Assessment of Gene Flow Through Detection of SexualCompatibility Between Transgenic Rice with barand Echinochloa crusgalli var.mitis

The possibility of gene flow between two varieties of transgenic rice with bar gene (Y0003 and 99-t) (male) and barnyard grass(Echinochloa crusgalli var. mitis ) (female) was studied by means of reproductive biology. The germination and growth of rice pollen grains on barnyard grass stigmas at 30 min, and 1-4 h after crossing by hand were observed with an optical microscope. The results were compared with the germination and growth of barnyard grass pollen grains at the corresponding time after self-pollination. The results showed that germination and growth of the pollen grains of the two varieties were similar on barnyard grass stigmas, but differed significantly from self-pollination of barnyard grass. Pollen grains germinated and pollen tubes penetrated stigmas normally, and the number of pollen grains being condensing or releasing their inclusions or having released them increased with the time after self-pollination. Pollen grains of transgenic rice on the stigmas of barnyard grass couldn't germinate or grow normally after crossing, neither could they penetrate the stigmas of barnyard grass. Therefore, it could be concluded that the sexual incompatibility between transgenic rice with bar gene and barnyard grass is due to the rice pollen being unable to penetrate the stigma of barnyard grass. Further proof of incompatibility lies in the fact that the emasculated barnyard grass pollinated with the rice pollen grains could not seed.

Verification of Glyphosate Resistance, Lepidopteran Resistance and Wide Compatibility of Male Sterile Line E1C4008S in Rice

Weeds and insect pests are two important biotic stresses resulting in yield loss in rice, and wide compatibility is the essential characteristic of breeding inter-subspecific hybrid rice. This study focused on glyphosate resistance, lepidopteran resistance and wide compatibility as well as identification of molecular and some agronomic characteristics of transgenic male sterile line E1 C4008 S. The results indicated that glyphosate resistance gene Epsps^# and lepidopteran resistance gene Cry1 ca^# were transferred into japonica wide compatibility male sterile line 4008 S by Agrobacterium-mediated method, and four independent transformation events named E1 C4008 S-1, E1 C4008 S-2, E1 C4008 S-3 and E1 C4008 S-4 were obtained, in which E1 C4008 S-3 and E1 C4008 S-4 were of single copy insertion. The EPSPS protein contents of E1 C4008 S-3 and E1 C4008 S-4 in different organs were significantly different both in descending order of leaf > stem > root, and ranged from 300.58 to 1410.69 μg/g at the tillering stage. The glyphosate tolerable concentration(dosage) of E1 C4008 S-3 and E1 C4008 S-4 reached at least 16 g/L(54.42 kg/hm^2), and the seeds of E1 C4008 S-4 can germinate normally on the medium containing 1 g/L glyphosate. The CRY1 C protein contents of E1 C4008 S-3 and E1 C4008 S-4 in different organs were significantly different both in descending order of leaf > stem > root, and ranged from 0.62 to 2.43 μg/g at the tillering stage. The larvae mortalities of rice leaf rollers fed on leaves of E1 C4008 S-3 and E1 C4008 S-4 for 5 d were 95.35% and 97.77%, respectively, while the average mortalities of silkworms fed with protein extracts from leaves of E1 C4008 S-3 and E1 C4008 S-4 reached 94.55% and 83.64%, respectively. The results suggested that wide compatibility and evaluated agronomic traits of E1 C4008 S-4 were not significantly changed by insertion of the exogenous genes. Overall, a novel male sterile germplasm with glyphosate resistance, lepidopteran resistance and wide compatibility was verified to be developed in rice.

Plant Regeneration from Protoplasts of Wide Compatible Rice

Rice selection 02428 and T984(Oryzasativa L.ssp.japonica)were germplasmresources with wide compatibility.Mature embryos of rice cultured on Lin-smaier and Skoog medium with 2.5 mg/l2,4-D,1.0 mg/l thiamine-HCL,3%(W/V)sucrose and 0.7%(W/V)agar,pH 5.8(LS2.5)were used for callus initiation.Cultures were

Genome-Wide Association Analysis and Allelic Mining of Grain Shape-Related Traits in Rice

Excavating single nucleotide polymorphisms (SNPs) significantly associated with rice grain shape and predicting candidate genes through genome-wide association study (GWAS) can provide a theoretical basis for discovery and utilization of excellent genetic resources in rice. Based on 16 352 SNPs, 161 natural indica rice varieties with various grain sizes in southern China were used for GWAS of grain shape-related traits, referring to grain length (GL), grain width (GW), 1000-grain weight (TGW), and grain length/width (GLW). Phenotypic statistics showed that coefficient of variation values for these four traits GL, GW, TGW and GLW were 9.92%, 9.09%, 20.20% and 16.38%, respectively. Each trait showed a normal distribution, and there was a certain correlation between these traits. Through general linear model correlation analysis, a total of 38 significant loci were identified, and a range of 100 kb upstream and downstream of the significant loci was identified as the candidate interval. On chromosome 3, GS3 and qGL3 were found to regulate GL. On chromosome 6, TGW6 and GW6a were found to regulate TGW. Also, some QTLs related to grain shape were found on chromosomes 5 and 9. Besides that, using sequenced 3K-germplasm resources, we found that there are 22 overlapped varieties between these two natural populations. Twenty-six SNPs and fourteen haplotypes were identified in five regions of GS3 genes. The detection of multiple candidate genes/QTLs within the candidate interval is beneficial for further excavation of superior rice genetic resources.

Selection of wide compatible restorer lines and their application in hybrid rice breeding

Since the middle of 1980’s, wide compatibility(WC) rice lines have been screened by ricebreeders in China and applied in hybrid ricebreeding program. Several WC lines such asPecos, T984, Lunhui 422, and 02428 withideal agronomic characters were identified. Weincorporated the WC gene into restorer linesby crossing these japonica WC lines with ob-tained indica lines. Some WC restorer lineswith indica-japonica medium type were ob-tained and their application value in intersub-specific hybrid rice breeding were evaluated. 1. Effect of crossing methods on selectionefficiencies of WC restorer lines

Genetic Improvement and Biological Characteristic Analysis of Dwarf Abortive TypeJaponicaCMS Lines with Wide Compatibility

From the view of methodology for genetic improvement ofJaponicacytoplasmic male sterile(CMS) lines,we introduced the breeding process of the dwarf-abortive type(DA type)JaponicaCMS line Chunjiang 95A with wide compatibility,and further investigated the biological characteristics of the DA type Chunjiang 95A and its isonuclear alloplasmic CMS line Dian-I type Chunjiang 95A.The results show that DA type Chunjiang 95A is a japonicalinous CMS line and mainly typical abortive,which is better in fertility stability than stained abortive typeJaponica CMS lines.The DA type Chunjiang 95A is featured with high stigma exsertion,good flowering habit,high outcrossing rate and wide compatibility.Its recoverability differs from stained abortive typeJaponicaCMS lines.Moreover,we discussed genetic improvement of the typical abortive type JaponicaCMS lines and the application prospects of heterosis.

Mining rice new germplasm containing S_5~n gene by functional molecular marker and sequencing

Inter-subspecific hybrids between indica and japonica varieties yield strong biological heterosis, but it is difficult to utilize the hybrids directly in commercial production due to sterility of F1. A special gene S5n may overcome the hybrids sterility, which is caused by the interaction between S5 loci. Recently, S5n had been cloned, and it was revealed containing a large DNA deletion sequence that made the gene nonfunctional, compared to S5i or S5j. We designed a pair of primers flanking the deletion sequence of S5n, and then applied to distinguish the varieties with S5n or non-S5n, convincing result suggested that the primers could be served as functional molecular marker to efficiently identify the new germplasm with S5n. Using the functional marker, we surveyed 197 varieties from China National Micro-core Rice Collection, and found ten of which represented S5n including following varieties: Haobuka, Sanbangqishiluo, Mubanggu, Xiaohonggu, Mowanggu’neiza, Laozaogu, Fanhaopi, Feie’nuo2, Baoxie-7B, Teqingxuanhui. Among them, two varieties Sanbangqishiluo and Laozaogu was previously reported to contain S5n gene. Further sequence analysis on the DNA sequence covering both sides of deletion in S5n of the 10 varieties confirmed that the detected sequences in above varieties was identical with those of varieties containing S5n, such as 02428 and Linglun. These results suggested that the gene in S5 locus of the ten varieties was also nonfunctional and it proved the presence of S5n gene.

Artificial Selection in Domestication and Breeding Prevents Speciation in Rice

Speciation has long been regarded as an irreversible process once the reproductive barriers had been established.However,unlike in natural populations,artificial selection might either accelerate or prevent speciation processes in domesticated species.Asian cultivated rice is a target crop for both domestication and artificial breeding;it contains two subspecies of indica and japonica,which usually produce sterile inter-subspecific hybrids due to reproductive barriers.In this study,we constructed the evolutionary trajectory of a reproductive isolation system S5,which regulates fertility in indica-japonica hybrids via three adjacent genes,based on the data of 606 accessions including two cultivated and 11 wild rice species.Although hybrid sterility haplotypes at S5 lead to establishment of a killer-protector reproductive barrier,origin of wide-compatibility haplotypes by complex hybridization and recombination provides an opposing force to reproductive isolation and thus prevents speciation during domestication.Analysis in a diallel set of 209 crosses involving 21 parents showed that the wide-compatibility genotypes largely rescued fertility of indica-japonica hybrids,indicating that the wide-compatibility gene would enable gene flow to maintain species coherence.This counteracting system indicates that combined effects of natural evolution and artificial selection may result in reversible processes of speciation in rice,which may also have implications for genetic improvement of rice.

Two sequence alterations,a 136 bp InDel and an A/C polymorphic site,in the S5 locus are associated with spikelet fertility of indica-japonica hybrid in rice

The rice indica/japonica hybrid shows strong heterosis.However,such inter-subspecific hybrid can＇t be directly used in rice production due to its low spikelet fertility.The S5 locus was proved to be associated with fertility of indica/japonica hybrid and its S5n allele from wide-compatibility variety （WCV） is capable to overcome fertility barrier.In the present study,we reported the causal sites in the S5 locus responsible for compatibility of indica/japonica hybrid.Fine-mapping of the S5 locus using the 11 test-cross families pinpoints a candidate S5 locus encoding aspartic protease （Asp）.Intragenic recombination within the Asp gene happened in a number of recombinants,resulting in chimeric S5j-S5n alleles.Just like S5n,the chimeric S5j-S5n allele displayed higher spikelet fertility when combined with the S5i allele.In the complementary test,however,the S5n allele from WCVs failed to enhance fertilities of the indica/japonica hybrids.Compared to both indica and japonica varieties,all nine WCVs from different resources are characterized with a 136 bp deletion in the Asp N-terminus,which probably renders the S5n allele non-functional.Furthermore,an A/C polymorphic site is detected 1,233 bp downstream of the Asp start codon.The heterozygous A/C site of the Asp gene in indica/japonica hybrid is believed to be the casual factor to cause partial sterility.The functional makers based on the two polymorphic sites will be broadly used in developing wide-compatibility rice varieties.

水稻籽粒锌含量全基因组关联分析及候选基因筛选

通过挖掘水稻籽粒中锌含量显著关联的QTL,并结合基因功能注释、单倍型和灌浆期籽粒相对表达量分析,筛选与水稻籽粒锌含量相关的候选基因。本研究以139份西北早粳稻核心种质资源为试验材料,结合重测序获得的1800920个高可信度SNPs,基于一般线性模型(GLM,general linear model)进行全基因组关联分析,对显著QTL区间内候选基因进行功能注释,并对进一步筛选后的基因进行单倍型和籽粒灌浆期相对表达量分析,以期筛选出与籽粒锌含量相关的候选基因和优势单倍型。结果表明,139份水稻中籽粒锌含量存在丰富的表型变异,且符合正态分布。全基因组关联分析共关联到12个显著SNP,分布在第1、2、4、10、11号染色体上,结合候选区间内的基因功能注释,推测定位在囊泡中的OsFRDL2可能与水稻籽粒锌元素富集相关;单倍型和灌浆期籽粒相对表达量分析表明,OsFRDL2在优势单倍型Hap5中表达量相对较高。因此推测OsFRDL2可能与水稻籽粒锌元素富集相关。研究结果为解析水稻籽粒锌元素富集机制和开发富锌水稻分子标记提供理论基础,为培育富锌水稻提供基因资源。

水稻籽粒γ-氨基丁酸含量的全基因组关联分析

【目的】挖掘与水稻籽粒γ-氨基丁酸含量显著相关的SNP位点及候选基因,为揭示水稻籽粒γ-氨基丁酸合成与积累的遗传基础和分子机制提供理论依据,为高γ-氨基丁酸含量的水稻品种选育奠定基础。【方法】以139份西北早粳稻核心种质为实验材料,测定其籽粒γ-氨基丁酸含量,结合重测序基因型数据,进行全基因组关联(GWAS)分析,根据单倍型分析和基因注释筛选候选基因,通过表达谱数据库和荧光定量PCR对预测基因进行表达模式分析。【结果】水稻籽粒γ-氨基丁酸含量表现出丰富的变异,变异系数为31.25%。全基因组关联分析检测到分别位于第6、8、9、11和12号染色体上共6个有代表性的SNP位点,筛选出已克隆与γ-氨基丁酸含量有关的OsBADH2和OsGABA-T2基因,预测出6个与γ-氨基丁酸含量有关的候选基因,将LOC_Os09g10720基因列为重点分析基因,并进一步验证了其参与水稻籽粒中GABA分解的可能性。【结论】检测到6个与水稻籽粒γ-氨基丁酸含量显著关联的SNP位点。筛出6个可能与水稻籽粒γ-氨基丁酸含量相关的候选基因,分别是LOC_Os09g09750、LOC_Os09g09760、LOC_Os09g10210、LOC_Os09g09820、LOC_Os09g10280和LOC_Os09g10720,其中LOC_Os09g10720作为重点候选基因。

Molecular evolution of rice S_5~n and functional comparison among different sequences

The wide compatibility gene, S 5 n , can overcome embryo sac sterility between indica and japonica subspecies of rice. Therefore, it is very important to characterize the features of the S 5 n sequence to reveal the origin and evolution of S 5 n . In this paper, 26 cultivated rice haplotypes and 22 wild rice accessions harboring S 5 n were used to sequence S 5 n . The results showed that 15 genotypes among the 48 materials were fully consistent with control cultivar 02428 (CK). The other 33 accessions had different degrees of variation in the S 5 n sequence. Variations in the coding region mainly occurred in the second exon and eight materials showed a 10-bp deletion at 1710–1719 bp, including wild (O. nivara) and cultivated rice, such as IRW501 and Yuetai B. S 5 n sequences were not biased and evolved neutrally. The 48 materials could be divided into 4 categories using a phylogenetic tree of the amino acid sequences. Most of the wild rice clustered together, and the cultivated rice clustered into another group. Eight cultivated rice and O. nivara (wild rice) clustered in another group, which were found to lack 10 consecutive bases in exon 2. Eight rice varieties with high numbers of differences in their S 5 n coding regions were crossed with testers (typically indica and japonica) to produced test cross F 1 populations. The F 1 s were examined for their ability to overcome indica-japonica hybrid sterility. The result showed that the embryo sac fertility of S 5 n -containing hybrids increased significantly compared with control hybrids, but there were no differences among the materials with divergent sequences, indirectly proving that S 5 n is a non-functional gene.

粳稻粒型相关性状全基因组关联分析及候选基因挖掘

为了促进优质粒型粳稻品种的培育,以295份国内外收集的粳稻品种组成的自然群体为试验材料,对2018-2019年粒长、粒宽、粒厚、长宽比和千粒质量等5个粒型相关性状进行表型分析,结合重测序获得的788,396个多态性SNP,利用TASSEL 5.0的MLM模型进行全基因组关联分析,针对2 a间共同检测到的且控制多个粒型相关性状的QTL区间内所有基因进行单倍型分析,结合前人研究结果和基因功能注释挖掘水稻优质粒型候选基因。结果表明,5个粒型相关性状均存在着广泛的表型变异,且均呈近似正态分布,粒型相关性状之间大多呈显著或极显著相关,在P≤5.46×10^(-6)阈值条件下,共检测到221个与水稻粒型相关性状显著关联的QTL,在水稻的12条染色体上均有分布,表型贡献率为8.62%~20.73%,其中,2018,2019年共同检测到7个QTL。进一步针对2 a间共同检测到且同时控制多个粒型相关性状的2个QTL区间内的所有基因进行单倍型分析,结合基因功能注释推测LOC_Os12g44290为水稻粒型的新候选基因。综上,利用全基因组关联分析对295个粳稻品种的粒型相关性状进行QTL定位及候选基因分析,为优质粒型的粳稻品种培育提供了理论基础。

贵州地方水稻广亲和资源筛选

【目的】探明贵州地方水稻品种资源的广亲和基因,为籼粳杂交水稻育种提供依据。【方法】利用S5^(n)、S5^(i)和S5^(j)序列存在136 bp差异,采用分子标记辅助筛选贵州地方水稻广亲和资源。【结果】利用分子标记qh在609份贵州地方水稻种质资源中筛选到糯旱谷⁃1、本地燕麦谷、旱谷、纳包糯旱稻2号、弼佑旱稻、双江旱稻、八百粒和旱禾⁃3共8份广亲和水稻资源。通过分子标记检测8份广亲和水稻资源S5位点OFR3、OFR4和OFR5的基因型,其中八百粒基因型为+⁃n,为广谱性广亲和品种。【结论】贵州地方水稻资源品种八百粒为广谱性广亲和品种,qh标记可用于广亲和基因资源筛选。

水稻粳型亲籼系粳型性的判别

选择均匀分布于水稻 1 2条染色体上的 1 0 0个微卫星标记对 6个籼型、6个粳型测验种和 1 0个待测粳型亲籼系的 DNA遗传多样性进行了分析 ,结果表明 ,测验种 DNA在籼粳分布上存在明显差异 ,表现在其粳型判别值 (Dj)的变异范围为 0 .1 2～ 0 .87,籼型测验种的 Dj值均低于 0 .2 5,粳型测验种的 Dj值均高于 0 .75,Dj 值可以用于粳型亲籼系粳型性的判别。根据 Dj值大小 ,待测粳型亲籼系 G2 4 1 6- 3、G2 4 1 7- 2 - 1、G30 0 4 - 4和 G30 0 5- 4 - 2属于偏粳型 ,G2 50 5- 1和 G2 60

水稻广亲和性遗传的主基因多基因混合模型分析

籼、粳亚种间的F1一般表现为半不育 ,这限制了籼、粳杂种优势的利用。广亲和基因的发现及其遗传研究有助于揭示这种半不育现象的遗传本质 ,使克服籼、粳亚种间F1的半不育成为可能。采用主基因 多基因混合遗传模型 ,分析了籼、粳杂交组合 30 37/ 0 2 4 2 8的P1、P2 、F1、B1、B2 和F2 六世代材料。研究结果显示 :广亲和性的遗传除受单个主基因控制外还受多基因的影响。在利用广亲和基因克服亚种间的半不育性时不仅要考虑主基因对育性的作用 。

具广亲和性的水稻隐性高秆细胞突变体

０２４２８ｈ系粳稻隐性高秆细胞突变体。来源于半矮秆广亲和种质０２４２８体细胞无性系变异。在１０３个Ｒ_２代株系中，有１株系在抽穗后分离出高、矮秆两种类型，高秆比矮秆高６１．４％，系因倒一和倒二节间伸长所致，以倒一节间伸长最为明显，占增长的６６．３％。在Ｒ_３、Ｒ_４代中矮秆类型继续分离出高秆和矮秆两种类型，分离比为３：１，高秆类型未见分离。显示所分离的高秆是供体亲本０２４２８的单基因突变。暂名为０２４２８ｈ。突变系０２４２８ｈ与供体亲本杂交，Ｆ_１的株高８８．８厘米，较０２４２８ｈ矮４２．２厘米，与０２４２８株高８７．６厘米近似；０２４２８ｈ与其它籼、粳品种杂交Ｆ_１的株高亦有类似结果。Ｆ_２的株高明显分离为两种类型，经ｘ￣２测验，矮秆与高秆的分离比符合３：１。说明０２４２８ｈ是受隐性单基因控制的高秆突变体。０２４２８ｈ与籼、粳稻品种杂交Ｆ_１的平均结实率分别为８０．６％和８３．１％，表明０２４２８ｈ仍保持着供体亲本的广亲和性。此外，本文对它的出现和在杂交稻育种中的利用进行了讨论。

水稻籼粳亚种间杂种优势利用研究进展

本文从水稻育种进步的宏观观点,回顾与展望了我国水稻杂种优势利用的历史进程。由早期追求以强大生物优势为主要育种目标的启蒙阶段进入三系法生态远缘优势与理想株型相结合的籼梗杂种优势间接利用阶段,实现了水稻杂种优势利用的第一次飞跃。现在又进入三系法、两系法籼粳有利基因交换与自由配组的籼梗杂种优势直接利用阶段,预期将产生水稻杂种优势利用的第二次飞跃。近年研究表明,双亲遗传距离与经济优势呈二次多项式关系,杂种优势含可利用与非可利用两个方面,广亲和系可以将亚种F_(?)结实率提高到正常水平,但它的稳定性受双亲遗传背景与环境互作影响。因此,采用“籼梗(爪)架桥”,亲缘渐渗,有利基因交换,亲本遗传改良以及因地制宜选择种植区,是籼粳亚种间杂种优势利用的主要方法。

分子标记辅助选择抗稻白叶枯病基因在选育水稻广亲和恢复系上的应用

以IRBB21为Xa21 基因供体,广亲和恢复系4183为受体,进行1次杂交并回交3次,逐代用分子标记检测手段,导入广谱抗白叶枯病基因Xa21 ,在保持广亲和恢复系4183优良经济性状的基础上,增强其抗白叶枯病的能力,育成抗白叶枯病的广亲和恢复系抗4183,其抗性达到了IRBB21的抗性水平,且保持了 4183 的广亲和性、恢复性及优良的经济性状。并就杂交水稻白叶枯病的抗性改良及育种对策进行了讨论。

秋稻品种‘Dular’广亲和基因效应的RFLP分析

利用ＲＦＬＰ标记对秋稻品种 Ｄｕｌａｒ的三交（南京１１／／Ｄｕｌａｒ／２５３３）的Ｆ２群体与籼粳品种的测交Ｆ１进行了单株带型鉴定，并对广亲和性的表达进行分析。结果表明：与第６染色体上标记ＲＧ２１３连锁的广亲和基因Ｓｎ５效应较大；不同位点广亲和基因具有明显的累加效应；位点内的互作导致了粳型配子的部分败育，同时还存在位点间的互作。