OsEF3, a homologous gene of Arabidopsis ELF3, has pleiotropic effects in rice

Heading date is an important agronomic trait in rice. A rice mutant with a late heading date and no photoperiodic sensitivity in long or short day conditions was obtained from rice T-DNA insertion mutants in Zhonghua11 (ZH11). Through isolation and analysis of the flanking sequence of the T-NDA insertion site, the target sequence of insertion was obtained and found to locate in AP003296, the sequence accession number of rice chromosome 1 of RGP ( http://rgp.dna.affrc.go.jp ). The putative amino acid sequences of this target gene are homologous to the Arabidopsis protein ELF3 encoded by an early flowering gene. The rice target gene orthologous to Arabidopsis ELF3 is named OsEF3 ; this encodes a putative nematode responsive protein-like protein. OsEF3 has pleiotropic effects in rice that differ from the effects of Arabidopsis ELF3 , which only affects biological rhythms. OsEF3 regulates heading date by influencing the BVG stage and does not affect photoperiodic sensitivity, which suggests that the OsEF3 gene may be involved in an autonomous pathway in rice. OsEF3 may affect root development and kilo-grain weight by delaying cell division or cell elongation.     

水稻T-DNA插入突变体库的筛选及遗传分析

T-DNA标签技术是分离和研究植物功能基因的有效方法,寻找T-DNA插入表型突变体是进一步开展研究的关键所在。文章对以ZH11、ZH15为受体亲本构建的4416份T,代标签系进行了表型鉴定,发现存在拟纯合突变和系内分离突变两种类型,突变表型涉及株高、生育期、叶形、叶色、分蘖力、植株松紧度、穗颈节、穗形、颖花、粒形、类病变、雄性不育、生长极性等14类性状。其中,株高、生育期、叶色、雄性不育有着相对较高的突变频率（超过1％）,株高和叶色的突变频率在品种及年度间表现稳定,而生育期、雄性不育波动较大,表明这类性状的表型易受到环境的影响。通过T1、T2连续世代的共分离分析,筛选出3个与穗部或颖花发育相关的T-DNA插入突变体,为分离相关功能基因奠定基础。随机选择42份有表型突变的标签系,通过质粒拯救和TAIL-PCR的方法分离其侧翼序列,从39个标签系中获得40条序列,其中25条为载体序列,14条与水稻基因组有很好的同源性,BlastN分析结果表明T-DNA有优先整合进植物功能基因内部的特性。     

Screening for and Genetic Analysis on T-DNA-inserted Mutant Pool in Rice

T-DNA tagging technique has provided a powerful strategy for identifying new functional genes in plants, and the key for success is the discovery of T-DNA-inserted mutants with changed phenotype. In this study, we screened 4 416 rice T₁ tagged lines generated by enhancer trap system integrated with GLL4/VP16-UAS elements from two transformed parents, ZH11 and ZH15. We found many lines showed obvious morphological mutations, including two types—fake-homozygous mutation and separating mutation. The mutation phenotype was related to 14 kinds of trait such as plant height, heading date, leaf shape, leaf color, tiller number, panicle shape, spikelet number, grain shape, disease-like mutation, male sterility, awn, and so on. Among them, plant height, heading date, leaf color and male sterility had a comparatively high mutation frequency (over 1%). The mutation frequency of plant height and leaf color had no significant change between different years or transformed parents, but the frequency of heading date and male sterility varied greatly, suggesting that environment had a great effect on the expression of latter two traits. By conducting continuously co-segregating analyses in T₁ and T₂ generation, we identified 3 T-DNA-inserted mutants with malformed panicle or spikelets, which would provide a base for cloning correlative functional genes. At the same time, we selected randomly 42 lines with mutation phenotype and obtained 40 flanking sequences from 39 tagged lines by plasmid rescue or TAIL-PCR, of which, 26 were vector backbone sequence, 14 had good identity to rice genome sequence. The BlastN result showed the T-DNA preferentially integrated into protein-coding region in plants.     

普通野生稻中增产相关QTL的发掘

普通野生稻(Oryza Rufipogon)是重要的遗传资源,发掘其优良等位基因将对水稻遗传改良产生重要影响。文章从以珍汕97为轮回亲本,普通野生稻为供体的BC2F1群体中选择一个与珍汕97表型明显不同的单株BC2F1-15,经过连续自交获得回交重组自交系BC2F5群体。均匀分布于12条染色体的126个多态性SSR(Simplesequence repeats)标记基因型分析,发现BC2F1-15单株在30%的标记位点为杂合基因型;利用该群体共检测到4个抽穗期、3个株高、4个每穗颖花数、2个千粒重和1个单株产量QTL。在第7染色体RM481-RM2区间,检测到抽穗期、每穗颖花数和产量QTL,野生稻等位基因表现增效作用;其他3个每穗颖花数QTL位点,野生稻等位基因也均具有增效作用。结果表明野生稻携带有增产相关的等位基因,这些有利等位基因无疑是水稻遗传改良可资利用的新资源。     

Global analysis of CCT family knockout mutants identifies four genes involved in regulating heading date in rice

Many genes encoding CCT domain‐containing proteins regulate flowering time. In rice (Oryza sativa), 41 such genes have been identified, but only a few have been shown to regulate heading date. Here, to test whether and how additional CCT family genes regulate heading date in rice, we classified these genes into five groups based on their diurnal expression patterns. The expression patterns of genes in the same subfamily or in close phylogenetic clades tended to be similar. We generated knockout mutants of the entire gene family via CRISPR/Cas9. The heading dates of knockout mutants of only 4 of 14 genes previously shown to regulate heading date were altered, pointing to functional redundancy of CCT family genes in regulating this trait. Analysis of mutants of four other genes showed that OsCCT22, OsCCT38, and OsCCT41 suppress heading under long‐day conditions and promote heading under short‐day conditions. OsCCT03 promotes heading under both conditions and upregulates the expression of Hd1 and Ehd1, a phenomenon not previously reported for other such genes. To date, at least 18 CCT domain‐containing genes involved in regulating heading have been identified, providing diverse, flexible gene combinations for generating rice varieties with a given heading date.     

Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice

In a screen for MADS box genes which activate and/or repress flowering in rice, we identified a gene encoding a MADS domain protein (OsSOC1) related to the Arabidopsis gene AtSOC1. AtSOC1 and OsSOC1 show a 97% amino acid similarity in their MADS domain. The rice gene contains a large first intron of 27.6 kb compared to the 1 kb intron in Arabidopsis. OsSOC1 is located on top of the short arm of chromosome 3, tightly linked to the heading date locus, Hd9. OsSOC1 is expressed in vegetative tissues, and expression is elevated at the time of floral initiation, 40-50 days after sowing, and remains uniformly high thereafter, similar to the expression pattern of AtSOC1. The constitutive expression of OsSOC1 in Arabidopsis results in early flowering, suggesting that the rice gene is a functional equivalent of AtSOC1. We were not able to identify FLC-like sequences in the rice genome; however, we show that ectopic expression of the Arabidopsis FLC delays flowering in rice, and the up-regulation of OsSOC1 at the onset of flowering initiation is delayed in the AtFLC transgenic lines. The reciprocal recognition and flowering time effects of genes introduced into either Arabidopsis or rice suggest that some components of the flowering pathways may be shared. This points to a potential application in the manipulation of flowering time in cereals using well characterized Arabidopsis genes.     

Roles of the Hd5 gene controlling heading date for adaptation to the northern limits of rice cultivation

During the diversification of cultivated rice after domestication, rice was grown in diverse geographic regions using genetic variations attributed to the combination of alleles in loci for adaptability to various environmental conditions. To elucidate the key gene for adaptation in rice cultivars to the northern limit of rice cultivation, we conducted genetic analyses of heading date using extremely early-heading cultivars. The Hd5 gene controlling heading date (flowering time) generated variations in heading date among cultivars adapted to Hokkaido, where is the northernmost region of Japan and one of the northern limits of rice cultivation in the world. The association of the Hd5 genotype with heading date and genetical analysis clearly showed that the loss-of-function Hd5 has an important role in exhibiting earlier heading among a local population in Hokkaido. Distinct distribution of the loss-of-function Hd5 revealed that this mutation event of the 19-bp deletion occurred in a local landrace Bouzu and that this mutation may have been selected as an early-heading variety in rice breeding programs in Hokkaido in the early 1900s. The loss-of-function Hd5 was then introduced into the rice variety Fanny from France and contributed to its extremely early heading under the presence of functional Ghd7. These results demonstrated that Hd5 plays roles not only in generating early heading in variations of heading date among a local population in Hokkaido, but also in extremely early heading for adaptation to northern limits of rice cultivation.     

Fine mapping of qHd1, a minor heading date QTL with pleiotropism for yield traits in rice (Oryza sativa L.)

Key message

A minor QTL for heading date located on the long arm of rice chromosome 1 was delimitated to a 95.0-kb region using near isogenic lines with sequential segregating regions.

Abstract

Heading date and grain yield are two key factors determining the commercial potential of a rice variety. In this study, rice populations with sequential segregating regions were developed and used for mapping a minor QTL for heading date, qHd1. A total of 18 populations in six advanced generations through BC₂F₆ to BC₂F₁₁ were derived from a single BC₂F₃ plant of the indica rice cross Zhenshan 97 (ZS97)///ZS97//ZS97/Milyang 46. The QTL was delimitated to a 95.0-kb region flanked by RM12102 and RM12108 in the terminal region of the long arm of chromosome 1. Results also showed that qHd1 was not involved in the photoperiodic response, having an additive effect ranging from 2.4 d to 2.9 d observed in near isogenic lines grown in the paddy field and under the controlled conditions of either short day or long day. The QTL had pleiotropic effects on yield traits, with the ZS97 allele delaying heading and increasing the number of spikelets per panicle, the number of grains per panicle and grain yield per plant. The candidate region contains ten annotated genes including two genes with functional information related to the control of heading date. These results lay a foundation for the cloning of qHd1. In addition, this kind of minor QTLs could be of great significance in rice breeding for allowing minor adjustment of heading date and yield traits.     

Understanding the genetic and epigenetic architecture in complex network of rice flowering pathways

Although the molecular basis of flowering time control is well dissected in the long day (LD) plant Arabidopsis, it is still largely unknown in the short day (SD) plant rice. Rice flowering time (heading date) is an important agronomic trait for season adaption and grain yield, which is affected by both genetic and environmental factors. During the last decade, as the nature of florigen was identified, notable progress has been made on exploration how florigen gene expression is genetically controlled. In Arabidopsis expression of certain key flowering integrators such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT) are also epigenetically regulated by various chromatin modifications, however, very little is known in rice on this aspect until very recently. This review summarized the advances of both genetic networks and chromatin modifications in rice flowering time control, attempting to give a complete view of the genetic and epigenetic architecture in complex network of rice flowering pathways.     

水稻基因组加倍对籽粒大小调控基因表达的影响

水稻是最重要的粮食作物之一,提高水稻产量一直是育种的主要目标。水稻四倍体相对于二倍体具有籽粒变大、粒重增加的特点,研究基因组加倍后籽粒大小基因的调控模式,在育种应用方面具有十分重要的意义。本文以二倍体 -四倍体水稻为材料,分析6个控制籽粒大小基因在幼穗发育中的表达差异,同时结合转基因实验,探讨基因剂量增加对基因表达水平和籽粒大小的影响。结果发现：基因组加倍后,水稻的发育进程不变,但株高增加,叶片变宽,籽粒变大,增大后的籽粒在籼稻表现为长、宽均增加显著,而在粳稻中长度比宽度增加更为明显。进一步分析控制籽粒大小基因的表达差异情况,发现这些基因的表达不仅受发育时期的影响,在籼粳亚种间也明显不同,即受遗传背景的影响。在基因组加倍的情况下,正调控基因GS5、HGW的表达普遍高于对应的二倍体;负调控基因GS3在籼稻D9311中趋于下调或沉默,而在粳稻DBl中趋于上调,GW2在D9311中上调,而在DBl中趋于沉默。通过转基因实验分析负调控基因GW2在二倍体Bl中的表达趋势,发现其在基因剂量线性增加的情况下,表达水平高于二倍体和四倍体,导致其籽粒变小。本研究结果有助于了解水稻中控制籽粒大小的基因在二倍体和四倍体中的表达模式,为高产育种提供理论依据。     

Identifying genetic components controlling fertility in the outcrossing grass species perennial ryegrass (Lolium perenne) by quantitative trait loci analysis and comparative genetics

Mutational load and resource allocation factors and their effects on limiting seed set were investigated in ryegrass by comparative mapping genomics and quantitative trait loci (QTL) analysis in two perennial ryegrass (Lolium perenne) mapping families sharing common genetic markers. Quantitative trait loci for seed-set were identified on chromosome (LG) 7 in both families and on LG4 of the F2/WSC family. On LG7, seed-set and heading date QTLs colocalized in both families and cannot be unequivocally resolved. Comparative genomics suggests that the LG7 region is syntenous to a region of rice LG6 which contains both fertility (S5(n)) and heading date (Hd1, Hd3a) candidate genes. The LG4 region is syntenous to a region of rice LG3 which contains a fertility (S33) candidate gene. QTL maxima for seed-set and heading date on LG4 in the F2/WSC family are separated by c. 8 cm, indicating distinct genetic control. Low seed set is under the control of recessive genes at both LG4 and LG7 locations. The identification of QTLs associated with seed set, a major component of seed yield in perennial ryegrass, indicates that mutational load associated with these genomic regions can be mitigated through marker-assisted selection.     

Transformation of rice with the Arabidopsis floral regulator LEAFY causes early heading

Onset of flowering, or heading date, is an important agronomic trait of cereal crops such as rice and early-heading varieties are required for certain regions in which rice is cultivated. Since the floral control gene LEAFY from Arabidopsis can dramatically accelerate flowering in dictoyledonous plants, the usefulness of LEAFY for manipulating heading date in rice has been tested. Constitutive expression of LEAFY from the cauliflower mosaic virus 35S promoter caused early flowering in transgenic rice, with a heading date that was 26–34 days earlier than that of wild-type plants. Early flowering was accompanied by a small yield penalty and some panicle abnormality. These observations suggest that floral regulatory genes from Arabidopsis are useful tools for heading date improvement in cereal crops.     

QTLs for heading date and plant height under multiple environments in rice

Both heading date and plant height are important traits related to grain yield in rice. In this study, a recombinant inbred lines (RILs) population was used to map quantitative trait loci (QTLs) for both traits under 3 long-day (LD) environments and 1 short-day (SD) environment. A total of eight QTLs for heading date and three QTLs for plant height were detected by composite interval mapping under LD conditions. Additional one QTL for heading date and three QTLs for plant height were identified by Two-QTL model under LD conditions. Among them, major QTLs qHd7.1, qHd7.2 and qHd8 for heading date, and qPh1 and qPh7.1 for plant height were commonly detected. qHd7.1 and qHd7.2 were mapped to small regions of less than 1 cM. Genome position comparison of previously cloned genes with QTLs detected in this study revealed that qHd5 and qPh3.1 were two novel QTLs. The alleles of these QTLs increasing trait values were dispersed in both parents, which well explained the transgressive segregation observed in this population. In addition, the interaction between qHd7.1 and qHd8 was detected under all LD conditions. Multiple-QTL model analysis revealed that all QTLs and their interactions explained over 80% of heading date variation and 50% of plant height variation. Two heading date QTLs were detected under SD condition. Of them, qHd10 were commonly identified under LD condition. The difference in QTL detection between LD and SD conditions indicated most heading date QTLs are sensitive to photoperiod. These findings will benefit breeding design for heading date and plant height in rice.     

Comparative genetics of flowering time

Analysis of genes controlling flowering time (heading date) contributes to our understanding of fundamental principles of plant development and is of practical importance because of the effects of flowering time on plant adaptation and crop yield. This review discusses the extent to which plants may share common genetic mechanisms for the control of flowering time and the implications of such conservation for gene isolation from the major cereal crops. Gene isolation may exploit the small genome of rice in map-based approaches, utilizing the conservation of gene order that is revealed when common DNA markers are mapped in different species. Alternatively, mechanisms may be conserved within plants as a whole, in which case genes cloned from the model dicot Arabidopsis thaliana provide an alternative route.     

Mapping of QTLs conferring extremely early heading in rice (Oryza sativa L.)

Two genes related to extremely early heading were identified in populations derived from crosses between Hoshinoyume, a variety adapted to the northernmost limit of rice cultivation (Hokkaido), and Nipponbare, a variety adapted to the temperate region of Japan. The segregations for heading date clearly revealed that a two-gene model determined the extremely early heading in the F₂ and BC₁F₁ populations under natural field conditions in Hokkaido. Using molecular markers corresponding to ten known quantitative trait loci (QTLs) for heading date, we carried out QTL analysis in the BC₁F₁ population and detected two QTLs, qDTH-7-1 and qDTH-7-2, both on chromosome 7, and observed epistatic interaction between them. We conclude that the recessive alleles of these two genes contribute to extremely early heading for the adaptation to Hokkaido environment and to stable rice production in Hokkaido. The relationships between the two QTLs identified in this study and known QTLs are discussed.     

Cytological analysis and genetic control of rice anther development

Microsporogenesis and male gametogenesis are essential for the alternating life cycle of flowering plants between diploid sporophyte and haploid gametophyte generations.Rice (Oryza sativa) is the world's major staple food,and manipulation of pollen fertility is particularly important for the demands to increase rice grain yield.Towards a better understanding of the mechanisms controlling rice male reproductive development,we describe here the cytological changes of anther development through 14 stages,including cell division,differentiation and degeneration of somatic tissues consisting of four concentric cell layers surrounding and supporting reproductive cells as they form mature pollen grains through meiosis and mitosis.Furthermore,we compare the morphological difference of anthers and pollen grains in both monocot rice and eudicot Arabidopsis thaliana.Additionally,we describe the key genes identified to date critical for rice anther development and pollen formation.