牡山羊草细胞质雄性不育小麦败育的生物学特性和细胞学研究

为明确牡山羊草细胞质雄性不育小麦的染色体组成及雄性败育特点,采用分子标记技术和酸性聚丙烯酰胺凝胶电泳技术对牡山羊草细胞质雄性不育小麦是否含有1BL/1RS易位染色体进行了鉴定。另外,为揭示牡山羊草细胞质雄性不育小麦败育发生的时期和败育的细胞学机理,以牡山羊草细胞质雄性不育小麦(Ju87B1-706A)和其同型保持系(706B)为供试材料,采用I2-K染色、醋酸洋红染色、DAPI染色和石蜡切片法对其花药形态特征及小孢子形成和发育过程进行细胞学观察。结果表明:Ju87B1-706A具有黑麦1RS的特异扩增条带(1 500bp),但缺少1BS的特异扩增条带(220bp),与其在A-PAGE结果的ω区与黑麦特异蛋白谱带的结果相对应,从而说明牡山羊草细胞质雄性不育小麦Ju87B1-706A为1BL/1RS雄性不育小麦类型;I2-K染色说明Ju87B1-706A的败育类型为典败和染败;Ju87B1-706A能正常进行减数分裂形成小孢子,到单核晚期时能形成正常的核但细胞皱缩,二核期细胞形态不规则能形成正常的精核但有些营养核不清晰,三核期精核呈圆形不能形成梭形的精核并发生部分空胞化;其单核晚期花药中绒毡层的延迟解离以及二核期三核期细胞团侵入药室造成小孢子败育,从而推测单核晚期是其雄性不育发生的关键时期,可能与绒毡层异常有关。     

黄淮南片麦区区试小麦品种(系)的遗传多样性及Pm21和1BL/1RS分子检测

为了解当前黄淮麦区区试品种(系)的遗传特征,利用分布于小麦基因组的96对SSR标记分析2016年参加黄淮南片麦区品种试验的78个小麦品种(系)的遗传多样性,同时,对Pm21基因和1BL/1RS易位系进行分子检测。结果表明,96对SSR引物中有80对(83.3%)在所有材料表现多态,共检测到307个等位变异,变幅为1~8,平均每个引物扩增3.84个等位变异;位点多态性信息含量(PIC)变幅为0.10~0.83,平均0.62;供试材料的遗传距离变幅为0.14~0.16,平均0.58。78个材料均不含有Pm21基因,69个(88.5%)材料属于1BL/1RS易位系。     

1BL/1RS小麦的分子和细胞学鉴定及黏类CMS育性恢复区域分布的分析

【目的】揭示黏类小麦细胞质雄性不育系育性恢复基因的区域分布及恢复材料的1BL/1RS情况,为黏类细胞质雄性不育系优良恢复源筛选提供理论依据,以推动黏类小麦雄性不育"三系"强优势组合的选配能不断得到新的材料保障。【方法】以8个黏类不育系和国内外一批小麦品种(系)为试材,结合分子和细胞学技术,进行1BL/1RS易位系鉴定,并利用中国国内法对其育性恢复程度进行分类。【结果】在参试的256份材料中,初步鉴定约20%的小麦品种(系)属于1BL/1RS易位系;育性表现半不育、高可育和全可育的品种(系)分别有86.15%、91.67%和100%为非1BL/1RS易位系,表现全不育和高不育的品种(系)中均有40%左右属于1BL/1RS易位系;恢复能力在50%以上的品种(系)在中国春麦区、长江中下游冬麦区、西南冬麦区和华南冬麦区的比例依次为60%、65.85%、68.42%和71.43%。【结论】黏类不育系优良恢复源大都为非1BL/1RS易位系,主要集中在中国春麦区和南方冬麦区。     

5种易恢复小麦雄性不育类型新保持系资源的建拓与研究

以10种不同山革草细胞质的异质小麦品种Chris为胞质供体,77(2)等一系列1B/1R易位系及两个核不育基因相同而胞质不同的正反杂交后代株(系)为核供体,采用杂交、置换回交和转育等方法,在单、偏、粘、易和二角型5种不育胞质源中,选育出易恢复性能极高的新保持系资源.测交和育性分析结果表明:(1)其不育系的F₁杂种恢复度均在90%以上.(2)单型不育性主要由一对主效隐性基因控制;偏、粘、易和二角型(1B/1R不育系)则是一种育性基因位点缺失,而仅由1RS片段与对应4种异质构成的互作不育.(3)单型不育系及F₁不产生单倍体;偏、粘、易和二角型1B/1R不育系产生单倍体是1B/1R易位染色体与4种异质专一互作后产生不育所带有的一种副效应;但从这4种异质中由新保持系资源培育出的无单倍体不育系可看出,单倍体性可随不育系核遗传背景不同而消失.此外,上述这5种异质不育系不但具有广泛的易恢复性,而且彼此间还存在着一定的互作关系,为简化制种程序提供了极方便的条件.     

Cloning of TaCYP707A 1 Gene that Encodes ABA 8′-Hydroxylase in Common Wheat（Triticum aestivum L.）

The plant hormone abscisic acid （ABA） regulates many important physiological and developmental processes in plants. The objective of this study was to clone the ABA 8′-hydroxylase gene in common wheat. In the present study, we used the eDNA sequence of barley HvCYP707A1 gene （GenBank accession no. AB239299） as a probe for BLAST search against the common wheat （Triticum aestivum L.） EST database in GenBank. All wheat ESTs sharing high similarity with the reference gene were subjected to contig assembly. Primers were designed based on the constructed contigs to clone the wheat CYP707A1 gene, designated as TaCYP707A1. The genomic DNA sequence of TaCYPTO7A1 gene comprised five exons and four introns, with a size of 2225 bp. The corresponding cDNA sequence of TaCYP707A1 was 1737 bp, containing an open reading frame （ORF） of 1431 bp, a 42-bp 5′-untranslated region （UTR） and a 264-bp 3′UTR, with 94.9% of identical sequences to HvCYP707A1 gene （AB239299）. The neighbor joining tree indicated that the deduced amino acid sequences of TaCYP707A1 gene was highly similar to those of barley and rice. The TaCYP707A1 gene was located on chromosome 6BL using a set of Chinese Spring nullisomic-tetrasomic lines and ditelosomic line 6BS. These results will be of high importance in understanding of molecular mechanism of ABA catabolism.     

几类异质非1BL/1RS小麦雄性不育系微效恢复基因累加效应的研究

为揭示非1BL/1RS小麦雄性不育系的恢复性遗传规律,特别是对恢复系的创制,以具有粘型、易型和偏型等细胞质的非1BL/1RS小麦雄性不育系,及对其有一定恢复力的小麦品种(系)132、83-3等为基础材料,以01-3-6为工具材料,采用特殊复合杂交法研究了这几类异质非1BL/1RS小麦雄性不育系微效恢复基因的累加效应。结果表明,以国际法表示时,不同核型材料中的粘型、偏型非1BL/1RS小麦雄性不育微效恢复基因均存在显著的累加效应,且在累加高代表现较为明显,以国内法表示时,则没有累加效应;不同核型材料对易型非1BL/1RS小麦雄性不育系育性恢复没有明显的规律性,以国内法计算的恢复度世代间变化幅度明显低于国际法;粘型、偏型非1BL/1RS小麦雄性不育系杂种F1产量的形成以小穗中部小花结实为主。     

小麦细胞质雄性不育系的分子细胞遗传学检测

利用 C-分带和 APAGE技术 ,对不同核型的具有粘果、易变、偏凸和二角山羊草细胞质的小麦雄性不育系进行了分子细胞遗传学标记检测。 APAGE分析发现 ,新不育系 5 - 1A和 7- 2 1A与 1BL/ 1RS易位型不育系77(2 )、82 2 2、偃师 9号、83(37) 6 5、80 (6 )同样均含有 1RS醇溶蛋白标记位点 Gld1B3。同时根尖染色体 C-分带显示5 - 1A和 7- 2 1A也含有 1RS端带。证明了 Gld1B3位点和 1RS端带可作为 1BL/ 1RS易位型不育系分子细胞遗传学标记。     

Structural Changes of 2V Chromosome of Haynaldia villosa Induced by Gametocidal Chromosome 3C of Aegilops triuncialis

Haynaldia villosa （2n=2X= 14, VV）, a relative of wheat, plays important roles in wheat improvement mainly owing to its disease resistance. Powdery mildew resistance gene Pm21 has been successfully transferred into wheat by Cytogenetic Institute, Nanjing Agricultural University, China, and is widely used in the current wheat breeding programs. In this research, our objective is to further transfer and utilize the beneficial genes such as eye-spot resistance, yellow rust resistance, and gene of the tufted bristles on the glume ridge （a remarkable morphology） mapped on 2V of Haynaldia villosa. A disomic addition line with gametocidal chromosome 3C ofAegilops triuncialis added in Norin-26 was crossed to the wheat-H, villosa disomic substitution 2V（2D） and the hybrid F1 was then self-crossed. Chromosome C-banding, genomic in situ hybridization （GISH）, and meiotic analysis in combination with molecular markers were applied to detect the chromosome variations derived from hybrids Fz and F3. To date, four translocations including one small segmental translocation T6BS·6BL-2VS, two whole arm translocations （preliminarily designed as T3DS·2VL and T2VS.7DL） and one intercalary translocation T2VS·2VL-W-2VL, one deletion Del. 2VS·2VL-, one monotelosomic Mt2VS, and one isochromosome 2VS·2VS line have been developed and characterized. One wheat SSR marker Xwmc25.120 tagging 2VS and one wheat STS marker NAU/STSBCD135-1 （2BL） tagging 2VL were successfully used to confirm the alien chromosome segments involved in the seven lines. The tufted bristles on the glume ridge appeared in lines T2VS-7DL, Mt2VS, 2VS-2VS as well as the parent DS2V（2D）, whereas in T3DS·2VL, this trait did not appear. The gene controlling the tufted bristles was located on 2VS. Gametocidal chromosome 3C ofAegilops triuncialis could successfully induce chromosome 2V structural changes.     

红麻线粒体基因NAD3和COB的RNA编辑与表达分析

以红麻细胞质线粒体近等基因系UG93A(不育系)、UG93B(保持系)及UG93A×福红992(恢复系)的F1代为材料,研究花药线粒体基因NAD3和COB的转录与表达。结果发现,在3种供试材料中,基因NAD3的CDS区在DNA水平和RNA编辑水平上均无差异;不育系与保持系的COB基因DNA编码序列无差异,但在RNA水平上发生了编辑,8个位点的编辑均发生于密码子的第一或第二位碱基,且导致氨基酸种类变化,主要为亲水性氨基酸转变为疏水性氨基酸;COB基因在UG93A中的RNA编辑频率低于UG93B;NAD3和COB基因在3种材料中的转录本大小基本相同,但在表达水平上表现为不育系显著低于保持系和F1代。由此推测NAD3和COB基因在红麻花药发育过程中有着重要的作用。     

普通小麦济麦21的LOX1基因cDNA片段克隆及分析

以普通小麦品种济麦21为材料,根据GenBank中已发表的LOX1基因序列设计引物,利用RTPCR技术获得一个小麦LOX1的基因片段,并对该片段进行测序和生物信息学分析。结果表明:克隆到的基因片段为384bp(GenBank登录号JX126806),GC含量为64.84%,推导氨基酸残基为127个,分子质量为11.15ku,等电点(pI)为4.93,与杜伦小麦、大麦、高粱、水稻的同源性分别为99.7%、94.8%、73.7%、56.2%。进化树分析表明,六倍体普通小麦LOX1基因与杜伦小麦LOX1基因(GenBank登录号HM126468)亲缘关系较近,先聚为一支,然后再与大麦的LOX1基因(GenBank登录号L35931.1)、高粱的LOX1基因(GenBank登录号GQ369443)聚为一类,最终与亲缘关系较远的水稻LOX1基因(GenBank登录号EU267789)聚类,这与物种亲缘关系的远近基本一致,表明所得LOX1基因片段可作为研究物种亲缘关系或遗传距离的参考标记之一。     

K型雄性不育小麦育性恢复基因的遗传特点及育性稳定性研究

利用2个K型小麦雄性不育系、21个恢复系及2个对照材料,组配杂交组合,经杂交、自交获得F1(AXR)和F2等世代材料,并考查其自交结实率,结合植物数量性状主基因+多基因混合遗传模型进行遗传分析,同时对部分组合的育性稳定性进行研究。结果表明:K型小麦雄性不育系的育性基因rf主要由雌配子传递,属配子体雄性不育类型;育性受2对加性-显性主基因+加性-显性多基因共同控制,且第1对主基因控制育性的作用强于第2对主基因;在F2群体中主基因的遗传率为62.44%,多基因遗传率为0,环境方差占表现型方差的37.56%,说明该类型小麦雄性不育性以主基因遗传为主,同时受多基因和环境的影响。育性稳定性研究表明,虽然K型细胞质雄性不育小麦的育性恢复年际间波动很大,但通过筛选可以获得恢复度高且稳定的恢复系,进而为杂交小麦的育种提供支持。     

新疆春小麦品种资源1BL/1RS易位和Bx7OE基因的分子检测

[目的]为新疆春小麦面筋品质改良提供有用材料和标记辅助选择的方法.[方法]选用4个STS标记对162份新疆春小麦品种资源1BL/1RS易位和Bx7亚基超量表达基因Bx7OE的分布进行检测,同时验证这4个标记的有效性.[结果]新疆春小麦品种资源中,1BL/1RS易位品种有24份,占14.8;,含有Bx7OE基因的品种有3份,占1.9;.在新疆春小麦地方品种、引进品种和自育成品种中,1BL/1RS易位和Bx7OE基因的分布频率也存在明显差异,其依次为7.5;、0;,24.4;、6.7;,14.1;和0;.[结论]新疆春小麦品种中1BL/1RS易位和Bx7OE基因的分布比例很低,这主要与新疆小麦育种中长期以来使用的亲本材料有直接关系.这四个特异性标记检测结果可靠稳定,可用于小麦品质育种中亲本评价和杂交后代优质基因聚合,可作为面筋强度辅助选择的有效工具.     

甘蓝型细胞质雄性不育杂交油菜亲本配合力与遗传力效应研究

选用甘蓝型油菜5个细胞质雄性不育系和5个优势恢复系,采用不完全双列杂交,配制25个杂交组合,对25个组合的10个主要数量性状进行了配合力和遗传力分析,结果表明,双亲的一般配合力效应依次对杂交F1代的一次有效分枝数、产量、株高、单株有效角果数和千粒重有明显影响;特殊配合力效应对二次有效分枝数、单株有效角果数和产量的表现有较大影响;5个不育系中A1的一般配合力效应值最高;5个恢复系中R4一般配合力效应值最高;产量特殊配合力好的组合是A5不育系×R1、A4不育系×R4.遗传力分析表明,产量、一次有效分枝数、单株有效角果数、株高和千粒重等的遗传力较高.     

The Selection of Transgenic Recipients from New Elite Wheat Cultivars and Study on Its Plant Regeneration System

In the protocol of wheat transformation, to use elite wheat cultivars as exogenous gene recipients can speed up the process of commercial field applications of transgenic wheat. However, it is necessary to screen wheat cultivars with good tissue culture response （TCR） continuously from plenty of elite wheat cultivars released for wheat transformation, and it is also important to find a plant regeneration system that is suitable for these cultivars. So, the TCR of mature and immature embryos of six wheat cultivars Chuannong 11 （CN11）, Chuannong12 （CN12）, Chuannong17 （CN17）, Chuannong18 （CN18）, Chuannong19 （CN19）, and Chuannong21 （CN21）, which possess superior agronomic traits, were investigated by using a good TCR wheat cultivar Bobwhite as control. The results indicated that only the immature and mature embryos of CN12, CN17, and CN18 exhibited good TCR compared with Bobwhite. No significant differences were observed between embryos of Bobwhite and of the three cultivars in TCR. Mature embryo-derived calli of CN12 were used as explants for transformation by particle bombardment of SAMDC gene. Seven transformants were obtained and the efficiency was 2.3%. This research supplies three new elite recipient cultivars for wheat transformation. The wheat plant regeneration system used in this research is different from those successful ones reported previously and it could be a reference for other wheat genotypes. Furthermore, Bobwhite and the three wheat cultivars were proved to be 1RS/1BL translocation, by methods of A-PAGE, C- banding, and genomic in situ hybridization （GISH）. These results imply that probably there is some relationship between 1RS/1BL translocation and TCR of wheat embryos. So this research gives us a hint that we should pay more attention to the 1RS/1BL translocations when we screen the wheat cultivars with good TCR and also that the mechanism of the effect of 1RS/ 1BL translocation on TCR is worthy of being investigated.     

Genetic analysis and SSR mapping of stem rust resistance gene from wheat mutant D51

Wheat (Triticum aestivum L.) stem rust caused by Puccinia graminis f. sp. tritici is one of the main diseases of wheat worldwide. Wheat mutant line D51, which forms a highly susceptive cultivar ‘L6239’ to the three races notated and cultured with immature embryos, shows resistance to prevailing races 21C3CPH, 21C3CKH, and 21C3CTR of P. graminis f. sp. tritici in China. In this study, the number and the expression stages of the resistance genes in mutant D51 were studied using inoculation identification and microsatellite (SSR) marker analysis. Two F₁ populations from the crosses of D51 × L6239 (60 individuals) and D51 × Chinese Spring (60 individuals), their F₂ populations (185 and 175 individuals respectively) at the seedling stage, and one F₂ population derived from the cross of D51 × L6239 (194 individuals) at the adult stage were inoculated with pathogen race 21C3CPH to test for resistance. All F₁ individuals of the two crosses were immune to stem rust at both seedling and adult stages. The response pattern of the three F₂ populations showed that the R:S segregation ratio was 3:1, suggesting that the stem rust resistance of D51 is controlled by a single dominant gene, and is expressed during the entire growth period. The identification of the stem rust resistance by the F₃ progeny test confirmed the credibility of the F₂ population test. Segregating populations and small population analyses were used to identify chromosomal regions and molecular markers linked to the gene by the SSR marker method. A total of 675 SSR markers and 185 individuals of the D516L6239 F2 population were used to search genetically linked markers to the target gene. Using Mapmaker 3.0 and Map-draw with Kosambi’s function and other options set at default values, molecular mapping revealed that the gene was located on chromosome 5DS, linked with and flanked by two SSR markers, Xgwm190 and Xwmc150, at 18.58 and 21.33 cM, respectively. It has been reported that only one stem rust resistant gene, Sr30, is located on the wheat chromosome 5DL, and that it has no resistance to 34C2MKK and 34C2MFK, while the parent L6239 of mutant D51 has no resistance to 21C3CPH, 21C3CTK and 21C3CTR, but has resistance to 34C2MKK and 34C2MFK. The results above indicate that the gene identified in the study might be a novel resistance gene to stem rust, tentatively designated as SrD51. __________ Translated from Acta Agronomica Sinica, 2007, 33(8): 1262–1266 [译自: 作物学报]     

小麦1BL/1RS易位系的生化和分子标记鉴定

本文利用A-PAGE方法对87份供试的小麦材料进行了黑麦碱(secalin)检测,并进一步利用低分子量麦谷蛋白Glu-B3的STS-PCR标记以及与抗条锈病基因Yr9紧密连锁的Xgwm582标记对上述材料进行了1BL/1RS易位检测。结果表明,在87份材料中鉴定出了一种新的改良1BL/1RS易位系,该类易位系的特征是:含有来自1RS的抗条锈病基因Yr9、不含黑麦碱、具有Glu-B3基因、比一般1BL/1RS易位系具有明显偏高的SDS-沉降值。本研究中选用的生化标记和分子标记可以有效检测出育种材料中的1BL/1RS、非1BL/1RS以及改良1BL/1RS易位系。     

Comparative analysis of genomes in Oryza sativa, O. officinalis and O. meyeriana with C 0 t-1 DNA and genomic DNA of cultivated rice

Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were applied to somatic chromosome preparations of Oryza sativa, O. officinalis and O. meyeriana with labeled probes of C ₀ t-1 DNA and genomic DNA from cultivated rice. The coverage percentage (%) and size (Mb) of C ₀ t-1 DNA in O. sativa, O. officinalis and O. meyeriana were 47.1 ± 0.16, 38.61 ± 0.13, 44.38 ± 0.13 and 212.33 ± 1.21, 269.42 ± 0.89, 532.56 ± 1.68, respectively. The coverage percentage and size of probe signals with genomic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0%, 93.6% and 634 Mb, 1 123 Mb respectively, in which there were 365 and 591 Mb in O. officinalis and O. meyeriana which came from O. sativa genomic DNA not from repetitive sequences of O. sativa, and the uncovered genome size in O. officinalis and O. meyeriana was 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of C ₀ t-1 DNA in O. sativa, O. officinalis and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during the evolution process. The repetitive sequence reduplication might be one of the important causes of genome enlargement in O. officinalis and O. meyeriana; the O. officinalis genome enlarged more slowly compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana formed by reduplication, rearrangement and gene selective loss during the evolution process. Translated from Scientia Agricultura Sinica, 2006, 39(6): 1083–1090 [译自: 中国农业科学]     

红麻雄性不育细胞质相关的cSNP位点发掘

在前期已获得红麻atp9基因编码区的基础上,以红麻细胞质雄性不育系P3A、保持系和F1代为材料,随机选取不同材料的3个atp9独立gDNA克隆子进行测序和序列比对。结果显示:具有不育细胞质的不育系和F1代与具有可育细胞质的保持系在第290位点处存在碱基差异,具有不育细胞质的材料均为碱基T,而具有可育细胞质的材料在相应位点为碱基A;atp9基因第290位点处T/A碱基转换的cSNP位点经已知细胞质的红麻种质资源的进一步检测,同时结合这些资源的田间育性进行分析发现,红麻种质资源UG93-2MS-1、UG93-2MS-2、UG93-2MS-3、UG93-2-22、KN250、KN142、ZB90在第290位点处为碱基T,能扩增出319bp条带,在田间对应的育性除品种UG93-2-22为可育外,其余均为不育或半不育;而福红992在第290位点处为碱基A,未能扩增出319bp条带,在田间对应的育性为可育。因此,位于atp9基因的第290位点处的T/A碱基转换的cSNP位点与红麻不育细胞质相关或连锁,为红麻不育细胞质鉴定提供了新的检测手段。     

提莫菲维小麦与光稃野燕麦易位系TGF314的顺序C-分带-基因组原位杂交鉴定

采用顺序C-带-基因组原位杂交（GISH）技术对提莫菲维小麦与光稃野燕麦远缘杂交后代中的外源遗传物质进行鉴定。GISH分析结果表明，在根尖细胞有丝分裂中期染色体具有2对清楚的杂交信号，说明该后代中可能含有稳定的外源遗传物质，且以易位的染色体片段形式存在。对该后代进行顺序C-带-GISH分析将杂交信号分别定位于提莫菲维小麦的7A和6G染色体短臂上，提莫菲维小麦的7A和6G染色体中含有光稃野燕麦的遗传物质。     

瑞华麦523PPO活性及1BL/1RS易位相关基因的分子检测

【目的】了解小麦品种瑞华麦523及其亲本郑麦9023和烟1604的PPO和1BL/1RS易位相关基因的遗传信息,旨在提高优质小麦品种选育的效率。【方法】利用小麦PPO活性基因的分子标记PPO18、PP0-B5、PPO16和PPO29及1BL/1RS易位的分子标记H20,检测小麦品种瑞华麦523及其亲本相关基因的等位变异及来源。【结果】小麦品种瑞华麦523在2AL染色体上PPO等位基因为PPO-A1a基因型,与亲本郑麦9023一样,为高活性PPO,不含亲本烟1604的低活性PPO;在2BL染色体上PPO等位基因为A型,与亲本一致,是高活性PPO;在2DL染色体上PPO等位基因为PPO-D1b基因型,与亲本郑麦9023一样,是一个高活性PPO,不含亲本烟1604的低活性PPO;利用分子标记H20检测表明,瑞华麦523和亲本郑麦9023一样,是一个非1BL/1RS易位系。【结论】瑞华麦523是一个具有高活性PPO、非1BL/1RS易位系的小麦品种,其小麦PPO和1BL/1RS易位相关基因主要来自母本郑麦9023,为该品种的推广应用和培育优质小麦新品种提供理论参考。