利用甲基磺酸乙酯(EMS)诱导小麦-华山新麦草染色体易位的研究

小麦(Triticum aestivum)种质24-3-1是通过染色体工程手段获得的一个高抗条锈病的小麦-华山新麦草二体异附加系。为了提高其遗传稳定性,促进华山新麦草(Psathyrostachys huashanica)优异基因的有效利用,对24-3-1进行甲基磺酸乙酯(ethylmethylsulfone,EMS)化学处理来诱导易位系。本研究利用细胞学和基因组原位杂交(genomic in situ hybridization,GISH)对其M2进行鉴定和易位系的筛选,并分析了不同EMS浓度对小麦-华山新麦草染色体易位的诱导效应。结果显示,在930个M2单株中共检测出了61个含有小麦-华山新麦草易位染色体的植株,易位频率为6.56%。其中7个单株检测出含有1条易位染色体,5个单株检测出含有1条易位染色体+1条华山新麦草染色体,20个单株检测出含有2条易位染色体,3个单株检测出含有3条易位染色体,26个单株检测出含有4条易位染色体。根尖细胞学观察和基因组原位杂交证明,含有2条易位染色体的单株为小麦-华山新麦草易位系;含有4条易位染色体的单株为小麦-华山新麦草易位-易位附加系。1.0%EMS为诱导小麦-华山新麦草染色体易位的最适浓度。本研究不仅为小麦特殊遗传材料的建立提供了重要的基因资源,同时也为小麦育种提供了新的种质。     

辐射诱导荆州黑麦染色体1R结构变异的研究

以60Co γ射线(12Gy)辐照普通小麦辉县红-荆州黑麦染色体1R二体添加系花粉,并授粉给辉县红,获得153粒辐射杂种M₁代种子。以Fluorescein-12-dUTP标记的荆州黑麦基因组DNA为探针,对其中33粒M₁代种子根尖细胞有丝分裂中期染色体进行GISH(genomic in situ hybridization)分析发现,23粒种子中的荆州黑麦1R染色体未发现明显变化,而另外10粒均发生了小麦和黑麦染色体易位,变异率为30.30%。电离辐射诱发产生的易位类型包括相互易位、大片段易位、小片段易位、整臂易位及端体等。这些易位染色体涉及1R染色体11个易位断点,其中位于长臂4个,短臂6个,位于着丝粒区1个,说明电离辐射可有效诱发目标染色体系列结构变异,为染色体缺失作图、重要性状基因定位和培育仅具目标基因的小片段易位提供了可能。     

分子标记和辐射技术在小麦-长穗偃麦草抗赤霉病易位系创建中的应用

为了打破小麦抗赤霉病育种种质资源狭窄的局限,丰富抗赤霉病遗传材料,本研究通过对中国春-长穗偃麦草二体异代换系DS7E/7A与扬麦16杂交后代进行辐射诱变,以产生小麦-长穗偃麦草易位系选择群体,并结合赤霉病抗性鉴定和长穗偃麦草染色体特异分子标记辅助选择,最终获得了5个小麦-长穗偃麦草抗赤霉病易位单株,创建了一条有效获得小麦-长穗偃麦草抗赤霉病易位系的技术路线。小麦-长穗偃麦草抗赤霉病易位系的成功创建不仅为小麦抗赤霉病育种提供了重要的中间材料,也将为小麦近缘种的抗赤霉病研究和利用提供理论与实践参考。     

小麦背景中披碱草部分染色体的FISH鉴定

为了建立小麦背景中粗穗披碱草(Elymus trachycaulus)和纤毛披碱草(Elymus ciliaris)染色体的跟踪鉴定方法,本研究利用寡聚核苷酸Oligo-pSc119.2-1和Oligo-pTa-535-1为探针的双色荧光原位杂交(FISH)和以(GAA)₈为探针的单色FISH,分别对4个小麦-粗穗披碱草附加系和5个小麦-纤毛披碱草附加系染色体进行分析。结果发现,经与小麦FISH核型比对后,建立了可用于追踪小麦背景中粗穗披碱草1S^t、5H^t、6H^t和7H^t染色体的标准FISH核型;而纤毛披碱草S^c和Y^c染色体FISH信号较弱。FISH检测发现,小麦-粗穗披碱草1S^t附加系自交自发变成了1S^t(1BS·3BL)代换系,且在其染色体组中检测到了一对T1BL·3BS易位染色体,同时发现5AS端部寡聚核苷酸Oligo-pSc119.2-1序列发生了删除。另外,在小麦-粗穗披碱草5H^t附加系中发现2B染色体短臂末端删除现象,形成了2B-del和2BS-4AS·4AL易位染色体,而另一条2B和4A为正常的完整染色体。这种染色体结构重排事件表明,部分小麦-远缘物种附加系细胞学并不稳定,因此,繁种前后应对材料进行单株细胞学鉴定。上述小麦-粗穗披碱草染色体结构变异体的获得,为研究染色体结构重排与基因转录表达和表型变化的关系提供了研究基础。     

小麦-黑麦1BL/1RS易位系7-1抗纹枯病的分子细胞学鉴定

黑麦(Secale cereale)是小麦(Triticum aestivum)的重要三级基因源,具有小麦改良所需要的多种优良性状。为丰富小麦杂交育种新的种质资源,利用墨西哥黑麦(2n=2x=14,RR)与普通优质小麦W770B(2n=6x=42,AABBDD)杂交,经过多年筛选,选出若干优良性状的衍生系,在小麦五叶期时用PDA培养基培养的带纹枯病菌的牙签,接菌到小麦芽鞘内,温室内培养,5周后鉴定纹枯病发病等级,计算病情指数,为了结果的准确性,经过多次牙签法鉴定,筛选出病情指数为PI=32.8%的抗病衍生系7-1。为明确衍生系7-1的遗传成分,本研究综合采用形态学、细胞遗传学、基因组原位杂交(genomic in situ hybridization,GISH)、特异序列扩增(sequence characterized amplified region,SCAR)分子标记、简单重复序列(simple sequence repeat,SSR)分子标记和醇溶蛋白分析。对7-1的根尖细胞和花粉母细胞的细胞遗传学观察表明7-1染色体结构和数目稳定2n=42=21Ⅸ;以黑麦DNA为探针的GISH分析观察表明7-1含有两个黑麦染色体臂;黑麦基因组SCAR标记分析表明,D15和P13LF/R能够在黑麦和7-1中扩增出黑麦特异条带,黑麦1RS SCAR标记分析表明,ω-sec-p1/ω-sec-p2、ω-sec-p3/ω-sec-p4和IB-267能够在黑麦和7-1中扩增出黑麦目的条带,说明7-1具有黑麦1RS染色体;筛选小麦每条染色体长短臂上的多对引物,在7-1中只有1BS上的引物Xgwm264和Xgwm11未能扩增出相应条带,其余染色体上的引物均扩增出了条带,说明7-1中缺失了小麦1BS染色体;醇溶蛋白分析表明7-1中扩增出了1RS黑麦碱条带,由此证实了小麦染色体1BS被黑麦染色体1RS所替换,该材料为小麦-黑麦1BL/1RS易位系。本研究中7-1为抗纹枯病的1BL/1RS易位系,为小麦纹枯病抗病育种提供了新的种质资源,拓宽了小麦育种材料。     

小麦-顶芒山羊草2M染色体系的鉴定与评价

为了研究小麦-顶芒山羊草新种质材料的染色体组构成及开发其在育种中的利用价值,本研究利用分子标记和荧光原位杂交对小麦-顶芒山羊草杂交新种质进行鉴定,并通过小麦主要病害生理小种接种和农艺性状调查等方法对鉴定的材料进行综合评价。分子标记和原位杂交结果显示,所鉴定材料分别为小麦-顶芒山羊草2M附加系、2M(2D)代换系、2AS-2ML.2MS易位系和2DS-2ML.2MS易位系;抗病性鉴定结果表明,含2M染色体的材料均高抗小麦条锈病,其小麦亲本则高感条锈病,表明2M染色体上可能含有抗条锈病新基因;农艺性状调查分析结果表明,2M染色质导入小麦,可影响其小穗数、穗粒数和穗粒重等产量性状。因此,在创制和利用抗条锈病的小麦-顶芒山羊草2M染色体小片段易位系时,应加强对上述农艺性状的考察,并利用当前主栽品种进行回交改良。本研究鉴定出的抗条锈病小麦-顶芒山羊草2M染色体系丰富了小麦抗病基因库,为小麦育种提供了新抗源。     

小麦-中间偃麦草抗条锈病渗入系的分子细胞学鉴定

小麦品系CH5383是源于中间偃麦草(Thinopyrum intermedium)的兼抗多种小麦病害的新种质。为明确其抗性来源和外源DNA片段的渗入位置,采用基因组原位杂交(GISH)和荧光原位杂交(FISH)对CH5383进行分析,GISH分析未发现外源信号,FISH结果观察到CH5383的3BL染色体端部与对照小麦(Triticum aestivum)品种中国春相比有明显的重组信号,初步推断CH5383染色体3BL端部可能有中间偃麦草DNA片段插入。用135对PLUG(PCR-based landmark unique gene)标记对CH5383、中国春和多个近缘物种进行扩增分析,发现位于3B染色体长臂端部的引物TNAC1383,能在CH5383和中间偃麦草中扩增出大约1 300 bp的特异DNA产物。从而进一步证实,CH5383是一个小麦-中间偃麦草小片段渗入系,携带抗条锈病基因的外源中间偃麦草DNA渗入片段位于3B染色体端部0.81-1.00区段。CH5383可以作为优异的小麦抗病育种新种质加以利用。     

小麦远缘杂交种质资源创新

小麦近缘种是改良小麦的一个重要基因库, 具有许多栽培小麦所不具备的优良特性。我们通过远缘杂交、染色体工程的方法创制了一大批不同类型的材料, 经基因组原位杂交GISH、多色FISH 和特异分子标记鉴定, 抗条锈病、白粉病、叶锈病鉴定, 品质、营养性状以及产量性状鉴定, 共选育出10 类可为育种家利用的抗病、优质、富含微量营养元素、氮高效、丰产性状优良的远缘杂交新种质和新不育系种质; 开发了414对黑麦基因组专化的EST 引物, 31 个黑麦染色体(臂)专化的EST 分子标记, 可应用于分子标记辅助育种, 或追踪检测小麦背景中的黑麦染色体或染色体片段; 进行了抗病新基因的遗传分析和分子标记定位工作。利用新种质, 选育出了一批表现突出的抗病、营养高效的小麦-黑麦、小麦-冰草远缘杂交新品系     

抗条锈病的小麦-非洲黑麦异代换系的分子细胞学鉴定

黑麦属(Secale)物种是改良小麦条锈病抗性的优良供体,为发掘和利用黑麦属野生种非洲黑麦(S.africanum)所携带的优异抗小麦条锈病基因,本研究在硬粒小麦(Triticum durum)-非洲黑麦双二倍体(基因组为AABBRaRa)和小麦(T.aestivum)杂交的高代材料中发现了一个免疫条锈病的株系HH41.HH41的体细胞染色体数目为2n=42.用小麦D基因组特异重复序列pAsl和秦岭黑麦(S.cereale)基因组总DNA作为探针的顺序原位杂交分析表明,HH41中一对小麦6D染色体被一对非洲黑麦6Ra染色体所代换.利用开发的基于表达序列标签的6R/6Ra特异分子标记也证实了HH41缺少6D特征带,具有6Ra特征带,是6Ra(6D)代换系.条锈菌生理小种(Puccinia striiformis Eriks.f sp.tritici)接种鉴定结果表明其抗条锈病性源自6Ra染色体.本研究还综合利用分子细胞学证据将来自非洲黑麦的6Ra染色体与栽培黑麦的6R染色体的多态性进行了比较,证实了6R(6D)代换系HH41是一种具有古老野生黑麦优异抗性的特殊珍贵材料,是创造异易位系、实现外源基因转移和改良小麦的重要资源.     

矮秆早熟小偃麦种质山农0057-2的选育与鉴定

利用普通小麦(Triticum aestivum)品种烟农15与中间偃麦草(Thinopyrum intermedium)杂交、回交和多代自交,从BC3F6中选育出一个矮秆、早熟新种质山农0057-2.综合应用形态学、细胞学、生物化学、分子生物学等方法对其进行鉴定.结果表明,山农0057-2平均株高64.2 cm,较烟农15降低14.6 cm;抽穗期和开花期均比烟农15提前4～5 d.其根尖染色体数目为2n=42,花粉母细胞减数分裂中期Ⅰ(PMC M Ⅰ)染色体构型为2n=0.24 Ⅰ+20.88 Ⅱ;它与烟农15的杂种F1在PMC M Ⅰ有80.49%的细胞形成21个二价体,未观察到多价体.高分子量麦谷蛋白亚基的SDS-PAGE鉴定结果表明,山农0057-2含有1条中间偃麦草的特异带;从202对SSR引物中筛选出2对引物(Xgwm234-5A/5BS和BARC172-7DL)能在山农0057-2中扩增出不同的中间偃麦草特异带,分别标记为Xgwm234300和BARC172400.山农0057-2是一个含有中间偃麦草染色体(片段)的种质材料,具有矮秆、早熟等优点,在小麦育种中具有重要的利用价值.     

含抗白粉病新基因普通小麦-黑麦1R二体异附加系的遗传学鉴定

黑麦（Secale cereale）含有丰富的优良基因,在小麦遗传改良中具有重要利用价值。为了鉴定普通小麦（Triticum aestivum）与奥地利黑麦杂交后代选育的抗白粉病品系N9436-1的黑麦遗传物质,对其进行了细胞学、基因组原位杂交、Giemsa-C分带、SCAR（sequence characterized amplified region）标记以及酸性聚丙烯酰胺凝胶电泳(A-PAGE)分析。结果表明,N9436-1形态学和细胞学稳定,2n=44=22Ⅱ,对白粉病免疫,携带奥地利黑麦的多小穗性状。以奥地利黑麦总基因组DNA为探针的原位杂交结果及Giemsa C-分带显示,N9436-1含有2条奥地利黑麦的1R染色体, SCAR标记鉴定及A-PAGE分析进一步证实N9436-1携带有黑麦遗传物质,表明N9436-1携带的抗白粉病基因不同于Pm8和Pm17,是新的抗白粉病基因,可作为白粉病抗源用于小麦抗病育种。     

Morphological and molecular cytogenetic characterization of partial octoploid Tritileymus

This study used cytological observation, genomic in situ hybridization (GISH), and seed storage protein electrophoresis to characterize a partial octoploid Tritileymus M842-1 derived from Leymus mollis (NsNsXmXm, 2n = 28) and common wheat cv. 7182 (Triticum aestivum L.), and its relationship with alien chromosomes and those from wheat. Mitotic observations showed that the chromosome configuration of M842-1 was 2n = 56 = 28II. Mitotic and meiotic GISH using two different probes for L. mollis and Psathyrostachys huashanica indicated that M842-1 contained 14 Ns genome chromosomes from L. mollis, which formed seven pairs of uniform bivalents, and confirmed the genomic composition as AABBDDNsNs. The separation of seed storage protein by polyacrylamide gel electrophoresis indicated that M842-1 expressed some L. mollis-specific glutenin and gliadin bands. The seeds of M842-1 were as large as those of wheat cultivars, while they also had a long spike and were covered with wax on the leaves and stems. Disease screening demonstrated that M842-1 was immune to stripe rust, highly resistant to powdery mildew, and moderately susceptible to wheat scab during the adult plant stage. This study demonstrated that the partial octoploid Tritileymus M842-1 could serve as a donor source in wheat breeding programs for the introduction of novel variation to promote high yield and disease resistance.     

Characterization of a new T2DS.2DL-?R translocation triticale ZH-1 with multiple resistances to diseases

In the present study, we report on a new triticale (×Triticosecale Wittm.) ZH-1, derived from the progeny of common wheat (Triticum aestivum L.) cv. MY15?×?Chinese Weining rye (Secale cereale L.). ZH-1, exhibiting shorter plant height and higher tillering ability compared to MY15, is immune to both powdery mildew and stripe rust and has stable fertility. Genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and C-banding revealed that the chromosome composition of triticale ZH-1 was 14 A-genome (1A-7A), 12 B-genome (1B-2B, 4B-7B), 12 R-genome (1R, 3R-7R), chromosomes 6D and T2DS.2DL-?R. Moreover, the PCR results of PLUG and EST-SSR markers also strongly supported the above stated chromosome composition of triticale ZH-1. In addition, the physical mapping of chromosome T2DS.2DL-?R showed a minute chromosomal fragment derived from rye was attached at the distal end of 2DL. The new triticale ZH-1 could be a valuable source for wheat improvement, especially for resistance to disease.     

Molecular cytogenetic characterization of the amphiploid between bread wheat and Psathyrostachys huashanica

A new wheat-Psathyrostachys huashanica amphiploid, PHW-SA, was characterized using molecular cytological tools to evaluate the potential utilization of P. huashanica for wheat improvement. PHW-SA pollen mother cells (PMCs) regularly revealed averagely 0.57 univalents, 24.51 ring bivalents, 3.19 rod bivalents and 0.01 trivalents per cell. Complete homologous chromosome pairing was seen in 81% of the PMCs, indicating a degree of cytological stability. P. huashanica C-banding karyotypes of PHW-SA displayed strong heterochromatin terminal bands at either or both ends. 24 P. huashanica chromosome arms showed telomeric bands. Using P. huashanica DNA as a probe and J-11 DNA as blocker, distinctive P. huashanica chromosomes, which were presented entirely greenish-yellow hybridization signals, were observed in a somatic metaphase of PHW-SA. GISH also revealed the chromosomal breakage and translocation occurring in the amphiploid PHW-SA, which may represent a centromeric fusion between P. huashanica and wheat chromosomes. Seeds storage proteins electrophoresis indicated that PHW-SA expressed some of P. huashanica specific gliadin and glutenin bands, and a few gliadin and glutenin bands of the parents disappeared and new bands appeared. The results indicated that the amphiploid PHW-SA could serve as novel germplasm sources for wheat improvement.     

辐射选育小麦易位系的研究

辐射处理六倍体小黑麦黑杂266×普通小麦克79F3-392的F0种子,通过一定的选育程序,选出了龙辐麦4号,龙辐10946和龙辐10877 3个易位系,经Giemsa-C带技术鉴定,其易位形式分别为6BS/6RL,2AS/2RL和7RL/7AL。酯酶同工酶和过氧化物同工酶的分析表明,3个易位系和黑杂266具有共同的特征酶带。试验证明,3个易位系具备双亲的优良特征特性,其中龙辐麦4号已由黑龙江省品种审定委员会审定推广种植。     

辐照甘薯切苗选育新品系

用~(60)Coγ射线辐照甘薯切苗,获得了抗黑斑病的突变系。经多点试验、区域试验和生产试验,突变无性系农大601(12-11-8)的春薯鲜产量和干物质含量均比原品种有所提高,且具有较强的抗黑斑病性能。采用聚丙烯酰胺电泳技术进行酯酶同工酶分析表明,农大601和亲本品种间存在一定差异。根尖细胞观察结果表明:辐照当代有明显的染色体畸变,但到第6无性代时,已和对照一样。因此说明变异不是染色体畸变造成,而是基因突变。     

A reevaluation of karyotype in cucumber (Cucumis sativus L.)

A karyotype analysis was conducted on the mitotic chromosomes of cucumber (Cucumis sativus L.; 2n = 2 × = 14), using improved chromosome preparation and C-banding techniques. Chromosome pairs were characterized on the basis of length and arm ratio value. The length of metaphase chromosomes ranged from 1.48 to 2.31 m, and the total length of the haploid chromosome complement was 13.06 m. The karyotype of this species was symmetric, and consisted of six metacentric and one submetacentric chromosomes. All chromosomes were defined by their unique C-banding patterns. The C-banded karyotype included centromeric, telomeric, and intercalary bands depending on chromosome pair leading to a precise identification of each pair. The C-banding patterns were distinctly different from observations made in previous study.