甘蓝型油菜细胞核 细胞质雄性不育三系的研究与利用 Ⅳ.隐性细胞核 波里马细胞质雄性不育三系的创建

甘蓝型油菜隐性细胞核雄性不育与波里马细胞质雄性不育可能具有完全不同的不育基因系统;采用有性杂交和连续回交的方法,将甘蓝型油菜隐性细胞核不育基因导入到含有波里马不育细胞质的基因型中,并得到甘蓝型油莱隐性细胞核 波里马细胞质雄性不育系RGCMS—S45A和RGCMS—117A及其相应的保持系S45B和117B;采用测交方法,从隐性细胞核雄性不育系中筛选出甘蓝型油菜隐性细胞核 波里马细胞质雄性不育保持系9个;波里马细胞质雄性不育的恢复系均是隐性细胞核 波里马细胞质雄性不育的恢复系。     

Tournefortii male sterility system in Brassica napus. Identification, expression and genetic characterization of male fertility restorers

A germplasm collection of 152 diverse rapeseed accessions from Canada, China, France, India, Poland and South Korea was assayed for identifying new fertility restorers and sterility maintainers for a Tournefortii (tour) cytoplasmic male sterility (CMS) system in rape‐seed. Only 16 (10.5%) genotypes showed complete fertility restoration following hybridization with tour CMS line NE 409A. Notable among these were GSL 8851, GSL 8953, Mokpo # 9, Mali, Buk‐wuk‐13, Kuju‐27 and Mokpo # 84. As many as 78 (51.3%) genotypes were perfect maintainers of sterility, the remaining 58 (38.2%) genotypes were classified as partial maintainers. To study the inheritance of fertility restoration, 20 CMS (tour) rapeseed lines were crossed with the four best fertility restorers, namely GSL 8851, GSL 8953, Kuju‐27 and Mokpo # 9, to obtain F₂ and test cross populations. Segregation data indicated that fertility restoration for tour CMS was governed by two genes, of which, one is stronger than the other (χ²_12:3:1). Differences in gene interactions were also observed (χ²_9:3:4) which could be explained on the basis of influence of female parent genotypes/or modified expression of the restorer gene(s) in different genetic backgrounds. Tests of allelism indicated that the restorer genes present in the four restorers evaluated were allelic.     

Frequency and fertility restoration efficiency of Rf3 and Rf4 genes in Indian rice

Identification of new parental lines is crucial for developing ecology‐specific hybrids with ideal agronomic performance. We screened a total of 570 different ecology‐specific Indian rice varieties for the presence of fertility restorer genes, Rf3 and Rf4 using tightly linked markers DRRM Rf3‐10 and RM6100, respectively. Among these varieties, 13% carried Rf3Rf3/Rf4Rf4, 31% carried rf3rf3/rf4rf4, 6% carried Rf3Rf3/rf4rf4 and remaining 50% carried Rf4Rf4/rf3rf3 allelic combinations. A mini set of 40 varieties with variable allelic combinations of fertility restorer genes were testcrossed with WA and Kalinga‐based CMS lines. All the 80 F₁s were evaluated for spikelet fertility and fertility restoration ability. Rf3Rf3/rf4rf4 genotypes mostly behaved as partial maintainers or partial restorers. In contrast, rf3rf3/Rf4Rf4 genotypes were partial or effective restorers. However, double dominant genotypes showed better fertility restoration than the genotypes containing Rf3 or Rf4 individually. Some of the genotypes showed unexpected restoration pattern implying occurrence of other fertility restorer(s) apart from Rf3 and Rf4. The perfect restorers and maintainers identified in this study can be directly used in hybrid rice breeding.     

高粱新胞质雄性不育遗传机理及三系配套的研究

研究了A1至A7胞质不育与1000余种品种资源的育性反应，从而建立了A1与A7种胞质不育恢保体系的创新体系，研究了A1，A2，A3，A5，A6胞质不育育性恢复基因的遗传机制，找到了A1，A2，A5，A6胞质不育的育性恢复基因的差异和有许多共同恢复系和保持系的原因，研究了A3甩质不育杂交种F2分离比率，分析了A3不育系育性恢复基因的遗传方式。     

Agro-morphological characterization of ovary culture-derived plants of rice (Oryza sativa L.)

Plants derived from unpollinated ovary culture of ten rice genotypes showed significant variability in agro-morphological characteristics. The ovary-derived plant (H1) populations were completely haploid, doubled haploid or haploid-doubled haploid mixture. Haploids had very drastic reduction in plant height, panicle length, grain length, breadth and number and spikelet fertility (0.0%–2.1%). Doubled haploids from the hybrid of UPRI 95–121 × UPRI 95–165 were normal with fertility ranging between 69.6% and 97.7%. A genetic segregation in ratio of 1:1 was observed for five pigmentation characters in the H1 population derived from hybrid UPRI 95–122 × UPRI 95–165. Plant height showed the largest coefficient of variability (28.5%) followed by the number of spikelets per panicle (24.2%), number of grains per panicle (22.0%), percent seed set (9.2%) and panicle length (9.0%). The range of variation in the H1 population from fully fertile hybrid PMS 2A (CMS) × IR 31802 (restorer) was similar to its corresponding F2 population for plant height, spikelet fertility and number of grains/panicle. A single clone of plants from the cultivar BG 1321 exhibited complete male sterility but normal female fertility when pollinated with other varieties. Ovary-derived plants from the CMS lines PMS 2A and IR 58025A showed stable male sterility and those from thermosensitive genetic male sterile line UPRI 95–140 showed thermosensitive genetic male sterility. These lines have potential in the hybrid breeding program and are being currently exploited. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of the Rf gene conferring fertility restoration of the CMS Dian-type 1 in rice by using simple sequence repeat markers and advanced inbred lines of restorer and maintainer

Cytoplasmic male sterility of Dian‐type 1 (CMS‐D1) was developed 30 years ago in Yunnan. A major gene conferring fertility restoration for the CMS‐D1 system was detected by microsatellite markers in advanced inbred lines consisting of 196 maintainers and 62 restorers developed in breeding programmes of hybrid rice involving the CMS‐D1 system. The gene was mapped between two simple sequence repeat markers, OSR33 and RM228, on chromosome 10, and was temporarily designated as Rf‐D1(t). The genetic distances of the gene to the two microsatellite markers were 3.4 and 5.0 cM, respectively. This linkage was confirmed by using an F2 population derived from a cross between a CMS‐D1 line and a restorer. This study also demonstrated that using OSR33 was reliable and efficient for identification of restoring lines in hybrid rice breeding with the CMS‐D1 system.     

Cytoplasmic Systems in Different Male-Sterile Lines of Rice

In order to characterize the cytoplasmic system in seven cytoplasmic-genic male-sterile lines (CMS; A lines) of rice, viz., V 20A, ‘Zhenshan 97A’, IR 46831A, ‘Madhu A’ (cms-WA), ‘Yar-Ai-Zhao A’ (cms-Gam), ‘Pankhari 203A’ (cms-TN) and Wu 10A (cms-bo) and their isonuclear maintainers (B lines), all possible crosses were made between CMS lines and maintainers (A × B) as well as between the maintainers themselves (B × B). Based on F₁ pollen and spikelet fertility the CMS lines V 20A, ‘Zhenshan 97A’, IR 46831 A, ‘Madhu’ A possessing cms-WA cytoplasm were found to be genetically different from ‘Pankhari 203A’ (cms-TN), Wu 10A (cms- bo) and ‘Yar-Ai-Zhao A’ (cms-Gam) cytoplasms. Cms-bo and cms-TN cytoplasms appeared to be identical. Since the cytoplasms of the A lines are different from those of the B lines, the nuclear genes operating to cause the sterility might also be different in (A × B) and (B × B) crosses.     

Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.)

The main objective of the present study was to identify mitochondrial DNA based marker, which can distinguish male sterile and fertile counterparts of the cytoplasmic male sterile (CMS) lines used in production of rice hybrids. Amplified fragment length polymorphism (AFLP) analysis in CMS lines: IR58025A & IR62829A and their respective maintainers: IR58025B & IR62829B identified a polymorphic DNA fragment of about 510 bp size that was present in both CMS (A) and absent in their maintainer (B) lines. Sequencing followed by database analysis of the polymorphic fragment indicated about 97% similarity with mitochondrial NADH gene subunits of rice, maize and wheat. Based on the variable sequence regions, a site specific primer pair (BF-STS-401) was designed. PCR analysis showed that BF-STS-401 could amplify a strong band of 464 bp size in CMS and a faint band of the same size in maintainer line. To act as a positive control and avoid possible errors in PCR, BF-STS-401 was multiplexed with a new primer pair (BF-STS-402), derived from mitochondrial atp9 subunit of rice, producing monomorphic amplification indiscriminately in both CMS and maintainer lines. Both the primer pairs in combination clearly differentiated CMS lines from their corresponding maintainer lines. This primer combination was validated in a set of diverse genotypes consisting of different sources of CMS lines, restorer lines, hybrids, varieties and mixed samples from private seed companies. Our results suggested that the multiplex primer pairs developed in this study can be effectively utilized to assess the genetic purity in commercial seed lots of CMS lines and hybrids of rice.     

Identification and characterization of new sources of cytoplasmic male sterility in rice

Search for male sterility-inducing cytoplasm in wild species of the genus Oryza was attempted with a view to diversify the base of the cytoplasmic genetic male sterility system currently used in the development of commercial rice hybrids. A total of 132 interspecific crosses were made involving accessions of four wild and two cultivated species, all belonging to the A genome. Wild accessions possessing sterility-inducing cytoplasms were identified following reciprocal and sterile F₂ backcross methods. Sterile segregants were pursued through substitution backcrosses to develop cytoplasmic male-sterile (CMS) lines. CMS lines were developed with the cytoplasm of either O. rufipogon (VNI) or O. nivara (DRW 21018, DRW 21001, DRW 21039, DRW 21030 and RPW 21111). Based on shape, staining, and abortive pattern of pollen and also on type of interaction with a set of restorers and maintainers for known cytoplasmic male steriles of WA source (V 20A), the newly-developed CMS lines were grouped into four classes. Of these, RPMS 1 and RPMS 2 showed gametophytic male sterility with a restorer reaction different from WA cytoplasmic male-sterile stocks.     

水稻质核互作雄性不育系选育的反向杂交法研究

不同的部分保持系可能存在不同的微效恢复基因,通过有性杂交,产生基因重组,能够获得完全保持系类型,但只有在不育细胞质中才能观察得到微效恢复基因是否存在以及它们的作用大小.反向杂交法以不育细胞质为选择背景,在杂交后代植株中直接观察到微效恢复基因的表达,获得的完全不育株,在一定程度上排除微效恢复基因,不育株再通过高温处理转换为可育后自交,来自不育株的微效恢复基因可以进一步排除掉,从而产生出没有(或很少)微效恢复基因的"亲本",利用该"亲本"高温处理后仍可转换为可育的特性,作为父本进行杂交或回交育种,在其后代中获得没有微效恢复基因的完全保持系.该研究为Cp 26不育细胞质源创造出了完全保持系.如果在田间鉴定出优异的完全不育株,对其进行单倍体育种(诱导孤雌生殖或花培),选择到没有(或很少)微效恢复基因的纯合不育株.再对其进行花培,筛选可育的突变体;或者利用纯合不育株的原生质体与一个已破坏细胞核的可育系原生质体融合,都可能得到具有纯合不育株细胞核和可育细胞质的保持系,而能够完善和改进反向杂交法.反向杂交法不但能够为所谓不能保持的不育细胞质源创造出保持系,而且有利于加强新不育系选育的目标性和预见性,提高不育系配合力和培育不同类型优异不育系.     

Identification and Inheritance of Fertility Restorer Genes for 'tour' CMS in Rapeseed (Brassica napus L.)

Eighteen genotypes of Brassica napus were crossed to a cytoplasmic male sterile (CMS) line of B. napus BO 15 carrying B. tournefortii cytoplasm (‘tour’ cytoplasm). Fourteen genotypes were found to be stable maintainers of the ‘tour’ CMS. Of the remaining four genotypes, GSL-1 and ‘Asahi-natane’ were found to be heterozygous and ‘Mangun’ and ‘Yudal’ were homozygous for the restorer gene. Analysis of the F₁ and F₂ progenies of (CMS) BO 15 בMangun’ and (CMS) BO 15 בYudal’ showed that fertility restoration is controlled by a single dominant gene. The availability of a number of stable maintainer lines and the simple inheritance pattern of fertility restorer gene makes ‘tour’ CMS a useful system for hybrid seed production in rapeseed.     

几个水稻不育系对白叶枯病的抗性

测定了８对不育系（Ａ）和保持系（Ｂ）以及一些杂交稻组合对６个小种的４０株白叶枯病菌的抗性，比较了不同类型不育系对６个小种群的抗性差异。结果表明，冈型、印尼水田谷型和大多数野败型不育系的抗性很差，ＢＴ型的抗性较高。菌株Ａｈ２８对冈４６Ａ和野败珍汕９７Ａ的致病力强。比较了６个恢复系（Ｃ）和８个杂交组合对不同小种的抗性程度。结果显示，杂交稻的抗病性主要受父本（恢复系）核基因的控制，但同时又受不育胞质的影     

Fine mapping of <Emphasis Type="Italic">Rf3</Emphasis> and <Emphasis Type="Italic">Rf4</Emphasis> fertility restorer loci of WA-CMS of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) and validation of the developed marker system for identification of restorer lines

The Wild Abortive (WA) system is the major cytoplasmic male sterility (CMS) source for hybrid rice production in indica rice and its fertility restoration is reported to be controlled by two major loci viz. Rf3 on chromosome 1 and Rf4 on chromosome 10. With the availability of the rice genome sequence, an attempt was made to fine map, develop candidate gene based markers for Rf3 and Rf4 and validate the developed marker system in a set of known restorer lines. Using polymorphic markers developed from microsatellite markers and candidate gene based markers from Rf3 and Rf4 loci, local linkage maps were constructed in two mapping populations of ~1,500 F₂ progeny from KRH2 (IR58025A/KMR3R) and DRRH2 (IR68897A/DR714-1-2R) hybrids. QTLs and their interactions for fertility restoration in Rf3 and Rf4 loci were identified. The identified QTL in both mapping populations together explained 66–72 % of the phenotypic variance of the trait suggesting their utility in developing a marker system for identification of fertility restorers for WA-CMS. Sequence comparison of the two candidate genes from the Rf3 and Rf4 regions in male sterile (A) and restorer (R) lines showed 2–3 bp indels and a few substitutions in the Rf3 region and indels of 327 and 106 bp in the Rf4 region respectively. The marker system identified in the present study was validated in 212 restorers and 34 maintainers along with earlier reported markers for fertility restoration of WA-CMS. Together DRCG-RF4-14 and DRCG-RF4-8 for the Rf4 locus and DRRM-RF3-5/DRRM-RF3-10 for the Rf3 locus showed a maximum efficiency of 92 % for identification of restorers.     

Isolation and genetic characterization of a fertility-restoring revertant induced from cytoplasmic male sterile rice

Summary A male fertile revertant was isolated from M₁ of a cytoplasmic male sterile indica rice line II-32A, the dry seeds of which were treated with ⁶⁰Co- rays at a dose of 290 Gy. The acquired revertant T24 was morphologically and agronomically similar to II-32B, the maintainer of II-32A. Testcrosses of the revertant with II-32A and Zhenshan 97A showed that the revertant was able to restore the fertility of CMS lines. Genetic analysis of the progenies between T24 and II-32A, Zhenshan 97A XieqingzaoA and DZhenshan 97A, which have different cytoplasms, showed that the fertility restoration of four CMS lines by T24 involved one nuclear gene, indicating that T24 was a result of the mutation of one nuclear gene. The mechanism of the restoration of CMS line by T24 was obviously different from other restorers such as Minghui 63 and 20964, which were shown to behave in two gene mode in fertility restoration. The discovery of the revertant T24 contributes to the understanding of CMS and fertility restoration of CMS in rice. The T24 and its parent II-32A may constitute a pair of near isogenic lines for the restoring gene, which should be valuable materials for molecular genetic analysis of CMS.     

普通小麦雄性不育系及其保持系花粉形态的扫描电镜观察

利用扫描电镜观察了4种细胞质类型普通小麦(T. aestivum)雄性不育系及其保持系共16个材料的花粉形态,结果表明:不同细胞质类型雄性不育系之间的花粉粒形状及花粉粒表面纹饰有一定的差异,但花粉粒的大小、花粉粒萌发孔特征与不育细胞质类型无相关关系。不育系与其保持系在花粉粒形状、花粉粒表面纹饰方面也存在着明显的差异。     

Effects of cytoplasm on heterosis and combining ability for agronomic traits in rice (Oryza sativa L.)

Summary Effect of sterilizing (WA) cytoplasm on heterosis and combining ability for days to flowering, plant height and grain yield in rice was studied in 70 crosses and their reciprocals produced by 10 cytoplasmic male sterile (A), their maintainer (B) and seven restorer (R) lines following line x tester design. The materials consisting of 140 hybrids (70 A/R and their reciprocal 70 R/B) plus 17 parental lines (10 B + 7 R) were evaluated under six environments, created by growing in three fields with different fertilizer doses (0, 60, 120 kg N/ha) and 2 seasons (dry and wet) during 1986 at IRRI farm. Reciprocal cross effect (A/R vs R/B) were highly significant for all the three traits. Interaction of reciprocal cross effects with environments were also highly significant for yield and days to flowering. Cytoplasmic effect for yield, days to flowering and plant height were estimated by comparing A/R and R/B combination and testing the significance of difference with LSD value. In order to avoid confounding effect of spikelet sterility on yields, twenty crosses showing normal spikelet fertility were selected. Both positive and negative cytoplasmic effects were observed for the three traits. The effects were modified by environments, except for plant height. Heterosis for all three traits was also affected by cytoplasm, however, manifestation of cytoplasmic effects was higher for heterosis for days to flower than in heterosis for yield and plant height. Effect of cytoplasm was more pronounced on general combining ability effects of parents than specific combining ability effects of crosses. Among the parents two CMS lines A4 (IR54752A) and A8 (IR22107-113-3-3A) and two R lines: R2 (IR46) and R7 (IR9761-19-1) showed consistent positive effect of cytoplasm on general combining ability. These lines have given several good heterotic combinations. The study indicated the usefulness of evaluating diverse cytoplasmic sources in various nuclear genotypes bred for hybrid rice breeding program.     

粳稻三系亲本的性状特征与杂种优势分析

收集具有代表性的BT型粳稻不育系、保持系及恢复系各16个,共配制229个杂交粳稻组合,就三系亲本的性状特征、粳稻的杂种优势、不同性状亲子间的相关性进行了研究。结果表明：(1) 恢复系和保持系的潜在库容相近,但恢复系分蘖性较弱、结实率和千粒重较低,单株产量显著低于保持系。恢复系的垩白度显著高于保持系,直链淀粉含量极显著低于保持系。改良外观品质和提高结实率是今后恢复系选育的主要目标。(2) 杂交粳稻普遍存在较强的中亲优势,多数性状的超亲优势不强,但在生长势上则表现非常明显的竞争优势。杂交粳稻大穗优势突出,但结实率多表现负向优势,是目前制约杂交粳稻产量优势发挥的主要限制因素。(3) 杂种的多数性状由不育系和恢复系共同决定,杂种与中亲值的相关性最好。比较而言,抽穗期、穗总粒数等性状与不育系关系较为密切,株高、穗实粒数、结实率、品质性状与恢复系关系较为密切。     

Marker‐assisted introgression of bacterial blight and blast resistance into IR 58025B,an elite maintainer line of rice

IR 58025A is a very popular wild‐abortive cytoplasmic male sterile (WA‐CMS) line of rice and is extensively used for hybrid rice breeding. However, IR 58025A and many hybrids derived from it possess mild aroma (undesirable in some parts of India) and are highly susceptible to bacterial blight (BB) and blast diseases. To improve IR 58025A for BB and blast resistance, we have introgressed a major dominant gene conferring resistance against BB (i.e. Xa21) and blast (i.e. Pi54) into IR 58025B, the maintainer line of IR 58025A. An introgression line of Samba Mahsuri (i.e. SM2154) possessing Xa21 and Pi54 genes in homozygous condition and fine‐grain type was used as donor parent, and backcross breeding strategy was adopted for targeted introgression of the resistance genes. PCR‐based molecular markers tightly linked to Xa21 and Pi54 were used for selection of BB‐ and blast‐resistant lines, while closely linked markers were used for identification of backcross‐derived plants devoid of Rf4 and aroma. At BC₂F₅, four backcross‐derived lines possessing resistance against BB and blast, devoid of aroma, high yield, short plant stature, long‐slender grain type and with recurrent parent genome recovery ranging from 88.8% to 98.6% were selected and advanced for further evaluation. The improved versions of IR 58025B, viz. SB54‐11‐143‐9‐44‐5, SB54‐11‐143‐9‐44‐98, SB54‐11‐143‐9‐44‐111 and SB54‐11‐143‐9‐44‐171, behaved as perfect maintainers when test‐crossed with WA‐CMS lines. Agronomically superior lines of improved IR 58025B are being converted to CMS line through backcrossing for developing high‐yielding and biotic stress‐resistant rice hybrids.     

An improved cytoplasmic male sterile (Diplotaxis berthautii) Brassica juncea: identification of restorer and molecular characterization

We report the development of an improved cytoplasmic male sterile (CMS) system of Brassica juncea carrying cytoplasm of the wild species Diplotaxis berthautii. Flowers of the CMS line are smaller than the euplasmic line but have improved nectaries. Anthers are slender and fail to extend to the level of stigma. Female fertility of the CMS line is comparable to the euplasmic line. Fertility restorers of Moricandia arvensis and D. catholica-based alloplasmic CMS systems of B. juncea were found capable of restoring male fertility to this new CMS line. The fertility restoration is monogenic and gametophytic. Southern analysis showed that the cytoplasm of the CMS line is different from euplasmic B. juncea and other CMS systems restored by the same restorer lines. Northern analysis of the CMS, fertility restored and euplasmic lines using eight mitochondrial gene probes revealed altered atpA expression associated with male sterility. Cleaved amplified polymorphic sequence (CAPS) markers were identified for the plastid gene psbB, which could be useful for a quick identification of this CMS line. S.R. Bhat and P. Kumar contributed equally to this work.     

新型不育胞质在栽培稻中的分布与鉴定

为了分析只含有不育基因orf216在栽培稻中的分布,鉴定具有新的细胞质雄性不育胞质类型的种质资源。选取栽培稻824个品系为研究对象,以细胞质雄性不育相关序列HL-Sp1的特异引物进行PCR扩增,初步筛选得到9个候选品系,利用细胞质雄性不育基因orfH79,orf216的特异引物进行PCR扩增完成进一步筛选,并用Southern杂交验证。结果表明,最终筛选出IR72892,IR43,IR735463个只含有不育基因orf216不含orfH79的新型细胞质雄性不育胞质类型的品系。通过分子标记辅助选择,已成功的从824个栽培稻种中筛选到新的具有细胞质雄性不育胞质类型的新品系,为后续不育系的培育和相关理论研究奠定基础。