云南地方稻种抗稻瘟病基因Pi ta和Pi b的鉴定

 根据抗病等位基因Pi ta、Pi b与pi ta、pi b感病等位基因在DNA序列上的差异,合成Pi ta引物YL155/YL87和YL183/YL87,Pi b基因引物Pi bdomF/Pi bdomR和Lys145F/Lys145R。利用这两套分子标记检测了176份云南地方稻种抗性基因Pi ta和Pi b的分布。结果表明,供试材料中72份含有Pi ta基因,占40.9%,其中99份粳稻中有28份含有Pi ta基因,占28.3%;77份籼稻中有44份含有该基因,占57.1%;抗性基因Pi ta在粳稻和籼稻品种中的分布频率存在明显差异,籼稻高于粳稻;Pi ta基因主要分布于玉溪、保山、临沧、思茅等地区。而176份供试材料中仅有33份含有Pi b基因,占18.8%,其中99份粳稻中有18份含有该基因,占18.2%;77份籼稻中有15份含有该基因,占19.5%;Pi b基因主要分布在滇西部的怒江、保山和昭通等地区。分别选取具有代表性的18及17份材料在温室进行Pi ta和Pi b基因抗性鉴定,结果与分子鉴定结果基本相符。     

稻瘟病抗性基因的鉴定及利用进展

20世纪60年代中期,日本率先开展了水稻品种抗稻瘟病基因分析的研究工作,鉴定了最初的8个抗性位点上的14个基因,并建立了一套抗稻瘟病基因分析用的鉴别体系（JDCs, Japanese differential cultivars）,随后,国际水稻研究所和中国等产稻国也逐渐开展了水稻稻瘟病抗性遗传的系统性研究。截至2007年12月,已至少报道了58个抗稻瘟病位点共67个主效基因。这些基因成簇地分布于除第3染色体外的所有水稻染色体上,其中,66个为显性基因,1个为隐性基因,包括Pi b、Pi ta、Pi z5、Pi zt、Pi 9、Pi d2、Pi 36和Pi 37等8个已被克隆的基因(Pi z5、Pi zt和Pi 9同为Pi z基因位点上的复等位基因)。还讨论了合理利用抗性基因等问题。     

辽宁省粳稻品种稻瘟病抗性基因分析

【目的】为明确辽宁省260份粳稻品种中稻瘟病抗性基因的类型,评价抗性基因利用价值,为辽宁省水稻抗病育种提供参考。【方法】选用9个稻瘟病抗性基因的特异引物分析辽宁省已审定的260份粳稻品种抗性基因种类、基因组合类型及地域分布情况,基于抗病基因检测条带的有无进行品种聚类分析,苗期人工喷雾接种、分蘖盛期和蜡熟期田间自然发病调查,评价9个抗性单基因系的抗病性。【结果】辽宁省粳稻品种中含有抗性基因数量从0～7个不等,抗性基因Pikh检出率最高,其次是Pikm和Pi1;所有参试品种均未检测到Pi9基因,仅在辽宁省农业科学院育成的辽粳421中检测到Pi40基因。参试的水稻品种中共有62个基因组合类型。基于抗性基因检测结果进行聚类分析,可以将260份粳稻材料分为11个类群。9个抗性单基因系中,Pi40抗性最好;其次是Pi9、Pigm和Pita。【结论】抗性基因Pi40、Pi9、Pigm和Pita在辽宁省粳稻品种选育和生产上具有重要的应用潜力;参试品种的80.38%集中在第Ⅴ、Ⅵ、Ⅶ和Ⅸ类群,没有明显的地域差异;参试品种抗病基因组合类型不够丰富,亲缘关系较近。     

4个抗稻瘟病基因分子标记的建立及在水稻亲本中的分布

建立稻瘟病抗性基因的分子标记对于培育抗稻瘟病水稻品种有重要意义。本文利用抗稻瘟病基因Pi9、Pi2、Pi5和Pita基因序列与日本晴等位基因的序列差异,建立4个基因的共显性分子标记M-Pi9、M-Pi2、M-Pi5和M-Pita,并用这4个分子标记检测24个抗稻瘟病单基因系和48份水稻亲本,结合48份水稻亲本对广西采集的稻瘟病菌ZB1和ZB13苗瘟抗性鉴定结果,验证4个分子标记的特异性和有效性。结果表明,M-Pi9仅在含Pi9和Piz的单基因系中检测出特异带;M-Pi2仅在含Pi2和Pizt的单基因系中检测出特异带;M-Pi5仅在含Pi5、Pi3和Pii的单基因系中检测出特异条带;M-Pita在含Pita和Pita2的单基因系中检测出特异带。具有M-Pi2特异条带的水稻亲本对ZB1和ZB13均表现出抗性,而具有M-Pi9、M-Pi5或M-Pita特异条带的水稻亲本对ZB13均表现出抗性。     

抗稻瘟病Pi2/9/z-t基因特异性分子标记的开发

通过对已克隆的抗稻瘟病基因Pi2、Pi9以及Piz-t进行序列比对,寻找各自特异的核苷酸差异,成功开发了基于PCR技术以及电泳检测技术的Pi2/Pi9/Piz-t以及Piz-t的基因特异性分子标记,能有效地将Pi2/Pi9/Piz-t与该位点上的其他抗性等位基因及感病等位基因区分开,这为分子标记辅助育种及抗病基因聚合提供有效的分子标记。用Pi2/Pi9/Pizt基因特异性分子标记对来自全国各稻区的共101份水稻品种和育种亲本进行分子检测,结果发现,除2个品种检测到Piz-t带型之外,大部分水稻品种不携带这3个抗性基因,这为有目的地开展品种的抗性改良提供了重要的参考信息。     

103个杂交稻亲本白叶枯病和稻瘟病抗性基因型检测

白叶枯病和稻瘟病是我国水稻生产的主要病害,选育抗病品种是一种经济有效的防治手段。明确抗性基因在水稻品种资源中的分布,将有助于应用分子标记辅助选择培育抗性品种。本研究针对13个重要抗病基因,包括8个白叶枯病抗性基因(Xa1、Xa4、xa5、Xa7、xa13、Xa21、Xa23和Xa26)和5个稻瘟病抗性基因(Pib、Pi2、Pi9、Pi25和Pita),应用这些基因的基因标记或连锁标记共29个,检测103份杂交稻亲本,初步确定了其等位基因分布和利用情况,为水稻基础材料的筛选及应用提供参考。     

稻瘟病抗性基因对安徽省稻瘟病菌种群抗性的影响

为分析抗稻瘟病基因对安徽省稻瘟病菌株的抗性水平的影响,采用10个水稻抗稻瘟病单基因品系对安徽不同地区的稻瘟病菌株进行致病力测定,同时对201份水稻品种的抗稻瘟病基因Pi9进行扩增,结合田间抗性表现,分析抗性基因Pi9的利用价值。抗性基因Pi9、Pik、Pizt等对安徽地区的稻瘟病菌株具有较好的抗性,抗性频率分别为66%、60%和54%,其中携带Pi9抗性基因的水稻品种的田间抗性表现也较好,说明Pi9可直接作为分子标记辅助安徽地区水稻品种选育抗原基因。     

82份籼粳稻骨干亲本抗稻瘟病基因的分子检测

北粳南移在江西省已推行11年,为进一步明确适合江西晚粳常规稻的稻瘟病抗性情况及粳渗籼骨干亲本的选择,本研究利用Pi37/Pi35/Pish、Pi5、Pi2、Pi9、Pia、Pi23、Pigm、Pi33/Pi42、Pi56、Pik/kg(t)/ks/kp/km/kh/43、Pikh共11个抗瘟基因的标记结合井冈山抗性谱的鉴定,对28份骨干粳稻和54份骨干籼稻进行分子标记与抗性分析。结果表明,粳稻中有1份携带其中的7个抗性基因,有5份携带6个抗性基因,有7份携带5个抗性基因,有12份携带4个抗性基因,有3份携带3个抗性基因,抗性频率最高的Pia为0.786,其次为Pikh和Pi9,频率均为0.643,Pigm和Pi37/Pish在这批粳稻材料中的抗性频率为0;籼稻中有8份携带其中的7个抗性基因,有10份携带6个抗性基因,有13份携带5个抗性基因,有17份携带4个抗性基因,有4份携带3个抗性基因,有2份携带2个抗性基因,抗性频率最高的Pi9为0.981,其次Pik/Pikh/Piks频率为0.833,Pigm、Pi37/Pish、Pikh、Pia、Pi5、Pi2、Pi23和Pi33/Pi42的频率分别为0.556、0.537、0.519、0.389、0.352、0.315、0.167和0.111,Pi56在这批籼稻材料中的抗性频率为0。明确这些材料抗瘟基因的分布,将为广谱持久抗性籼粳材料创制骨干亲本的选择与配组指明了方向。     

水稻冷胁迫响应基因OsSADMC功能标记的开发和利用

通过对95份水稻种质资源进行苗期耐冷性鉴定,筛选出10份苗期耐冷(存活率≥60%)和9份冷敏感的材料(存活率为零),并对水稻冷胁迫响应基因OsSADMC进行了表达分析,发现对照和冷胁迫处理条件下该基因在耐冷品种中的表达量都显著高于冷敏感品种。对耐冷材料丽江新团黑谷(LTH)和冷敏感材料IR29的基因编码区域进行了克隆和测序分析,在两个品种间鉴定了10个SNP位点,其中第749碱基处存在的一个碱基变异(C突变为T),从而使丝氨酸变为亮氨酸。根据其中一个SNP位点设计了OsSADMC的功能标记SADMC-CAPS1,该标记可以准确地鉴定出19份耐冷性不同水稻种质资源OsSADMC的基因型和耐冷性。     

黄淮麦区部分骨干品种重要基因等位类型分析及全基因组优异位点挖掘

为了从分子水平上了解黄淮麦区部分骨干品种(尤其是西农系列品种)的春化和光周期特性、矮秆基因、抗赤霉病基因类型及全基因组优异位点的分布,以西农979、西农511等近年黄淮麦区主栽小麦品种(共64份)为材料,采用分子标记及小麦35K芯片对供试品种进行检测。结果表明,13份材料含有显性春化基因 Vrn-D1(20.3%),3份材料含有显性基因 Vrn-B1(4.7%),未检测到显性基因 Vrn-A1和 Vrn-B3;除中国春和宁春45外,其余62份材料均含光周期不敏感基因 Ppd-D1a;9份材料携带矮秆基因 Rht-B1b,28份材料携带矮秆基因 Rht-D1b,35份材料携带矮秆基因 Rht8;15份材料同时含有 Rht-D1b和 Rht8;苏麦3号和兰考198含抗赤霉病基因位点 Fhb1。芯片检测结果发现,西农系列品种亲缘关系较近,共含有1 049个特异SNP,集中在2A和6B染色体上,这些位点可能是决定西农系列品种区别于其他品种的重要遗传位点;所有参试材料共含有1445个相同的SNP位点,集中在2D和3B染色体上。     

水稻抗性基因Pi对福建省稻瘟病菌优势菌群的抗性分析

【目的】抗病品种的合理利用和布局是实现水稻抗瘟持久化的关键因子之一。为明确水稻24个抗性基因在福建省的抗病性及其应用前景,【方法】首先分别采用全国稻瘟病菌生理小种鉴别品种和CO39近等基因系来鉴定2012-2015年间从福建省各稻区种植的普感品种丽江新团黑谷上采集的347株稻瘟病菌单孢菌株的生理小种类型和致病型,再测定24个抗性基因对福建省稻瘟病菌的抗性。【结果】根据全国稻瘟病菌生理小种鉴别品种对菌株的抗感反应,可将供试稻瘟病菌的生理小种划分为6个群36个生理小种,其中ZA、ZB和ZC为主要种群,ZC15、ZD7和ZB15为优势生理小种。根据CO39近等基因系的接种结果,将供试稻瘟病菌划分为17个致病类型,其中I34.1为优势致病型。供试的24个抗性基因对347株福建省稻瘟病菌菌株的抗性频率不同,抗谱为9.80%~89.91%。其中,Pi-k~m、Pi-7(t)、Pi-9(t)、Pi-k~p、Pi-k、Pi-k~h、Pi-z~5和Pi-ta(1)等8个抗性基因的抗病性较强,抗谱均高于70.00%。【结论】说明这8个抗性基因在福建省具有较好的应用前景,育种时可以考虑联合利用这些抗性基因。同时,实验结果表明,这8个抗性基因对主要生理小种的抗谱和主要致病型的抗谱均值高于69.00%,与其对所有测试菌株的抗谱吻合。说明利用稻瘟病菌的优势种群或优势致病型来鉴定水稻品种的抗病性是可靠的。     

Development of new markers to genotype the functional SNPs of SSIIa, a gene responsible for gelatinization temperature of rice starch

Gelatinization temperature (GT) is an important quality predictor that determines the cooking quality of rice. GT is genetically controlled by the starch synthase IIa (SSIIa) gene. Two functional single nucleotide polymorphisms (SNPs) inside the SSIIa have already been found to be responsible for the variation of GT. One of these, GC/TT SNP at 4329/4330 bp, could be genotyped by four primers in a single PCR ( Bao et al., 2006a), but another one, G/A SNP at 4198 bp, has not been detected by a PCR-based marker. Here, we developed cleaved amplified polymorphic sequence (CAPS) and derived cleaved amplified polymorphic sequence (dCAPS) markers to detect these SNPs. A dCAPS marker that the PCR products were cleaved by the BseR I restriction endonuclease was designed to detect GC/TT SNP. Both CAPS and dCAPS markers were designed to detect G/A SNP using the restriction endonuclease Nla III and Tsp45 I, respectively. All the markers developed were co-dominant. It was known that the A allele of G/A SNP was rare among rice germplasm, but it was still in use by rice breeders. 11 rice accessions including landrace and breeding lines with A allele of G/A SNP were detected. The F₂ individuals from two crosses were used to analyze the co-segregation between the SNP alleles and the GT. The segregation ratio of two SNPs did not conform to the expected Mendelian ratio of 1:2:1, but the SNPs were co-segregated with GT. The markers developed in the present study would be useful in molecular breeding for the improvement of the quality of rice grain.     

Chromosome Segment Substitution Lines: A Powerful Tool for the Introgression of Valuable Genes from Oryza Wild Species into Cultivated Rice (O. sativa)

Wild species of rice (genus Oryza) contain many useful genes but a vast majority of these genes remain untapped to date because it is often difficult to transfer these genes into cultivated rice (Oryza sativa L.). Chromosome segment substitution lines (CSSLs) and backcross inbred lines (BILs) are powerful tools for identifying these naturally occurring, favorable alleles in unadapted germplasm. In this paper, we present an overview of the research involving CSSLs and BILs in the introgression of quantitative trait loci (QTLs) associated with the improved performance of rice including resistance to various biotic and abiotic stresses, and even high yield from wild relatives of rice and other unadapted germplasm into the genetic background of adapted rice cultivars. The CSSLs can be used to dissect quantitative traits into the component genetic factors and evaluate gene action as single factors (monogenic loci). CSSLs have the potential to uncover new alleles from the unadapted, non-productive wild rice accessions, develop genome-wide genetic stocks, and clone genes identified in QTL studies for functional genomics research. Recent development of high-density single-nucleotide polymorphism (SNP) arrays in rice and availability of custom-designed medium- and low-density SNP arrays will enhance the CSSL development process with smaller marker-defined segment introgressions from unadapted germplasm.     

Waxy gene haplotypes: Associations with apparent amylose content and the effect by the environment in an international rice germplasm collection

Apparent amylose content (AAC), the key determinant of rice end-use quality attributes, is primarily controlled by the Waxy gene which codes for granule bound starch synthase (GBSS). We examined the combination of sequence variation in the Waxy gene and environmental effects, and their associations with AAC using 171 rice accessions originating from 43 countries. The combination of two single-nucleotide-polymorphism (SNP) markers in the Waxy gene allows for the identification of three marker haplotypes in this gene. The first SNP is at the leader intron splice site (In1 SNP), and the second polymorphism is in exon 6. The haplotypes explained 86.7% of the variation in AAC and discriminated the three market classes of low, intermediate and high AAC rice from each other. The environment affected the AAC of all haplotypes. Higher air temperature during grain development associated with a decrease in AAC of low and intermediate AAC-types, but with an increase in AAC of high AAC-type. The association of AAC with several Waxy RM190 microsatellite-(CT_n) alleles in combination with the In1 SNP was also examined. In conclusion, the Waxy haplotypes studied appear to be useful markers for selecting the AAC of breeding lines developed from the world's rice germplasm.     

Strategy for Use of Rice Blast Resistance Genes in Rice Molecular Breeding

Rice blast is one of the most destructive diseases affecting rice production worldwide. The development and rational use of resistant varieties has been the most effective and economical measure to control blast. In this review, we summarized the cloning and utilization of rice blast resistance genes, such as Pi1, Pi2, Pi9, Pi54, Pigm and Piz-t. We concluded that three main problems in the current breeding of rice blast resistance are: availability of few R(resistance) genes that confer resistance to both seedling and panicle blast, the resistance effect of pyramided lines is not the result of a simple accumulation of resistance spectrum, and only a few R genes have been successfully used for molecular breeding. Therefore, novel utilization strategies for rice blast R genes in molecular breeding were proposed, such as accurately understanding the utilization of R genes in main modern rice varieties, creating a core resistant germplasm with excellent comprehensive traits, screening and utilizing broadspectrum and durable resistance gene combinations. Lastly, the trends and possible development direction of blast resistance improvement were also discussed, including new genes regulating resistance identified via GWAS(genome-wide association study) and improving rice blast resistance using genetic editing.     

Polymorphism in the GBSS gene affects amylose content in US and European rice germplasm

The present study was conducted to investigate the relationship between waxy allelic forms and amylose in European and US rice germplasm. These allelic forms were defined according to the single-nucleotide polymorphisms (SNP) found in the leader intron 5′ splice site (G → T), exon 6 (A → C) and exon 10 (C → T). The combination of these three SNPs accounted for 89.2% of the variation in apparent amylose content in a pedigree of 85 US rice varieties and 93.8% of the variation among 279 accessions in a European germplasm collection. The allelic forms TAC and TCC were found in low amylose varieties. All varieties with intermediate levels of apparent amylose had the GCC allele. High levels of apparent amylose varieties had either the GAT and GAC allele. The sequence AGTTATA in the intron 1 distinguished the low amylose varieties from the other classes regardless of any other base changes. Intermediate amylose varieties can be distinguished from those with high apparent amylose by changes in either exon 6 or exon 10. However the simplest interpretation of the data is that the tyrosine/serine change in exon 6 is responsible for the lower levels of Granule bound starch synthase (GBSS) protein and thus lower levels of amylose in intermediate vs. high amylose verities.     

辽宁省粳稻育成品种遗传基础分析

以辽宁省1980～2000年审定的71个粳稻品种为分析材料,通过对其系谱、细胞核构成及细胞质来源进行了分析。结果显示,在系谱结构上,日本粳稻丰锦、福锦、黎明、Pi5、三好共衍生出54个水稻品种,占品种总数的76.06%。在细胞核构成上,外来种质资源占辽宁省粳稻育成品种平均细胞核比例的71.86%,其中日本粳稻资源比例达到67.06%。在细胞质来源上,外引19个细胞质家族共衍生出55个水稻品种,占品种总数的77.46%,其中日本粳稻15个细胞质家族,衍生水稻品种数占育成品种总数的46.48%。表明,外引种质资源对辽宁省粳稻品种的基础性作用,日本粳稻资源对辽宁省粳稻品种具有较大的遗传贡献,在一定程度上辽宁省粳稻育成品种遗传背景较为单一。同时,探讨了拓宽辽宁省粳稻品种群体遗传基础及水稻种质资源利用的措施。     

籼型水稻中稻瘟病抗性基因分布及抗性研究

稻瘟病严重威胁着水稻的安全生产。目前,培育抗性品种是控制稻瘟病危害最经济有效的途径之一。本研究利用11个主效稻瘟病抗性(R)基因(Pi2、Piz-t、Pi9、Pi54、Pik-m、Pid3、Pib、Pit、Pi5、Ptr和Pita)的分子标记对48个常规稻、15个不育系和129个杂交稻进行了检测。结果表明,在常规稻中,Ptr和Pi5的分布频率均为35.42%,Pib、Pi2、Piz-t、Pit的分布频率介于20.0%~30.0%之间,其余均在15%以下;在不育系中,Pita的分布频率为40.0%,其余均在20%以下;杂交稻中,Pita、Pib、Pi54、Ptr和Pi5的分布频率在40.31%~55.04%之间,其余均在20%以下;Pita在各个品系中均广泛存在,频率介于35.42%~51.16%。田间抗性评价表明,R基因较多的品种具有较高的抗性。综上所述,在参试的水稻材料中,不育系中存在抗性基因较少,所有类型材料中广谱抗性基因分布较少,抗性基因聚合可以有效提高稻瘟病田间抗性。     

Analysis of Blast Resistance Genes and Neck Blast Resistance of japonica Rice Varieties/Lines Recently Developed in Jiangsu Province

【Objective】Evaluating the breeding potential of the known blast resistance (R) genes harbored in rice varieties is one of prerequisites for developing rice blast resistant varieties and R genes-based control of the disease epidemic.【Method】A total of 195 new japonica varieties/lines from Jiangsu Province were genotyped using functional markers corresponding to 14 rice blast R gene loci. In order to identify gene or gene combination with breeding potential against neck-blast, 158 japonica rice varieties were then artificially inoculated with blast strains as well as 17 advanced backcrossing lines containing Pigm gene in two different genetic backgrounds at booting stage. 【Result】Most varieties carried 2 to 5 R genes, and none of new varieties contained Pigm; the distribution frequencies of Pib, Pita and Pikh were relatively high and all exceeded 45%, the rest 10 genes were under 30%; Pid3, Pid2, Pia and Pb1 had apparently higher frequency in lines from regional tests than that in authorized and released varieties. Three genes with effects ordered as Pia > Pi3/5/i > Pita significantly contributed to neck-blast resistance, and significantly positive interaction effects were found between Pia and Pita, and between Pi3/5/i and Pita. Combination effect of Pia+Pita was significantly higher than that of Pi3/5/i+Pita, and the varieties harboring Pia+Pita reached resistant or highly resistant levels. The resistance of 17 advanced backcrossing lines carrying Pigm was all significantly improved as compared with corresponding controls, and all reached resistant level or even higher. 【Conclusion】 The results suggested that Pigm and the combination of Pia+Pita have critically important breeding potential in japonica rice breeding against neck blast in Jiangsu Province.