Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight(BB) diseases,the two most prevalent diseases jeopardizing rice production.Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food.An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions.Only one pair of parents,DH146 × TM487,showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes,Xa21,xa13 and xa5,thus it was used in the marker-assisted selection(MAS).F2 individuals of DH146 × TM487 were genotyped using flanking markers of RM3330 and sequence tagged site(STS) marker SA7 for Pi25.The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21,xa13 and xa5 identified by the markers pTA248,RM264 and RM153,respectively in subsequent generations.Finally,after selection for agronomic traits and restoration ability among 12 pyramided lines,we acquired an elite restorer line,R8012 including all four target genes(Pi25+Xa21+xa13+xa5).Hybrid Zhong 9A/R8012 derived from the selected line showed stronger resistance to blast and BB,and higher grain yield than the commercial checks uniformally in experimental plots,2007 state-wide yield trial and 2008 nation-wide yield trial.This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes.The resultant restoring line and its hybrid would play an important role in securing rice production in China.     

Application of Functional Markers to Identify Genes for Bacterial Blight Resistance in Oryza rufipogon

Field resistances of nine accessions of common wild rice (Oryza rufipogon Griff.) and one rice variety (IR24) were evaluated by using nine strains of bacterial blight pathogen (Xanthomonas oryzae pv. oryzae) from the Philippines. IR24 was highly susceptible to all the strains, and six common wild rice accessions resisted all the nine strains, with a resistance frequency of 67%. The accessions Yulin and Wanning were only susceptible to PXO280 and PXO71, respectively. The accession Gaozhou was susceptible to the three strains PXO79, PXO99 and PXO339, whereas resistant to the other six strains. It could be concluded that there is at least one resistance gene in each common wild rice accession. The functional markers of the genes xa5, xa13, Xa21 and Xa27 were used to detect the presence of these resistance genes in the nine tested wild rice accessions, and it was found that four wild rice accessions contained heterozygous xa13. Among the nine common wild rice accessions, five were homozygous for Xa27 and three homozygous for xa27, and the accession Laibin contained neither xa27 nor Xa27. In addition, there were no xa5 and Xa21 in all of these accessions.     

利用功能标记鉴定普通野生稻中的白叶枯病抗性基因

 以9个菲律宾白叶枯病菌小种对供试的9份普通野生稻（Oryza rufipogon Griff.）及1份高感白叶枯病材料IR24进行抗性鉴定,发现IR24对所有的菌株都表现为高感, 6份野生稻材料对9个菌株表现全抗,占参试野生稻总数的67%。取自广西玉林的1份材料只感PXO280(P8),海南万宁的1份材料感PXO71(P4),广州高州的1份材料对PXO79、PXO99和PXO339感病,而这几份材料对其余菌株都表现为抗病,说明每份材料至少含有1个抗性基因。利用已克隆的白叶枯病抗性基因xa5、xa13、Xa21和Xa27的功能标记检测,结果表明9份供试普通野生稻中都不含抗性基因xa5、Xa21;5份为显性Xa13纯合体,4份为隐性抗病xa13杂合体;5份为抗病显性Xa27纯合体,3份为隐性xa27纯合体,1份材料中xa27和Xa27都不存在。     

疣粒野生稻抗白叶枯病新基因的初步鉴定

以10个菲律宾白叶枯病菌小种和1个中国白叶枯病菌小种为供试菌系,以高感白叶枯病水稻品种IR24及携有不同抗白叶枯病基因的近等基因系IRBB1等16个材料作为参照,对粳稻品种8411/疣粒野生稻体细胞杂交获得的两个抗白叶枯病新种质SH5和SH76进行了白叶枯病抗谱鉴定。结果表明SH5和SH76在苗期的抗谱较广,并且与已知抗病基因的抗谱不同,但与IRBB5(xa5)和IRBB7(Xa7)相似。分别用xa5和Xa7的分子标记2F_1R和M5进行检测,确定SH5和SH76中不含有xa5和Xa7基因。初步推测SH5、SH76可能含有一个新的抗病基因或者一个连锁的基因簇群。     

水稻白叶枯病抗性基因鉴定进展及其利用

 1982~1987年，日本热带农业研究中心（TARC）和国际水稻研究所（IRRI）合作采取统一研究方案，创建了国际水稻白叶枯病抗性鉴别系统。统一了命名，删去了重复（包括与Xa3相同的Xa4b、Xa6和xa9）。截至2005年6月，经国际注册确认和期刊报道的水稻白叶枯病抗性基因共30个，其中 Xa22(t)、Xa26(t)、xa26(t)、Xa27(t)、xa28(t)、Xa29(t)和3个Xa25(t)为暂定名基因，有待订正。在30个基因中，21个为显性基因（Xa），9个为隐性基因（xa）；13个表现全生育期抗性，15个为成株期抗性，Xa21和Xa25(t) (O. minuta)两个基因在分蘖后期表达抗性。已被定位的抗性基因有17个，即第11染色体上有7个，Xa3、Xa4、Xa10、Xa21、Xa22(t)、Xa23和Xa26；第4染色体上有4个，Xa1、Xa2、Xa12和Xa14;第5染色体上有2个，xa5和xa13；第6染色体上有Xa7和Xa27；第12染色上有Xa25(t)；第1染色体上有Xa29（t）。包括Xa1、xa5、Xa21、Xa26五个基因已被克隆。并讨论了合理利用抗性基因等问题。     

Identification of QTLs underlying resistance to a virulent strain of Xanthomonas oryzae pv. oryzae in rice cultivar DV85

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice in China. A strongly virulent Xoo strain, designated Z-173, is widely distributed across China and Southeast Asia. Indica rice DV85 is known to carry the two resistance genes, xa5 and Xa7. However, their effectiveness against Z-173 is unknown. Using a recombinant inbred line (RIL) population derived from a cross between DV85 and the susceptible cultivar Kinmaze, we have identified the quantitative trait loci (QTLs) responsible for the resistance of DV85 to Z-173. Following 2 years of phenotyping, three QTLs associated with the resistance were detected. These were linked to RFLP markers X362, X292 and G1091 on chromosomes 3, 5, and 6, respectively. Qxa-5 and Qxa-6 probably correspond to xa5 and Xa7, respectively. Both the xa5 and Xa7 resistances are stable over different years, and act independently of one another in determining resistance. The effect of xa5 was larger than that of Xa7. Efficient ways to improve the resistance to Z-173 are discussed.     

Genetic Improvement of Rice for Bacterial Blight Resistance: Present Status and Future Prospects

The production and productivity of rice has been challenged due to biotic and abiotic factors. Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae, is one of the important biotic stress factors, which reduces rice production by 20%–50%. The deployment of host plant resistance is the most preferred strategy for management of BB disease, and breeding disease resistant varieties remains a very economical and effective option. However, it is difficult to develop rice varieties with durable broad-spectrum resistance against BB using conventional approaches alone. Modern biotechnological tools, particularly the deployment of molecular markers, have facilitated the cloning, characterization and introgression of BB resistance genes into elite varieties. At least 46 BB resistance genes have been identified and mapped from diverse sources till date. Among these, 11 genes have been cloned and characterized. Marker-assisted breeding remains the most efficient approach to improve BB resistance by introducing two or more resistance genes into target varieties. Among the identified genes, xa5, xa13 and Xa21 are being widely used in marker-assisted breeding and more than 70 rice varieties or hybrid rice parental lines have been improved for their BB resistance alone or in combination with genes/QTLs conferring tolerance to other stress. We review the developments related to identification and utilization of various resistance genes to develop BB resistant rice varieties through marker-assisted breeding.     

Improvement of Bacterial Blight Resistance in Rice Cultivars Jyothi and IR50 via Marker-Assisted Backcross Breeding

Abstract

In pathogen population analysis of 208 Xanthomonas oryzae pv. oryzae(Xoo) strains that were assembled from different parts of India, 21 pathotypes were identified on the basis of disease reactions on near-isogenic lines (NILs) and 13 pathotypes, on rice differentials. Rice cultivars, Jyothi and IR50, which are high yielding but highly prone to bacterial blight (BB) caused by pathogen populations of Xanthomonas oryzae pv. oryzae in India, were chosen. To improve the BB resistance of these two varieties, a pyramid line, NH56, containing four R-genes, Xa4, xa5, xa13, and Xa21, was selected as the R-donor based on resistance to existing pathogen population. The four R-genes were successfully transferred to cultivars through a traditional backcross method and their presence was documented with marker-aided selection (MAS). Thirty BC₄F₂ plants derived from JxNH56 (cv. Jyothi) and 45 BC₄F₂ plants derived from IR50xNH56 (cv. IR50) had all four resistance genes (Xa4, xa5, xa13, and Xa21), which should be useful resistance donors for breeding other BB-resistant elite indica varieties.     

四川主要水稻恢复系抗白叶枯病基因的分子检测

用分别与水稻抗白叶枯病基因Xa4、Xa7、Xa21和Xa23紧密连锁的PCR标记MP12、M3、pTA248和RM206对四川省新选育的以及主要应用的39份恢复系进行了分子检测，结果表明，这些恢复系均不含有Xa7（M3）和Xa21（pTA248）抗性等位点，17份携有榭（MP12）抗性等位点，少数（6个）携有Xa23（RM206）抗性等位点。在杂交水稻恢复系抗性改良中，应采用分子标记辅助选择（MAS）加快引入利用除工卅外的其它儿个显性抗性基因。     

Marker-Assisted Pyramiding of Genes Conferring Resistance Against Bacterial Blight and Blast Diseases into Indian Rice Variety MTU1010

Two major bacterial blight(BB) resistance genes(Xa21 and xa13) and a major gene for blast resistance(Pi54) were introgressed into an Indian rice variety MTU1010 through marker-assisted backcross breeding. Improved Samba Mahsuri(possessing Xa21 and xa13) and NLR145(possessing Pi54) were used as donor parents. Marker-assisted backcrossing was continued till BC2 generation wherein PCR based functional markers specific for the resistance genes were used for foreground selection and a set of parental polymorphic microsatellite markers were used for background selection at each stage of backcrossing. Selected BC2F1 plants from both crosses, having the highest recoveries of MTU1010 genome(90% and 92%, respectively), were intercrossed to obtain intercross F1(ICF1) plants, which were then selfed to generate 880 ICF2 plants possessing different combinations of the BB and blast resistance genes. Among the ICF2 plants, seven triple homozygous plants(xa13xa13Xa21Xa21Pi54Pi54) with recurrent parent genome recovery ranging from 82% to 92% were identified. All the seven ICF2 plants showed high resistance against the bacterial blight disease with a lesion lengths of only 0.53–2.28 cm, 1%–5% disease leaf areas and disease scoring values of ‘1' or ‘3'. The seven ICF2 plants were selfed to generate ICF3, which were then screened for blast resistance, and all were observed to be highly resistant to the diseases. Several ICF3 lines possessing high level of resistance against BB and blast, coupled with yield, grain quality and plant type on par with MTU1010 were identified and advanced for further selection and evaluation.     

Association Mapping and Marker Development of Genes for Starch Lysophospholipid Synthesis in Rice

Phospholipids are a major kind of lipids in rice grains and have fundamental nutritional and functional benefits to the plant. Their lyso forms(lysophospholipids, LPLs) often form inclusion complexes with amylose or independently influence the physicochemical and functional properties of rice starch. However, the genetic basis for LPL synthesis in rice endosperm is largely unknown. Here, we performed a preliminary association test of 13 LPL compositions among 20 rice accessions, and identified 22 putative main-effect quantitative trait loci responsible for all LPLs except for LPC14:0 and LPE14:0. Five derived cleaved amplified polymorphic sequences and one insertion/deletion marker for three LPL-synthesis-related candidate genes were developed. Association analysis revealed two markers significantly associated with starch LPL traits. These results provide an insight into the genetic basis of phospholipid biosynthesis in rice and may contribute to the rice quality breeding programs using functional markers.     

水稻品种五丰占2号的白叶枯病抗性遗传分析

研究了五丰占2号的白叶枯病抗性遗传及在回交世代中的抗性表现。结果表明,用白叶枯病菌株浙173（Ⅳ型）接种,五丰占2号表现中抗,IRBB5表现抗;五丰占2号的白叶枯病抗性受微效多基因控制,基因效应分析表明,该性状符合加性-显性模型,以加性效应为主;隐性主效基因xa5控制的IRBB5对白叶枯病菌株浙173的遗传符合隐性主基因的分离比。对白叶枯病菌株浙173的抗性反应与xa5基因的PCR检测结果一致。在五丰占2号2/IRBB5 B1F1群体中,基因型Xa5Xa5与Xa5xa5的分离比为1∶1;在五丰占2号2/蜀恢162 B1F1群体中,白叶枯病抗性达到五丰占2号水平的植株数占群体总数的68.3%。如果要将IRBB5中的xa5基因与五丰占2号的微效基因聚合,用五丰占2号回交1次是必要的。     

水稻白叶枯病菌小种分化的监测

通过每10年1次对病原菌致病力分化的监测发现,国内稻区病菌小种的组成基本稳定,仅个别小种有所变化。目前流行的优势小种仍为C1和C2群,即携带有[i]Xa3[/i]抗性基因的品种仍可广泛种植;但能够侵染[i]Xa4[/i]抗性基因的C4、C5群小种的数量有所增加,即将达到流行的阈值。为便于与国内外小种鉴别的结果相比较,选用IRRI推出的几个近等基因系材料作为鉴别品种,将中国的白叶枯病菌区分为8个小种(C1~C8)     

Identification of Major Locus Bph35 Resistance to Brown Planthopper in Rice

An introgression line RBPH660, derived from wild rice Oryza rufipogon, showed stable resistance to brown planthopper(BPH). Segregation analysis indicated BPH resistance of RBPH660 was controlled by multiple genes/QTLs. By using the bulked segregant analysis(BSA)-seq method, two genomic regions harboring QTLs resistance to BPH were identified from 1.20 to 16.70 Mb on chromosome 4 and from 10.20 to 12.60 Mb on chromosome 9 in RBPH660, respectively. A major resistance locus, designated as Bph35 accounting for 51.27% of the phenotypic variation with a LOD score of 42.51, was mapped to the candidate region of chromosome 4 between In Del(insertion-deletion) markers PSM16 and R4 M13. For fine mapping of Bph35, one simple sequence repeat and three newly developed In Del markers were used to screen the recombinants. Finally, the Bph35 locus was delimited in the region from 6.28 to 6.93 Mb and there were 18 predicted protein-encoding genes with a total of 114 non-synonymous single nucleotide polymorphism(SNP) variant sites between the resistant and susceptible parents. Out of these genes, Os04 g0193950, encoding a putative NB-ARC(nucleotidebinding adaptor shared by APAF-1, R proteins and CED-4) and LRR(leucine-rich repeat) domain protein with nine non-synonymous SNP substitutions in its coding sequence regions, might be the candidate gene for Bph35. These findings would facilitate the map-based cloning of the Bph35 gene and development of resistant varieties against BPH in rice.     

水稻抗白叶枯病基因Xa3/Xa26家族成员的表达模式分析

 水稻抗白叶枯病基因Xa3/Xa26是一个多基因家族的成员。为了检测该基因家族中是否可能存在其他抗病基因和这些成员可能发挥功能的部位,系统研究了Xa3/Xa26基因家族成员的表达模式。将籼稻品种明恢63、特青、93 11和粳稻品种日本晴中Xa3/Xa26基因家族的13个成员（MRKa、Xa3/Xa26、MRKc、TRKa、TRKb、TRKc、9RKa、9RKb、9RKe、NRKa2、NRKe、NRKf1和NRKf3）的启动子与编码绿色荧光蛋白（GFP）的报告基因进行融合表达,通过农杆菌介导的遗传转化方法将不同启动子调控的报告基因分别导入水稻。通过检测GFP表达,分析这些家族成员的组织特异性表达模式,发现Xa3/Xa26基因家族成员的表达模式基本一致,主要集中在水稻各组织的维管束细胞及其周围细胞中。Xa3/Xa26基因家族的表达模式与抗维管束病原菌（如白叶枯病菌）基因的功能非常吻合。这种表达模式为寄主进化过程中,从该家族不断产生新的抗病基因提供了重要信息。     

携有抗白叶枯病新基因Xa23水稻近等基因系的构建及应用

 1993～1998年,构建了携有抗稻白叶枯病新基因Xa23的近等基因系,命名为CBB23。以全生育期高度感病并具有改良株型的籼稻品种JG30为轮回亲本，与携有Xa23的抗性供体H-4杂交，JG30/H-4的F1通过回交、自交直至B5F4，各世代均用与目标基因Xa23相对应的专化小种菌系P6人工接种鉴定，同步进行株型和农艺性状选择，直至抗性稳定和农艺性状类似其轮回亲本。比较了CBB23 (携Xa23) 和IRBB21 (携Xa21) 对20个菌系包括10个菲律宾小种、3个日本小种和7个中国病原型代表菌系的抗谱，Xa23抗所有20 个菌系； Xa21 抗19个菌系，对菲律宾10号小种则高度感病。用近等基因系CBB23构建了JG30/CBB23组合的F2分离群体，通过SSR标记筛选，初步将Xa23定位于水稻第11染色体。进一步筛选出3个与Xa23更紧密连锁的AFLP标记。其中的APKj23与Xa23之间的图距约为1.0 cM。并报道了在抗病育种中已有效的应用了携有Xa23的近等基因系 CBB23。     

稻米淀粉理化特性相关的水稻淀粉分支酶基因标记开发

 基于籼稻93 11和粳稻日本晴基因组间的序列差异,成功发展了水稻淀粉分支酶基因(Sbe)9个分子标记。利用这些标记对102份非糯材料进行了基因型检测,并分析了Sbe1、Sbe3基因标记基因型多态性对稻米淀粉理化特性的影响。Sbe1、Sbe3基因标记多态性位点对揭示非糯材料间稻米淀粉理化特性的差异不显著,说明淀粉分支酶基因这些等位性变异位点对非糯稻米的蒸煮食味品质影响不显著;在鉴定籼稻品种时,有6个标记的鉴定准确率达80%以上。还对标记开发的意义和分子标记辅助选择育种进行了探讨。     

InDel和SNP标记在水稻图位克隆中的应用

在水稻功能基因的图位克隆中,常需要高密度的分子标记,而目前公布的各种标记的密度还远未达到令人满意的程度。InDel和SNP标记是新型的分子标记类型,基本可以满足精细定位目的基因的需要。InDel和SNP标记可以较容易地通过生物信息学的方法获得,而且经济实用、特异性高、稳定性好。介绍了设计InDel和SNP标记的方法,并以一个水稻卷叶基因的研究为实例,介绍了在基因精细定位研究中利用这两种标记的方法和提高标记设计成功率的注意点。     

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.     

利用基因组文库加速Xa23基因定位的染色体步移

 用距离水稻抗白叶枯病基因Xa23 0.8 cM的EST标记C189，扩增Xa23的近等基因系CBB23的基因组片段（0.8 kb）为探针,筛选水稻明恢63的TAC文库和广陆矮4号的PAC文库，对获得的7个阳性克隆用酶切法和Tail PCR法进行末端片段分离，获得15个末端片段， 用于感病亲本金刚30和抗病亲本CBB23之间的多态性检测，发现7个末端片段在双亲间有多态性，分别为69B、70N、81N、45B、45N、84N和84B。用这些末端片段作RFLP标记，对金刚30/CBB23的F2群体进行检测和连锁分析,结果表明这些标记与Xa23的遗传距离依次为0.4、0.4、0.4、0.5、0.6、0.6和1.1 cM。虽然69B、70N和81N与Xa23的遗传距离均为0.4 cM，但序列比对揭示69B与70N的物理距离为35 kb，与81N为95 kb，69B距Xa23最近。三者与Xa23的遗传距离虽然相同，但物理距离存在很大差异。这些末端片段标记加密了Xa23基因一侧的遗传图谱，并使其遗传距离缩短到0.4 cM，加速了Xa23的定位进程，为Xa23的分离克隆奠定了重要基础。讨论了这种染色体步移方法的适用条件。