haplotypes, Hap1 (represented by 64 varieties) and Hap2 (represented by one variety) were found. The results will lay a foundation for the application of the useful allelic variations or haplotypes of gene GS5 to the rice yield and quality breeding program.

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Effect of Different Mechanical Transplantation Methods and Density on Yield and Its Components of Different Panicle-typed Rice Ya-jie HU, Hai-jun QIAN, Wei-wei CAO, Zhi-peng XING, Hong-cheng ZHANG, Qi-gen DAI, Zhong-yang HUO, Ke XU, Hai-yan WEI, Bao-wei GUO 2016, 30(5): 493-506.  DOI: 10.16819/j.1001-7216.2016.6015 Abstract ( )   HTML ( )   PDF (1184KB) ( )   In order to apply suitable panicle-typed cultivar and plant density under different mechanical transplantation methods, and clarify high-yield formation characteristics of different panicle-typed rice, an experiment was conducted in Xinghua test point of Yangzhou University during 2013 and 2014. Three panicle-typed rice including large panicle type (LPT, Yongyou 2640 and Yongyou 8), medium panicle type (MPT, Wuyunjing 24 and Ningjing 3) and small panicle type (SPT, Huaidao 5 and Huaidao 10) were field-grown. We compared three mechanically transplanted methods including mechanically transplanted pot seedling (A), mechanically transplanted carpeted seedling in row spacing of 30 cm (B), mechanically transplanted carpeted seedling in row spacing of 25 cm (C) at different plant densities, namely plant spacing of 12 cm, 14 cm and 16 cm (marked as 1, 2, 3) in A and plant spacing of 10 cm, 11.7 cm, 13.3 cm, 14.8 cm, 16 cm (marked as 1, 2, 3, 4, 5) in B and C. Yield and its formation, panicle traits were investigated. The main results were as follows: 1) for mechanically transplanted pot seedling, with declining plant density, yield of LPT increased and then reduced, peaking in A2 treatment; yield of MPT and SPT reduced, peaking in A1 treatment. For mechanically transplanted carpet seedling, with reducing plant density, yield of LPT increased and then reduced in B, peaking in B4 treatment, yield of LPT increased in C, peaking in C5 treatment; yield of MPT increased and then reduced in B and C, peaking in B3 and C4 treatment, respectively; yield of SPT reduced in B, peaking in B1 treatment, yield of SPT increased and then reduced, peaking in C2 treatment. In the same plant density, yield of mechanically transplanted pot seedling was significantly higher than mechanically transplanted carpet seedling, the range of increased yield followed a tendency of LPT>MPT>SPT, with no significant difference in B and C. For B and C, in the same plant spacing, yield of LPT was higher in B than C; yield of MPT was higher in B than C in plant spacing of 10 cm, 11.7 cm, 13.3 cm, but it was opposite at plant spacing of 14.8 cm, 16 cm; yield of SPT was higher in C than B in plant spacing of 11.7 cm, 13.3 cm, 14.8 cm, 16 cm. 2) With plant density reduced, panicle number reduced and spikelet number per panicle increased in different panicle-typed rice under different mechanically transplanted methods. At the same plant density, there was no significant difference in panicle, grain filled percentage and 1000-grain weight in different mechanically transplanted methods, but spikelet number per panicle was significantly higher in A than B or C, following a trend of LPT>MPT>SPT. For B and C, number of panicle was lower in B than in C, but it was opposite in spikelet number per panicle. 3) Panicle length, grain density, grain weight per panicle, number of primary and secondary rachis branches, number of grains of primary and secondary rachis branches were increased with plant density reduced, it was opposite in the ratio of No. of primary rachis branch to No. of secondary rachis branch and ratio of No. of grains of primary rachis branch to No. of grains of secondary rachis branch. In the same plant density, panicle length, grain density, grain weight per panicle, number of primary and secondary rachis branches, number of grains of primary and secondary rachis branches in A than in B or C. Therefore, mechanically transplanted pot seedling of LPT could reduce plant density, it was beneficial to enlarge panicle type and increase total spikelet number and yield; for MPT and SPT, higher density should be adopted to increase panicle and spikelet number per panicle for high yield. For mechanically transplanted carpet seedling of LPT, B method and wider plant spacing should be applied to expand panicle type and acquire higher yield; for MPT, B method and medium density should be applied to coordinate panicle and spikelet number per panicle to increase total spikelet number; for SPT, C method and narrow plant spacing should be applied to increase number of panicle to achieve high yield.

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Characteristics of Stem Growth and Formation of Grain of indica Hybrid Rice in Different Planting Methods and Their Correlation with Meteorological Factors Qing-lan TIAN, Bo LIU, Hong SUN, Sha HE, Xiao-yuan ZHONG, Min ZHAO, Wan-jun REN 2016, 30(5): 507-524.  DOI: 10.16819/j.1001-7216.2016.6012 Abstract ( )   HTML ( )   PDF (2093KB) ( )   The objective of this study is to explore the growth pattern and differences of stem and panicle of indica hybrid rice under various planting methods in panicle differentiation stage, and to investigate the effects of planting methods on grain formation. A split plot experiment was carried out in 2014 with two factors, including variety (Yixiangyou 2115 and F you 498) and planting method (mechanized direct-seeding, MD; mechanized transplanting, MT; hand transplanting, HT).The Growth pattern of stem and panicle, the formation characteristics of grain were researched, and the relationship between meteorological factors in panicle differentiation stage and the growth of stem and panicle were analyzed. The results were as follows: 1) Length of first to third elongated internodes from the base stablized 20 d, 16 d and 12 d before heading,respectively, indicating that reducing length and increasing roughness of basal first and second internodes should be done 16 d before heading. Dry panicle weight and ratio of panicle to stem increased rapidly 12 d before heading. 2) Dry weight ratio of panicle to stem was significantly positively correlated with rachis branches and spiketets from 16 d before heading to heading date. The ability that panicle competing assimilates with stem directly affected the formation of grain. 3) MT had higher dry weight ratio of panicle to stem in middle and late differentiation stage of panicle, MT also had higher dry matter accumulation of panicle than MD and HT in heading stage. So MT had an advantage in the number of grains. MT also had higher crop growth rate of population and higher dry matter accumulation per stem and population than MD and HT after jointing. MT also had higher leaf area index in heading, and its grain leaf ratio was higher than MD, so MT contributed to more grains and spikelets, longer panicle length. F you 498 with large panicle had higher dry matter weight of panicle in heading than Yixiangyou 2115 with middle panicle, and it had higher dry matter weight ratio of panicle to stem than Yixiangyou 2115, these were the important reason for that F you 498 had higher number of grains. Denser grain of F you 498 compared to Yixiangyou 2115 resulted from its larger leaf area index and grain to leaf ratio.4) MT had the longest growth duration,while MD had the shortest. MT had a longer panicle differentiation stage, higher accumulated temperature and illumination hours, providing a good temperature and light condition for the formation of grain in MT. Length and dry weight of stem and length of panicle were greatly influenced by meteorological factors. Suitable temperature and a certain amount of accumulated temperature before booting was conducive to the elongation of stem and panicle and filling of stem. Given the differences in growth under different planting methods and of varieties, we should take measures to regulate growth of stem and panicle and the distribution of assimilates in an appropriate time. In order to achieve the goal of increasing grain yield, the dry weight ratio of panicle to stem should be increased on the basis of cultivating strong stalk.

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Morphological and Physiological Responses of Rice Seedlings to Water Temperature Under Complete Submergence Jing XIANG, Hui-zhe CHEN, Yu-ping ZHANG, Yi-kai ZHANG, De-feng ZHU 2016, 30(5): 525-531.  DOI: 10.16819/j.1001-7216.2016.5183 Abstract ( )   HTML ( )   PDF (618KB) ( )   To reveal the effects of floodwater temperature on morphology and physiology of rice seedlings, rice genotypes with different submergence tolerance were used: IR64Sub1 (tolerant), IR42 (sensitive), Gangyou 725 and Taiyou 398 (main cultivar). Seedlings were submerged twenty-five days after sowing at two water temperature for 0, 3, 6, 9 and 12 days. The results showed that the oxygen concentration in the 20℃ floodwater was higher than that in 30℃ floodwater, the oxygen concentration decreased drastically in 30℃ floodwater than 20℃ floodwater with in creasing submergence days. The shoot elongation percentage was higher when seedlings were submerged in 30℃ floodwater than 20℃ floodwater condition. The same response was found in the tolerant variety IR64Sub1, but the shoot elongation percentage was the lowest among four varieties. The difference in shoot elongation of IR42 between two floodwater temperature was biggest after 9-day and 12-day submergence. Non-structural carbohydrate concentrations and leaf SPAD value were lower under 30℃ floodwater temperature than 20℃temperature floodwater condition. This findings help to understand the cause of high survival of rice submerged in low temperature floodwater.

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Amylose Content in Good Eating Quality Rice Under Different Nitrogen Rates and Sowing Dates Shu YAO, Xin YU, Li-hui ZHOU, Tao CHEN, Qing-yong ZHAO, Zhen ZHU, Ya-dong ZHANG, Chun-fang ZHAO, Ling ZHAO, Cai-lin WANG 2016, 30(5): 532-540.  DOI: 10.16819/j.1001-7216.2016.6068 Abstract ( )   HTML ( )   PDF (555KB) ( )   The amylose content in good eating quality rice was investigated under four nitrogen application levels including high(450 kg/hm2), medium(300 kg/hm2), low(150 kg/hm2) and zero(CK) levels, different sowing dates and short daylight treatment by using the semi-glutinous new japonica rice varieties (strains) with different amylose content in 2013 and 2014. The results showed that the level of nitrogen fertilizer has a significant effect on amylose content of good eating quality japonica rice. The amylose content was decreased with the increasing level of nitrogen fertilizer. The amylose content under the high nitrogen level was the lowest in the two years. Sowing date has a significant influence on amylose content of good eating quality japonica rice. The amylose content was decreased with the sowing date postponed. The effect of sowing date on amylose content was related to the temperature during 6-15 days after heading, and high temperature resulted in the increase of amylose content. This was also confirmed under the short daylight treatment.

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Molecular Characterization of Ustilaginoidea virens T-DNA Insertion Mutant B2510 Hui DING, Mi-na YU, Ya-hui WANG, Jun-jie YU, Xiao-le YIN, Hui-wen BO, Xing HUANG, Yong-feng LIU 2016, 30(5): 541-551.  DOI: 10.16819/j.1001-7216.2016.5198 Abstract ( )   HTML ( )   PDF (1537KB) ( )   To isolate the gene concerned with molecular pathogenic process of Ustilaginoidea virens (U. virens) , T-DNA integration flanking sequence and the mutant genes of a mutant strain B2510 were analyzed. Compared with the wild-type U. virens strain P1, the pathogenicity of the mutant strain B2510 in the field was significantly decreased. The growth rate of B2510 on MM medium was slower than that of P1, but was not significantly different on PSA and TB3 medium which were nutritionally endowed. The mutant strain B2510 did not produce conidiophores in PS broth medium. Genomic Southern bolt analysis confirmed that there were double T-DNA events inserted in genome of mutant strain B2510. The flanking U. virens sequences of T-DNA obtained by TAIL-PCR were adjacent in the wild type and with no sequences lost, and only a few bases of T-DNA were changed. The T-DNA insertion site was in the promoter region of UV8b_1412 and downstream 3'region of UV8b_1386, respectively. RT-PCR analysis confirmed that the expression of both of two genes in B2510 were significantly decreased. The genes which were affected by T-DNA insertion may be associated with pathogenicity and participate in the regulation of pathogenic process of U. virens in rice.

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Virulence Characteristics of Nilaparvata lugens(Stål) Reared on Resistant Rice Variety IR56 Yu ZHENG, Jia-chun HE, Pin-jun WAN, Feng-xiang LAI, Yan-qun SUN, Jing-jing LIN, Qiang FU 2016, 30(5): 552-558.  DOI: 10.16819/j.1001-7216.2016.6016 Abstract ( )   HTML ( )   PDF (609KB) ( )   Rice varieties with the planthopper resistance gene Bph3were initially released and promoted in the Southeast Asia in the early 1980s. These varieties are still resistant to the brown planthopper [BPH, Nilaparvatalugens (Stål)], which has attracted much attention of rice breeders in China. A local BPH population that was forced to feed on IR56 with Bph3 gene for over 40 generations (IR56p) was used to study the virulence characteristics from perspectives of insects (development and reproduction) and rice variety reaction (SSST test). The results were as follows: 1) IR56p reared on resistant rice IR56 (IR56p-IR56) showed significantly increased emergence rate (ER), newly emerged adult body weight (BW), adult longevity (AL), number of eggs laid (NE), honeydew excretion (HE), and adult weight gain (WG), as well as markedly decreased nymphal duration (ND), compared to TN1 population (TN1p, a normal population reared on a susceptible rice variety TN1) on rice IR56 (TN1p-IR56). In contrast, IR56p-IR56 showed similar ER, NE and egg hatchability(EB), compared to TN1p or IR56p reared on rice TN1 (TN1p-TN1 or IR56p-TN1), despite significantly lower ND, AL, HE, WG and longer ND. The difference in BW and ND between IR56p-IR56 and TN1p-TN1 (or IR56p-TN1) was less than half of that between IR56p-IR56 and TN1p-IR56. 2) SSST test showed that the resistance of rice IR56 to IR56p was Grade 7, higher than Grade 3 of IR56 to TN1p. The resistance of TN1 to IR56p and TN1p was Grade 9 (total loss). It is concluded that the virulence of IR56p to IR56 is much higher than that of TN1p to IR56. Although the virulence of IR56p to IR56 was lower than that of IR56p or TN1p to TN1, most evaluations were similar among the three groups. In addition,the resistance of another Bph3-containing variety RathuHeenati to IR56p was Grade 1 (high resistance). Such an unexpected result implies that other BPH resistance genes contributes to the resistance of RathuHeenati to IR56p.

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Allelopathic Response to Different Temperature Conditions of Wild Rice (Oryza longistaminata) and Its Descendants Gao-feng XU, Shi-cai SHEN, Fu-dou ZHANG, Yu-hua ZHANG 2016, 30(5): 559-566.  DOI: 10.16819/j.1001-7216.2016.5158 Abstract ( )   HTML ( )   PDF (458KB) ( )   Rice allelopathy is considered as a complex phenomenon of chemical ecology, and temperature plays an important role in regulating allelopathic potential. Former studies have demonstrated that the wild rice (Oryza longistaminata) has high allelopathic potential.Therefore,understanding the allelopathic responses to temperature of wild rice and its descendants is very important to finally exploit and utilize allelopathic genes of wild germplasm resources.The root exudates of different allelopathic potential rice accessions(OL, RD23, F1, RL159, RL169 and RL219)were respectively collected by the resin of AG 50W-X8,followed by the evaluation of their allelopathic potential to barnyardgrass.The results demonstrated: 1) Allelopathic response to temperature,leaf ages and temperature×leaf age were significantly different among different allelopathic potential rice accessions. The allelopathy was significantly affected by temperature, leaf age and temperature×leaf age of OL, F1, RL159 and RL169, but low allelopathic rice accessions of RD23 and RL219 followed an opposite trend. 2) Regardless of leaf ages,the root exudates have weakest inhibitory ability to the growth of barnyardgrass at low temperature(15℃). The effects of allelopathic rice accessions OL, F1, RL159 and RL169 were greater than low allelopathic ones at low temperature(15℃) condition. 3) High temperature was conducive to the allelopathic enhancement of allelopathic rice, but the allelopathy was not always enhanced with increasing temperature. The allelopathy was strengthened with increasing temperature of allelopathic rice accessions OL, F1, RL159 and RL169 when they were at the 6-leaf stage and 8-leaf stage, but at the 2-leaf stage and 4-leaf stage, the allelopathy was enhanced first and then decreased with increasing temperature, and the allelopathy was strongest at 25℃. 4)Allelopathic responses to leaf ages were significantly different among different allelopathic potential rice. The allelopathy is the highest at the 2-leaf stage for allelopathic rice accessions OL, F1, RL159 and RL169 except for 15℃, then the allelopathy weakened with the increasing rice leaf ages.This finding suggests that allelopathic enhancement by temperature conditions at different leaf ages were worthwhile in future for the understanding of allelopathic responses to associated weeds.