Mobilization of the active MITE transposons mPing and Pong in rice by introgression from wild rice (Zizania latifolia Griseb.)

Hybridization between different species plays an important role in plant genome evolution, as well as is a widely used approach for crop improvement. McClintock has predicted that plant wide hybridization constitutes a "genomic shock" whereby cryptic transposable elements may be activated. However, direct experimental evidence showing a causal relationship between plant wide hybridization and transposon mobilization has not yet been reported. The miniature-Ping (mPing) is a recently isolated active miniature inverted-repeat transposable element transposon from rice, which is mobilized by tissue culture and gamma-ray irradiation. We show herein that mPing, together with its putative transposase-encoding partner, Pong, is mobilized in three homologous recombinant inbred lines (RILs), derived from hybridization between rice (cultivar Matsumae) and wild rice (Zizania latifolia Griseb.), harboring introgressed genomic DNA from wild rice. In contrast, both elements remain immobile in two lines sharing the same parentage to the RILs but possessing no introgressed DNA. Thus, we have presented direct evidence that is consistent with McClintock's insight by demonstrating a causal link between wide hybridization and transposon mobilization in rice. In addition, we report an atypical behavior of mPing/Pong mobilization in these lines, i.e., the exclusive absence of footprints after excision.     

Extensive Microsatellite Variation in Rice Induced by Introgression from Wild Rice (Zizania latifolia Griseb.)

Background

It is widely accepted that interspecific hybridization may induce genomic instability in the resultant hybrids. However, few studies have been performed on the genomic analysis of homoploid hybrids and introgression lines. We have reported previously that by introgressive hybridization, a set of introgression lines between rice (Oryza sativa L.) and wild rice (Zizania latifolia Griseb.) was successfully generated, and which have led to the release of several cultivars.

Methodology

Using 96 microsatellite markers located in the nuclear and organelle genomes of rice, we investigated microsatellite stability in three typical introgression lines. Expression of a set of mismatch repair (MMR) genes and microsatellite-containing genes was also analyzed.

Results/Conclusions

Compared with the recipient rice cultivar (Matsumae), 55 of the 96 microsatellite loci revealed variation in one or more of the introgression lines, and 58.2% of the altered alleles were shared by at least two lines, indicating that most of the alterations had occurred in the early stages of introgression before their further differentiation. 73.9% of the non-shared variations were detected only in one introgression line, i.e. RZ2. Sequence alignment showed that the variations included substitutions and indels that occurred both within the repeat tracts and in the flanking regions. Interestingly, expression of a set of MMR genes altered dramatically in the introgression lines relative to their rice parent, suggesting participation of the MMR system in the generation of microsatellite variants. Some of the altered microsatellite loci are concordant with changed expression of the genes harboring them, suggesting their possible cis-regulatory roles in controlling gene expression. Because these genes bear meaningful homology to known-functional proteins, we conclude that the introgression-induced extensive variation of microsatellites may have contributed to the novel phenotypes in the introgression lines.     

中国菰资源研究现状及应用前景

中国菰(Zizania latifolia(Griseb.)Turcz.ex Stapf)是一种多年生禾本科稻亚科稻族菰属挺水植物。研究表明,菰具有现代水稻栽培品种所需要的许多优良性状,在生态治理方面表现出应用潜力;菰米营养价值高于普通稻米,且兼具保健功效。菰的这些特性使其在水稻种质改良、重金属离子污染土壤治理以及功能性食品开发方面表现出诱人的前景。近年来,菰属植物的研究工作取得了一定的进展,主要涉及菰的起源进化、菰的经济价值、菰与生态环境的关系、菰遗传多样性分析、菰组学及育种应用等五方面。因文献报道的时间跨度大,涉及面广,目前未见详尽的有关该物种研究进展的专门论述。本文从上述5个方面进行总结,促进对菰的了解,为这种重要野生资源的研究、保护和开发利用提供借鉴,进而加速该物种的研究进程。     

Extent and pattern of DNA methylation alteration in rice lines derived from introgressive hybridization of rice and Zizania latifolia Griseb

We have reported previously that introgression by Zizania latifolia resulted in extensive DNA methylation changes in the recipient rice genome, as detected by a set of pre-selected DNA segments. In this study, using the methylation-sensitive amplified polymorphism (MSAP) method, we globally assessed the extent and pattern of cytosine methylation alterations in three typical introgression lines relative to their rice parent at ∼2,700 unbiased genomic loci each representing a recognition site cleaved by one or both of the isoschizomers, HpaII/MspI. Based on differential digestion by the isoschizomers, it is estimated that 15.9% of CCGG sites are either fully methylated at the internal Cs and/or hemi-methylated at the external Cs in the rice parental cultivar Matsumae. In comparison, a statistically significant increase in the overall level of both methylation types was detected in all three studied introgression lines (19.2, 18.6, 19.6%, respectively). Based on comparisons of MSAP profiles between the isoschizomers within the rice parent and between parent and the introgression lines, four major groups of MSAP banding patterns are recognized, which can be further divided into various subgroups as a result of inheritance of, or variation in, parental methylation patterns. The altered methylation patterns include hyper- and hypomethylation changes, as well as inter-conversion of hemi- to full-methylation, or vice versa, at the relevant CCGG site(s). Most alterations revealed by MSAP in low-copy loci can be validated by DNA gel blot analysis. The changed methylation patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. Sequencing on 31 isolated fragments that showed different changing patterns in the introgression line(s) allowed their mapping onto variable regions on one or more of the 12 rice chromosomes. These segments include protein-coding genes, transposon/retrotransposons and sequences with no homology. Possible causes for the introgression-induced methylation changes and their implications for genome evolution and crop breeding are discussed.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Z. Y. Dong and Y. M. Wang have equally contributed to the work.     

菰核基因组SSR引物的开发及其在稻族植物中的通用性检测

利用数据库中已有的部分菰(Zizania latifolia Turcz.)核基因组序列,采用in silico方法开发其SSR引物,并选取我国不同纬度的5个菰野生种群,对合成的64对引物进行筛选。结果显示:64对引物中有15对至少在一个种群中表现出多态性;共发现84个等位基因,每个位点平均有5.6个等位基因。在5个种群中,观察杂合度为0.000~0.941,预期杂合度为0.072~0.625。种群间的基因流(Nm=0.576)水平较低导致了种群间表现出较高的遗传分化(FST=0.432)。进一步对稻族其他物种的通用性检测发现,15个多态位点中,有8个位点在亚洲栽培稻(Oryza sativa L.)中得到扩增,有9个位点在普通野生稻(O.rufipogon Griff.)中得到扩增。     

Morphological changes and resource allocation of Zizania latifolia (Griseb.) Stapf in response to different submergence depth and duration

Plants around ponds, rivers and lakes are subjected to long-term partial or complete submergence. When they are flooded, water level affects the plants simultaneously with duration of submergence. Separate and interactive effects of water level and duration on the growth of the herbaceous perennial Zizania latifolia (Poaceae) were investigated by exposing the plants in greenhouse water tanks to submergence in different water depths and for different time-spans. The plants exhibited great shoot elongation upon submergence and prolonged flood duration, and the basal tiller number of the species decreased with higher water levels. Submergence treatment advanced the flowering date and increased the inflorescence number. Plant total biomass did not differ among all the treatments, while the root:shoot ratio decreased with increased water level, prolonged duration of submergence and their interaction. The high plasticity in morphology and shifts in reproductive strategy and biomass allocation enabled the Zizania plants to survive the compound effect of flooding height and duration. This may explain the occurrence of this species in habitats subjected to long-term flooding. The results obtained in this experiment will contribute to understanding the impact of flooding dynamics on plants and the ways of adaptation responses to prolonged waterlogging.     

Abundant genetic diversity of the wild rice Zizania latifolia in central China revealed by microsatellites

Wild rice Zizania latifolia is a perennial emergent aquatic plant widely distributed across China. Wild populations of Z. latifolia are important to aquatic ecosystems and are valuable genetic resources for breeding. However, they have been faced with significant habitat losses in recent decades. For 10 nuclear microsatellites, high levels of genetic diversity (H_E = 0.572–0.636) were found across seven surveyed populations from central China. The main factors responsible for that were its long life history and predominant outcrossing reproductive system. Low genetic differentiation among populations was found based on Wright's F_ST = 0.098. Similarly, AMOVA analysis showed only 7.73% of the total molecular variation was attributed to inter‐population differentiation. The weak population structure of Z. latifolia could be due to high gene flow mediated by water or birds (Nm = 2.30, M = 2.18). Importantly, most populations exhibited mutation‐drift disequilibrium, suggesting a recent population decline. Based on the results, wild populations of Z. latifolia are expected to lose genetic diversity and increase genetic structure in future generations. Therefore, conservation management is urgently needed to maintain the genetic resources of Z. latifolia.     

Production of a highly asymmetric somatic hybrid between rice and Zizania latifolia (Griseb): evidence for inter-genomic exchange

 A highly asymmetric and fertile somatic hybrid plant was obtained via protoplast fusion in an intergenric combination. Gamma-ray-irradiated Zizania latifolia (Griseb). Turcz. ex Stapf mesophyll protoplasts were electrofused with idoacetamide-inactivated rice protoplasts derived from a 2-month-old suspension cell culture. Two of the six putative hybrid calli regenerated plants. Cytological observation showed that the somatic chromosome numbers of both plants were the same as the rice parent (2n=24). Nevertheless, the hybrid nature and inter-genomic exchange events of one of the plants, i.e. SH6 (SH for somatic hybrid), were confirmed by Southern analysis using both total genomic DNA and moderate-copy, Z. latifolia-abundant DNA sequences as probes; in both cases, parental specific and/or new intergenomic recombinant hybridization fragments were detected. In both plant and seed morphology, the hybrid (SH6) was distinct from its rice parental cultivar, as well as from the wild donor species, Z. latifolia. Received: 15 August 1998 / Accepted: 30 September 1998     

Isolation and characterization of a set of disease resistance-gene analogs (RGAs) from wild rice, Zizania latifolia Griseb. I. Introgression, copy number lability, sequence change, and DNA methylation alteration in several rice-Zizania introgression lines.

Eight resistance-gene analogs (RGAs) were isolated from wild rice, Zizania latifolia Griseb., by degenerate primers designed according to conserved motifs at or around the nucleotide-binding site (NBS) of known NBS-containing plant resistance genes. The 8 RGAs were classified into 6 distinct groups based on their deduced amino acid sequence similarity of 60% or greater. Gel-blot hybridization of each of the RGAs to 4 rice - Z. latifolia intro gression lines indicated an array of changes at either introgressed Zizania RGAs or, more likely, their rice homologs. The changes included dramatic increase in copy number, modification at the primary DNA sequence, and alteration in DNA methylation patterns.     

基于SSR和ISSR的鄱阳湖流域野生菰资源的遗传多样性分析

用SSR和ISSR标记对鄱阳湖流域30个野生菰居群的遗传多样性与遗传结构进行了分析。筛选出的19对SSR引物共扩增出多态性条带253条,平均多态性条带比率(PPB)为91.67%,Nei's基因多样性指数(He)和平均Shannon信息指数(I)分别为0.2712和0.4144,遗传相似性系数(GS)为0.5590~0.8368,遗传距离(GD)的变化范围为0.1632~0.4410;筛选出的14个ISSR标记引物共扩增出83条条带,平均多态性条带比率(PPB)为78.29%,Nei's基因多样性指数(He)和平均Shannon信息指数(I)分别为0.2386和0.4174,遗传相似性系数(GS)为0.5132~0.9342,遗传距离(GD)变化范围为0.0658~0.4868。根据SSR和ISSR基因型数据,采用UPGMA法分别在阈值为0.698和0.728时可将30个野生菰居群聚为3类。可能受人为、水流、动物活动、风等多种因素的影响,居群间的亲缘关系与地理分布无明显相关性。本研究表明,鄱阳湖流域野生菰居群间SSR和ISSR基因型的多样性丰富,居群间的这种遗传差异或变异,对该地区乃至更大范围内野生菰的遗传进化、基因资源的开发利用和种质资源的保护有着重要意义。     

Responses of early recruitment processes with rhizome to flooding depth and salinity in Manchurian wild rice (Zizania latifolia)

Aquatic Ecology - Environmental stress, such as intense rainfall, drought and salinization, is predicted to increase not only in intensity, but also in frequency under future climate scenarios....     

Decomposition of aboveground and belowground organs of wild rice (Zizania latifolia): mass loss and nutrient changes

Decomposition of aboveground and belowground organs of the emergent macrophyte Z.latifolia was investigated using a litterbag technique for a period of 359 days in a freshwater marsh in Japan. Aboveground parts were classified into: leaves, sheaths and stems. Belowground parts were classified into: horizontal rhizomes (new rhizome, hard rhizome, soft rhizome) and vertical rhizome (stembase). The decay rate (k) was 0.0036 day⁻¹, 0.0033 day⁻¹ and 0.0021 day⁻¹ for leaves, sheaths and stems, respectively. For belowground parts, the decay rate varied considerably from 0.0018 day⁻¹ to 0.0079 day⁻¹, according to differences in the initial chemical compositions of rhizomes. After 359 days of decay, new rhizomes lost 94% of their original dry mass, compared with a loss of 48–84% for the other rhizomes. There was a significant positive relationship between litter quality and decay rate for horizontal rhizomes. For the new rhizomes, which had an internal nitrogen content of 24.2 mg N g⁻¹ dry mass, the mass loss was 40% higher than that of soft rhizomes, which had an internal N content of 9.8 mg N g⁻¹ dry mass. Over the period of 359 days, the nitrogen concentration in all rhizome types decreased to levels lower than initial values, but the phosphorus concentration remained constant after an initial leaching loss. Most nitrogen and phosphorus were lost during the first 45 days of decay. Changes in carbon to nitrogen (C:N) and carbon to phosphorus (C:P) ratios basically followed inversed trends of the nitrogen and phosphorus concentrations.     

Hypoxic metabolism in wild rice (Zizania palustris): enzyme induction and metabolite production

Alcohol dehydrogenase (ADH, EC 1. 1. 1. 1), lactate dehydrogenase (LDH, EC 1. 1. 1. 27) and alanine aminotransferase (AlaAT, EC 2. 6. 1. 2) activity in wild rice ( Zizania palustris L.) root tissue increased after 4 days of exposure to hypoxic stress. The activities of ADH and AlaAT also increased in leaf tissue under these same conditions, whereas LDH activity did not. Isozyme banding patterns indicate that wild rice has at least two functional Adh genes, only one of which is hypoxically induced in root and leaf tissue. The isozyme profile of LDH also indicates the presence of two functional Ldh genes in wild rice. Two bands of AlaAT activity are visible on native electrophoretic gels of root and leaf tissue. Neither of these bands appears to increase in activity in hypoxic samples, even though spectrophotometric assays indicate an increase in AlaAT activity. Ethanol accumulation was the highest of all the metabolites measured. Alanine and malate also accumulated under hypoxic conditions but only to about one-fifth the level of ethanol. Succinate, aspartate and lactate showed no observable changes throughout the induction period. These results show that wild rice differs from domesticated rice ( Oryza sativa L.) in its metabolic responses to anaerobic stress. The possible role of these responses in conferring flood tolerance is discussed.     

The effect of sediment chemistry on the successful establishment of wild rice (Zizania palustris L.) in northern Saskatchewan water bodies

Sediment chemistry of productive and non-productive wild rice sites was measured over 3 consecutive growing seasons Although productive sites differed from non-productive sites in terms of pH, N and potential N mineralization, redox potential was considered to be the major factor which distinguished these site classes. Productivity of wild rice in commercial stands could not be consistently correlated to sediment nutrients. However, growth appeared to be restricted in strongly reduced sediments and sediment Eh is considered to be a rapid, accurate method of estimating site potential. Good growth is expected if Eh is above –150 mB while poor growth is expected when Eh drops below –200 mV. Eh values of sediment samples changed as storage time increased. It is therefore recommended that Eh is recorded on-site.     

Construction of introgression lines carrying wild rice (Oryza rufipogon Griff.) segments in cultivated rice (Oryza sativa L.) background and characterization of introgressed segments associated with yield-related traits

Introgression lines (ILs) are useful tools for precise mapping of quantitative trait loci (QTLs) and the evaluation of gene action or interaction in theoretical studies. A set of 159 ILs carrying variant introgressed segments from Chinese common wild rice (Oryza rufipogon Griff.), collected from Dongxiang county, Jiangxi Province, in the background of Indica cultivar (Oryza sativa L.), Guichao 2, was developed using 126 polymorphic simple sequence repeats (SSR) loci. The 159 ILs represented 67.5% of the genome of O. rufipogon. All the ILs have the proportions of the recurrent parent ranging from 92.4 to 99.9%, with an average of 97.4%. The average proportion of the donor genome for the BC₄F₄ population was about 2.2%. The mean numbers of homozygous and heterozygous donor segments were 2 (ranging 0–8) and 1 (ranging 0–7), respectively, and the majority of these segments had sizes less than 10 cM. QTL analysis was conducted based on evaluation of yield-related traits of the 159 ILs at two sites, in Beijing and Hainan. For 6 out of 17 QTLs identified at two sites corresponding to three traits (panicles per plant, grains per panicle and filled grains per plant, respectively), the QTLs derived from O. rufipogon were usually associated with an improvement of the target trait, although the overall phenotypic characters of O. rufipogon were inferior to that of the recurrent parent. Of the 17 QTLs, 5 specific QTLs strongly associated with more than one trait were observed. Further analysis of the high-yielding and low-yielding ILs revealed that the high-yielding ILs contained relatively less introgressed segments than the low-yielding ILs, and that the yield increase or decrease was mainly due to the number of grain. On the other hand, low-yielding ILs contained more negative QTLs or disharmonious interactions between QTLs which masked trait-enchancing QTLs. These ILs will be useful in identifying the traits of yield, tolerance to low temperature and drought stress, and detecting favorable genes of common wild rice.     

Plant regeneration from protoplasts of wild rice (Oryza rufipogon Griff.)

Embryogenic callus initiated from basal segments of micropropagated shoots of Oryza rufipogon were used to initiate cell suspension cultures. After approximately 3 months these cultures were capable of yielding large numbers of protoplasts which underwent sustained division in agarose-solidified medium at a frequency comparable to that observed with Japonica rice protoplasts in previous studies. O. rufipogon plants were reproducibly regenerated from the protoplast-derived callus and are currently being grown to maturity. This is the first report of plant regeneration from protoplasts of a wild species of Oryza.Abbreviations BAP 6-benzyalamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2[N-morpholino] ethanesulphonic acid - NAA -naphthalene acetic acid - PAR photosynthetically active radiation - SCV settled-cell volume     

Factors influencing the production of sclerotia in the wild rice (Zizania aquatica) pathogenSclerotium hydrophilum

Sclerotium hydrophilum was shown to be auxoheterotrophic for thiamine, with the addition of this vitamin being required for the induction of sclerotia on defined media, but riboflavin and pyridoxine also have a positive effect. In the absence of thiamine, an increase in glucose concentration lead to a decrease in the yield of sclerotia; however, the addition of thiamine negate this inhibition and, instead, as the glucose concentration increased a higher proportion of sclerotial initials matured. Overall it was found that thiamine, specifically the pyrimidine component of thiamine, is crucial for initiating sclerotium production, while glucose stimulates maturation. The effect of light on sclerotium production was found to be complex and dependent on the growth medium. Light is not required for either the induction or maturation of sclerotia, but continuous irradiation of developing cultures with either white light or black light induces an endogenous rhythm whereby sclerotia are formed every 48h. When exposed to alternating light/dark regimes mycelium that formed in the light does not mature sclerotia, but dark-formed mycelium does, even if it is subsequently exposed to light.