【作者】 王林生；

【导师】 陈佩度；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 赤霉病是温暖湿润麦区的一种灾害性病害,随着全球气候变暖和少、免耕栽培方式的扩大应用,赤霉病为害有扩展和加重趋势。培育和利用赤霉病抗性品种是一种经济有效的策略。在很长一段时期抗赤霉病育种使用的抗源局限于少数几个品种及其衍生系,遗传基础狭窄。大赖草(Leymus racemosus,2n=4x=28)是一种与小麦亲缘关系较远的多年生植物,具有耐盐、抗旱、抗多种病害等多种优良特性,尤其高抗小麦赤霉病,将其抗性基因导入普通小麦,对于拓宽小麦赤霉病抗性基础具有重要意义。南京农业大学细胞遗传所已获得了对赤霉病有较高抗性,分别具有大赖草Lr2(即7Lr)、Lr7、Lr14(即5Lr)染色体的异附加系。本研究在此基础上,以小麦-大赖草抗赤霉病异附加系为基础材料,采用60Co-γ射线分别辐射处于减数分裂期的大孢子母细胞和成熟花粉,创造更为丰富的抗赤霉病结构变异材料,尤其是小片段易位系,为小麦抗赤霉病育种提供新的种质资源。1、利用800rad剂量60Co-γ射线处理处于减数分裂期的小麦-大赖草二体异附加系DA5Lr植株,抽穗后去雄套袋,授予中国春的花粉。从79株M1中,得到6株易位,6株端体,染色体结构变异率15.2%。M1根尖细胞染色体GISH分析,鉴定出1株含1条5Lr染色体和1条外源小片段插入易位染色体的植株,让其自交,M2选育出了纯合的小片段插入易位系。顺次C-分带GISH表明,易位染色体可能涉及小麦A、D组染色体。进一步用A组专化探针BAC676D4和D组专化探针pAs1与其杂交,表明该染色体涉及A组染色体；再用5SrDNA探针与其杂交,发现易位染色体短臂上有明显的5SrDNA结合位点,结合小麦C分带标准带型,该易位染色体涉及的小麦染色体为1A,该易位命名为Ti1AS·1AL-5LrL-lAL。对该易位系花粉母细胞减数分裂期染色体进行GISH分析,中期Ⅰ染色体构型：17.47(?)W+0.90(?)Ti+2.53(?)W+0.10(?)Ti；杂合小片段插入易位减数分裂GISH表明,易位染色体TilAS·1AL-5LrL-lAL与小麦染色体1A能联会成稳定的二价体,且外源插入片段与着丝粒之间小麦非姊妹染色单体间可发生交换,观察到了交换的花粉母细胞(后期Ⅰ、末期Ⅰ和后期Ⅱ)和四分体类型。易位杂合体自交,后代可分离出纯合易位、杂合易位和正常小麦3种类型,其比例符合1:2:1。赤霉病抗性鉴定结果,具有外源插入易位染色体的植株,表现出较好的赤霉病抗性。本研究获得的WLS5-2和WLS6-3两个易位系,均由大赖草5Lr染色体长臂和大部分短臂及1小片段小麦染色体组成的易位类型(W?-5LrS·5LrL),但两易位系稳定性较好,结实率较高,对赤霉病抗性较好,可用来进一步创造小片段易位。对M1根尖细胞染色体C分带和GISH分析,得到1株涉及5Lr两个臂的2条易位染色体的植株。将其与二体附加系DA5Lr杂交,观察F1中具有1条5Lr和2条易位染色体植株花粉母细胞减数分裂行为,发现该F1植株双线期由2条易位染色体和1条5Lr及1条小麦染色体联会成‘十字形’结构,中期Ⅰ形成‘链形’或四体环结构,从而证明两易位染色体为相互易位染色体。C分带表明,易位涉及的小麦染色体可能为A、D组染色体,将其分别与pSc119.2和pAs1专化探针杂交,易位染色体中的小麦染色体片段上显现出较强的pAs1杂交信号,根据pAs1标准染色体分子核型,结合C分带结果,表明该小麦染色体为7D,相互易位染色体命名为7DS-5LrL和5LrS-7DL。配子传递分析表明,2条相互易位染色体常常一起传递,自交后代较难获得各自的纯合易位个体,需通过回交再自交才有可能获得赤霉病抗性较好的7DS-5LrL纯合易位系。本研究也得到了5Lr#1L端体异附加系。该端体异附加系遗传稳定性好,高抗赤霉病,可作为进一步利用的重要材料。2、研究发现在中国春背景下,大赖草5Lr、WLS6-3(W?-5LrS·5LrL)和WLS5-2(W?-5LrS·5LrL)易位染色体及端着丝粒染色体5Lr#1L表现出明显的花粉优先传递性。它们通过雄配子的传递率分别为88.4%、86.7%、70.0%和92.5%,显著高于其理论值50.0%,5Lr长臂上可能具有花粉优先传递基因。花粉育性鉴定结果,这种优先传递不同于杀配子染色体效应,属于一种新的花粉优先传递类型。3、利用1200rad60Co-γ射线辐射处理普通小麦-大赖草单体附加系MA7Lr的成熟花粉,授予已去雄的绵阳85-45,从57株M,中,得到9株端体,6株易位,结构变异率26.3%。对M1植株根尖细胞染色体顺次C-分带和GISH分析,鉴定出大赖草7Lr#1S端体植株,从其自交后代中选育出一对7Lr#1S端着丝粒染色体代换了1对7A染色体的端二体代换系。筛选出共显性EST-SSR分子标记-CINAU31.对该代换系花粉母细胞减数分裂期染色体进行分子原位杂交和染色体配对分析,MI的染色体构型为17.50(?)W+2.19(?)W+0.42(?)7Lr#1S+1.08 I7Lr#1S+0.69 IW,2条端着丝粒染色体在M I配成二价体的PMC占观察总数的59.7%。在后期Ⅰ和末期Ⅰ端着丝粒染色体7Lr#1S常出现特殊的染色体行为,可观察到2条端着丝粒染色体移向一极、端着丝粒染色体落后、端着丝粒染色体的姊妹染色单体提前分离等方式,从而产生了3种不同类型的四分体,且在一些四分体中有数目不等的微核出现。但由于端二体代换系产生的7Lr#1S雌雄配子有较高的传递率,保证了该端二体代换系较好的遗传稳定性,其自交后代中84%的植株为端二体代换。两年大田、温室赤霉病接种鉴定,对赤霉病表现出较高的抗性,表明该端着丝粒染色体携有赤霉病抗性的主效基因。因此,端二体代换系可作为外源基因定位、功能分析、端体细胞学行为研究的良好材料,同时也可作为进一步创造小片段易位的重要资源。经根尖细胞染色体C分带和GISH分析,从M2中选育出了一个涉及小麦4B染色体短臂和大赖草7Lr短臂的易位系,属顶端易位类型。易位断点在小麦4B染色体C带S1.6区,易位系命名为T4BL·4BS-7LrS。对M2植株进行根尖细胞顺次C-分带和GISH分析,还得到1株2条易位染色体均纯合的植株(2n=44)。C-分带结果显示,易位涉及的小麦染色体可能是A、D组染色体。其中1条涉及外源片段有较强的端带和FISH信号,为大赖草7Lr的短臂,而另1条外源片段较短,C-分带带纹不明显,且具有外源着丝粒,是1条具有较小7Lr片段的易位染色体。进一步用A、D组专化探针BAC-676D4和pAs1与其纯合易位根尖细胞染色体制片杂交,2条易位小麦染色体有较强的pAs1杂交信号,比较pAs1标准染色体分子核型,涉及的小麦易位染色体为2D,易位断点在小麦2D染色体C带L1.3区,两易位染色体命名为T2DS·2DL-7LrS和T2DL-7Lr。更多还原

【Abstract】 Scab is a disastrous disease for common wheat (Triticum aestivum) in warm and humid areas and exhibits a trend of rapid expansion and aggravation along with the global climate warming and introduction of less- or non-tillage. Development and utilization of resistant cultivars in wheat production has been considered as the most economical and effective strategy. However, the scab-resistance source of wheat in current breeding programs is particularly narrow since most of the resistant cultivars released in past decades originated from only a few varieties, e.g. Sumai 3 and Frontana. Leymus racemosus (2n=4x=28), a perennial and distant relative species of wheat, possess preferred characteristics for wheat improvement, such as salt and drought tolerance, diseases resistance. More recently, this species is becoming a new and potential source for wheat scab resistance. The introduction of new resistance genes from relative species to wheat to broaden the genetic base of breeding materials is essential for an effective and successful breeding program. T.aestivum-L. racemosus alien addition lines of Lr2 (7Lr), Lr7 and Lr14 (5Lr) with high resistance to scab were developed by Cytogenetics Institute, Nanjing Agricultural University. In present study, the microsporocytes and pollens of T. aestivum-L. racemosus alien addition lines were irradiated by 60Co-γray to produce various chromosome structural change materials with scab-resistance, especially small fragment translocation line, and provide new genetic resources for breeding of the scab-resistance wheat.1. Plants of the T. aestivum-L.racemosus addition line DA5Lr during meiosis were irradiated by60Co-y-rays 800R (l00R/min) and were emasculated and bagged after heading. The emasculated flowerets were pollinated by pollens of T. aestivum cv. Chinese spring after 2-3 days. Six T. aestivum- L.racemosus translocations and six telosomics were selected from seventy-nine M1 plants. The frequency of plant with structural aberrance of alien chromosome was 15.2%.In M1 progenies, a plant with a chromosome 5Lr and a small alien fragment intercalary translocation chromosome was identified by chromosome GISH at metphase of root tip cells. In its self-crossing progenies, the homozygous plant with small fragment intercalary translocation was selected. The sequential C-banding and GISH showed that the translocation chromosome might be involved in the chromosome of A or D genome of wheat. The wheat segment of translocation chromosome has only dispersible signals of BAC 676D4 when 676D4 specific to A genome and pAsl specific to D genome was used as probe in double color FISH, indicating that the translocation chromosome was involoved in chromosome of A genome. The obvious site of 5SrDNA on the short arm of the translocation chromosome was observed after FISH using 5SrDNA as probe. Combination the results of C-banding and in situ hybridization, this translocation chromosome was involved in 1A and was designated as TilAS-lAL-5LrL-lAL. Chromosome configuration of the homozygous translocation line at MI of PMCs after GISH was 17.47(?)w+0.90(?) Ti+2.53(?)w+0.10(?)Tu. In heterozygous plants, the intercalary translocation chromosome with small alien fragment paired with 1A as stable bivalent. Meanwhile, non-sister chromatids exchange occurred in the region between alien intercalary fragment and centromere was observed and could be reflected through the segregation of translocation chromosome exchanged or non-exchanged at anaphase I, telophase I and anaphaseⅡ, and tetrad types. The self-crossing progenies of translocation heterozygote produced three types of plants with homozygous, heterozygous translocation and no translocation chromosome respectively. Their proportion fits 1:2:1 statistically. The evaluation of scab resistance showed that plants with alien intercalary translocation chromosome had high resistance to wheat scab.Two translocation lines, WLS5-2 and WLS6-3, both involved in the long arm and partial short arm of 5Lr, one small fragment of unknown wheat chromosome (W?-5LrS-5LrL) were obtained and showed high genetic stability, seed set and scab resistance.One plant with two translocation chromosomes involved in two arms of 5Lr, respectively, was detected in M1 by C-banding and GISH analysis. The plant was crossed with disomic addition DA5Lr. There were one 5Lr and two translocation chromosomes in the F1 plants. The shape of cross and ring or chain quadrivalent were observed at diploma and MI respectively, indicating that two translocation chromosomes were reciprocal translocation chromosomes. The results of C-banding showed that translocation chromosomes were involved in the chromosome of A or D genome. After in situ hybridization using BAC 676D4 and pAsl as probe, only signals of pAsl on wheat segment of two translocation chromosomes were observed. According to the pattern of pAsl and C-banding of these chromosomes, it could be considered the translocation occurred between 5Lr and 7D. The two reciprocal translocation chromosomes were designated as T7DS-5LrL and T5LrS-7DL repectively. Gemate transmission analysis showed that two reciprocal translocation chromosomes were usually transferred together, and homozygous lines only involved in one translocation chromosome were difficultly obtained. The homozygous translocation line 7DS-5LrL with scab-resistance should be selected through backcross and then self cross.In addition, an alien telosomic addition line 5Lr#1L was also obtained. It had high genetic stability and scab-resistance, and is becoming a useful resource.2. The results of gamete transmission analysis showed that L.racemosus chromosome 5Lr, translocation chromosome TW-5LrS-5LrL(in WLS6-3) and TW-5LrS-5LrL (in WLS5-2), and telocentric chromosome 5Lr#1L appeared obviously pollens preferential transmission. Their transmission rate was 88.4%,86.7% and 92.5% through male gamete respectively, significantly higher than theoretical value of 50%, and suggesting a pollen preferential transmission gene on long arm of 5Lr. Because the pollens without 5Lr chromatin showed high fertility, the preferential transmission in these materials was different from the effect of gametocidal chromosome, and should belong to a new type of preferential transmission.3. The pollen of T. aestivum-L. racemosus monosomic addition line MA7Lr was treated by irradiation with 1200 rad 60Co-y-rays prior to pollinating to emasculated wheat cv. Mianyang85-45. Nine telosomics and six T.aestivum-L.racemosus translocations of fifty-seven progenies plants were obtained. The frequency of plant with structural aberrance of alien chromosome was 26.3%.Nine plants with a telocentric chromosome 7Lr#1S were identified by the sequential C-banding and GISH of root tip cells chromosome in M1 plants, and one ditelosomic substitution line 7Lr#1S was selected from self-crossing progenies and confirmed by chromosome C-banding and GISH. Furthermore, a co-dominant EST-SSR marker CINAU 31 was employed to identify this substitution line. A pair of chromosome 7A of common wheat were found to be replaced by a pair of telocentric chromosome 7Lr#1S, and further investigation showed that chromosome configuration of the substitution line at MI of PMCs after GISH was 17.50(?)W+2.19(?)W+O.42(?)7Lr#1S+1.08 I 7Lr#1s+0.69 I w. Two telocentric chromosomes paired as a bivalent in 59.7%of PMCs. Abnormal chromosome behaviors of telocentric chromosomes were observed in part of PMCs at anaphase I and telophase I, including the moving of two telocentric chromosomes to the same pole, lagging and earlier separation of their sister chromatid. All these abnormal behaviors can be grouped into three distinct types of tetrads according to different numbers of 7Lr#1S in their daughter cells and various micronucleus in some tetrads. However, due to the high transmission frequency of the female and male gametes with a 7Lr#1S,84% of the self-crossing progeny plants had ditelosomic substitution. The substitution line showed high resistance to wheat scab in a successive two-year test both in the greenhouse and field; hence, the line will be particularly valuable for alien gene mapping, small fragment translocation induction and telosomic cytological behavior analysis.An alien translocation line was selected from M2 progenies. The translocation occurred between chromosome 4B of wheat and 7LrS of L.racemosus. It belonged to a distal translocation by C-banding and GISH analysis. The breakpoint of translocation chromosome located in S1.6 region of chromosome 4BS of wheat, and was designated as T4BL-4BS-7LrS.One plant (2n=44) with two homozygous translocation chromosomes involved in 7Lr was detected in M2 by the sequential C-banding and GISH analysis. The results of C-banding showed that the translocation chromosomes were involved in the chromosome of A or D genome. After in situ hybridization using BAC 676D4 and pAsl as probe, only signals of pAs1 on wheat segment of two translocation chromosomes were observed. According to the pattern of pAsl and C-banding of these chromosomes, it could be considered the translocation occurred between 7Lr and 2D. The two translocations were designated as T2DS-2DL-7Lr and T2DL-7Lr repectively. The breakpoint of translocation chromosome located in L1.3 region of chromosome 2D of wheat更多还原