【作者】 戴剑；

【导师】 洪德林；

【作者基本信息】 南京农业大学 ， 种子科学与技术， 2011， 博士

【摘要】 种子的真实性和纯度是种子质量检验的最主要的质量指标之一。植物新品种特异性(Distinctness)、一致性(Uniformity)和稳定性(Stability)的测试(简称DUS测试)是农业部植物新品种保护办公室对申请品种授予新品种权进行实质审查最重要的工作内容。水稻是我国主要的粮食作物,杂交稻具有杂种优势,产量高,抗性好,其种植面积已占我国水稻面积的一半。我国是世界上最大杂交水稻种子生产国和消费国,申请水稻新品种保护的品种数量在逐年增加,是我国目前品种权申请保护的主要对象之一。然而,目前种子的真实性和纯度检测和植物新品种DUS测试均是田间种植方法鉴定。该方法周期长,费工占地,用于测试的标记性状有限,不能满足种子市场销售,新品种及时授权的需要,研究快速、简便、准确的DNA分子标记技术鉴定水稻品种对于杂交水稻种子质量检测和新品种授权具有重要意义。为运用SSR分子标记快速、准确鉴定江苏省地区范围内生产上的杂交水稻种子的真实性和纯度,需要考虑以下几个重点问题：快速、高效的DNA提取方法是分子标记鉴定品种的颈瓶技术,传统的DNA提取方法技术难度大、操作步骤繁琐,是分子标记品种鉴定技术不能普及的主要问题之一；两系杂交稻母本的不育性易受光照时间和温度的影响,两系杂交稻会因不同年份或地区光、温条件的变化导致母本自交结实而严重影响种子纯度,但在海南的种植鉴定中,不育系有时表现可育,给形态鉴定带来一定的困难；长江中下游地区是籼、粳杂交稻和常规稻均有种植的地区,在该地区的杂交稻制种田中经常出现籼稻不育系与粳稻父本或粳稻不育系与籼稻父本杂交的一种籼粳亚种杂交组合“大青棵”,这种杂株植株高大,抽穗期晚,杂交稻中大青棵是严重影响企业形象和产量的杂株类型,但海南种植鉴定时,大青棵受光温影响不一定表现植株高大、抽穗期晚等特征,因此难以准确鉴别。为此,本论文拟从以下4个方面开展研究：一是改进DNA提取方法和步骤,研究快速、高效,并获得高质量的DNA提取方法。二是对生产上推广的4个两系杂交稻和1杂交粳稻的7亲本进行多态性分析,并应用SSR多态性标记鉴定两系杂交组合。三是建立近几年来生产上常用的33份籼粳杂交稻亲本DNA指纹数据库,并对这些亲本进行遗传相似性分析,为杂交稻种子纯度鉴定提供依据。四是选用了该地区的常用籼粳不育系与粳籼父本配制了55个籼粳亚种间杂交组合,考察了F1代株高、抽穗期和结实率等主要农艺性状,筛选用于鉴定本试验所配制的籼粳杂交大青棵组合类型杂株的SSR分子标记,为该地区杂交稻中可能出现的大青棵的鉴定提供了依据。获得的主要结果如下：1.改进了DNA提取方法。本研究应用了常规方法的DNA提取方法,同时也研究应用了改进了DNA提取方法。在改进的DNA提取方法中,无需使用液氮,而将幼苗叶片放在真空冷冻干燥仪冷冻干燥2-3天,每个试管里盛放一小钢珠球,使用研磨仪粉碎叶片组织。由于样品分别在单独的试管里进行研磨,避免了交叉污染。在试验过程中,使用96孔联体试管板,而不是单个离心试管,使用排枪代替单枪操作,CTAB方法提取DNA。通过多次试验比较,这种方法是一种快速、高效并获得高质量的DNA的提取方法。按照该方法操作,每人每天可提取上千个DNA样品。2.通过4对SSR引物组合可以鉴定两优培九、两优108、培矮64S/E32和两优1224个生产上常用两系杂交稻。选用52对SSR引物对以培矮64S为母本的4个两系杂交稻组合的5个亲本DNA多态性进行了分析,并以1个杂交粳稻86优8号作为对照。结果表明：46对引物能在7个亲本间扩增出多态性条带。8对引物能将4个两系杂交稻与对照杂交粳稻区分开且在86优8号的亲本中具有多态性,可区分两优培九、两优108、培矮64S/E32、两优122和86优8号F1及其亲本的SSR引物分别为34、32、31、30和14对,有16对SSR引物均可用于区分4个两系杂交组合Fl和亲本,RM206和RM286引物可区分本试验的5个组合和各自的亲本。应用其中一对引物RM505对两优培九进行鉴定验证,结果表明该引物能准确区分两优培九及其亲本。根据本试验中引物的多态性和特异性的结果,有多种途径可鉴别这4个两系杂交组合。可通过RM13(或RM206、RM286等)、RM224(或RM337)、RM234(或RM252、RM505和RM565)和RM25(或RM217、RM248和RM585)等4对引物将两优培九、两优108、培矮64S/E32和两优122分别加以鉴别。3.建立了33个籼、粳杂交稻亲本的SSR指纹图谱数据库,发现了6对可以用于鉴定籼、粳类型的SSR特征标记。选用分布在12条染色体上的84对SSR引物对5个粳型核质互作雄性不育系、4个籼型核质互作雄性不育系和1个温敏雄性不育系、14个籼型父本和9个粳型父本共33份材料进行遗传相似性分析,并建立SSR指纹图谱数据库。结果表明：在33份亲本中能够扩增出多态性的引物为54对,占所用引物的64.3%。33份亲本间的遗传相似系数变异范围为0.40～0.99。在遗传相似系数0.66处,33份亲本被聚为3个类群,培矮64S和冈46A为第1类群,17个籼稻亲本为第Ⅱ类群,14个粳稻亲本为第Ⅲ类群。在籼、粳亚种内,不育系和可育品种又分为不同的亚群,基于SSR分子标记的聚类分析结果与与育种家提供的籼粳分类信息基本一致,进一步表明SSR指纹图谱可以用于鉴定品种。利用18对引物能将33份亲本区分开,RM264能将Ⅱ-32A、协青早A、冈46A和K17A4个籼型不育系与籼型可育品种区别开,RM432能将5个粳型不育系与粳型可育品种区别开。6对引物RM6、RM13、RM16、RM240、RM247和RM248均为鉴别籼、粳亚种类型的特异引物,利用这6个标记可以鉴别籼型不育系串入粳稻花粉或粳型不育系串入籼稻花粉所产生的籼粳杂株类型。4.发现了可以用于本研究大青棵鉴定的SSR分子标记。有6对引物均可用于以六千辛A为母本的大青棵的鉴定,RM9或RM218可用于以Ⅱ-32A为母本的大青棵的鉴定,RM50和RM11等2个引物组合可用于以9522A为母本的大青棵的鉴定。研究了55个籼稻不育系与粳稻父本、粳稻不育系与籼稻父本杂交组合的主要农艺性状及其SSR分子标记特征,以具备株高135cm以上、抽穗期121天以上特征的组合作为大青棵类型。结果表明：38个粳型不育系与籼稻父本的杂交后代中,以9522A为母本的杂交组合中只有9522A/紫尖籼-1是大青棵组合,以六千辛A为母本的8个组合均为大青棵组合；17个籼型不育系和粳稻父本的杂交后代中,以Ⅱ-32A为母本的杂交组合中,仅有Ⅱ-32A/C57,其余籼稻不育系为母本与粳稻父本的杂交组合均不是大青棵。与杂交F1代株高显著相关的标记的引物有RM9、RM283、RM429、RM515和RM483,其中RM9、RM283、RM429和RM515的标记与株高极显著相关；与抽穗期显著相关的标记的引物有RM9、RM44、RM206、RM152、RM276、RM228、RM515. RM211、RM432、RM454,其中RM9、RM44、RM206、RM152、RM515、RM211、RM432、RM454的标记与抽穗期极显著相关。根据是否同时具备籼型和粳型特异性标记来鉴定是否是籼粳亚种间杂交组合。在以9522A为母本的杂交组合中,大青棵可以通过RM50和RM11(或RM25、RM152、RM228、RM251、RM252、RM286、RM302、RM415)2个引物组合加以鉴定。以Ⅱ-32A为母本的4个组合中,Ⅱ-32A/C57大青棵可以通过RM9或RM218加以鉴定。因本研究中以六千辛A为母本所配制的组合均是大青棵,可以通过RM9、RM152、RM279、RM413、RM415和RM4296对引物中的任一个与其他粳稻不育系为母本的组合加以鉴别。本研究通过配制55个籼稻不育系与粳稻父本、粳稻不育系与籼稻父本杂交组合和F1代农艺性状的调查,偶然发现3726A/明恢63新组合结实率较高,达87.1%以上,株高适中(122 cm),抽穗期(109天)适宜,是否合适生产上用种有待进一步验证。更多还原

【Abstract】 The authenticity and genetic purity of seed is the most major indicator of seed quality testing. Testing of distinctness, uniformity and stability of new varieties of plants is the important content for examining the factors of application varieties and deciding if they can be granted the right of new varities in the office for the protection of new varities of plants, MOA. Rice is the major crop in our country. Hybrid rice has heterosis, high product, good resistance. There is almost half of rice area for planting hybrid rice. Our country is the biggest country for producing and consuming the seed of hybrid rice in the world. The number of rice to be applied variety protection is increasing every year. But the method for testing authenticity and genetic purity of seed and DUS testing for new varieties of plants is still the method of identification relying on planting in fields. It is long time for one growth cycle period and it costs much labor and occupies much area of field. The marker characters for testing are limit. This method can not meet the demand for seed market and new varieties authorization in time. Thus, there is important significance to study rapid, simple, accurate method of DNA molecular marker to identify rice varieties for testing hybrid seed quality and granting the new variety authorization. In order to rapidly, accurately identify the authenticity and genetic purity of hybrid rice seed in the area scope of Jiangsu Province by using SSR molecular marker, severl key problems need to be considered as the follows:rapid, high effective DNA extraction method is an bottleneck technology for identifying varieties by using molecular marker. Traditional DNA extraction method has much difficulty of technology, tedious operating procedure. These disadvantages are one of main problems which prevent it from popularization; The sterile character of female parent of two-line hybrid rice is influenced easiliy by illumination time and temperature. The genetic purity is seriously influenced by selfing of female parent because of different conditions of illumination and temperature in different years and different regions. But sterile line is fertile sometime during planting identification in Hainan. It brings difficulties to identification according morphological characters; It is the area where there are both Indica and Japonica hybrid and conventional rice in the area of middle and lower Yangtze river. There contantly are one kind of subspecies hybrid combination between Indica and Japonica named "Daqingke"which is generated by Indica sterile lines and Japonica male parents or Japonica sterile lines and Indica male parents in the producing seed of hybrid in this area. This kind of offtype has high height of plant and late heading date. The corporate image and production were seriously influenced if there was Daqingke in hybrid rice. But it is difficult to identify Daqingke because Daqingke may not have the characters of high height plant and late heading date while planting in Hainan for identification. Thus, it was suggested to be studied as the following four aspects:the first is to improve the method and procedure of DNA extraction and do research for the rapid, high effective and high quality DNA extraction method. The second was to analyze the polymorphism of 7 parents of 4 common two-line hybrid and one Japonica hybrid and use the polymorphic SSR markers to identify two-line hybrid. The third is to establish DNA fingerprint data of 33 Indica and Japonica parents of hybrid common used during recent years in production and analyze the genetic similarity coefficients in order to offer the basis for identifying hybrid seed. The fourth is to screen SSR markers for identifying Daqingke offtype among 55 hybrid combination of Indica and Japonica which were made by common Indica, Japonica sterile line and janponica and Indica male parents in this study after the major characters of plant height, heading date and seed setting rate were investigated. It offered the basis for identifying Daqingke which might generate in hybrid rice in this area. The major results are obstained as follows:1. A rapid, high effective and high quality DNA extraction improved method was applied in this study. Conventional DNA extraction method and improved DNA extraction method were both used in this study. In the procedure of improved DNA extraction method, the tissue of seedling leave were frozen and dried for 2-3 days in the vacuum freezing machine, then, was ground instead of using liquid nitrogen. There is one small steel ball in each tube. The tissue was ground by grinding meachine. Because each sample was ground in separate tube, it prevented the cross contamination from each other. The plates of 96 connected tubes were used instead of seperated tubes, multiple-pipettes instead of single pipette during the whole procedure and CTAB method were used to extract DNA for the method. It was verified to be a rapid, high effective and high quality DNA extraction method through many times testing and comparing. According to this method, one person can extract about 1000 DNA samples per day.2. Four two-lines hybrids could be identified by one group of four SSR primers. DNA polymorphism of five parents of four two-line hybrid rice with Peiai64S as female parent were analyzed by 52 pairs of SSR primers with one Japonica hybrid rice named 86you8 as the control. The results are as follows:46 SSR primers could amplify polymorphism among 7 parents.8 pairs of SSR primers which could amplify polymorphism among the parents of 86you8 could distinguish the 4 two-line combinations from the control. There were 34,32,31,30 and 14 pairs of SSR primers which could be used to distinguish the combinations (F1) and the parents of Liangyoupeijiu, Liangyou108, Peiai64S/E32, Liangyou122 and 86you8, respectively.16 SSR primers could distinguish all of the 4 two-line hybrid rice and their parents. RM206 and RM286 could distinguish the five combinations and their parents in this study. RM505 was tested to identify Liangyoupeijiu and it’s parents. The result showed that it could distinguish Liangyoupeijiu and it’s parents accurately. There were several kinds of methods to distinguish the 4 two-line combinations according to SSR polymorphism and specificity in this study. The four two-line combinations of Liangyoupeijiu, Liangyou108, Peiai64S/E32 and Liangyou122 could be distinguished by the group of four pairs of primers including RM13 (or RM206, RM286 and so on), RM224 (or RM337), RM234 (or RM252, RM505 and RM565) and RM25 (or RM217, RM248 and RM585).3. The fingerprint of 33 parents based on SSR markers was established in this study.6 primers could be used to identify Indica and Japonica. Genetic similarity and SSR fingerprint was studied by 84 pairs of primers distributed on 12 chromosomes in rice using 33 parents including 5 CMS lines of Japonica,4 CMS lines and 1TGMS line of Indica,14 Indica male parents,9 Japonica male parents in rice. Fifty four of the 84 SSR primers showed polymorphism among the 33 parents, accounting for 64.3% of the primers used. Genetic similarity coefficients among 33 parents ranged from 0.40-0.99. The 33 parents were classified three groups at the genetic similarity coefficient of 0.66. The first group included Peiai 64S and Gang 46A. The second group included 17 Indica parents and the third group included 14 Japonica parents. The sterile lines and fertile varieties could be classified into subgroups among Indica or Japonica, which was coincident with the groups and subgroups determined by their pedigree analysis. It futher suggested that SSR fingerprint could be used to identify varieties. All of the 33 parents could be distinguished each other by 18 pairs of the primers. RM264 could distinguish the 4 CMS lines (II-32A, XieqingzaoA, Gang46A and K17A) and the fertile varieties in Indica varieties used. RM432 could distinguish the 5 CMS lines and the fertile varieties in Japonica varieties used. The primers of RM6, RM13, RM16,RM240, RM247 and RM248 could distinguish Indica varieties and Japonica varieties. These 6 pairs of primers could be used to identify the off-type plants produced by CMS pollinated by Japonica in Indica rice seed producing field, or by CMS pollinated by Indica in Japonica rice seed producing field.4. Several SSR markers were discovered to identify Daqingke in this study. The major agronomy characters and the characters of SSR molecular marker of 55 hybrid combination of Indica and Japonica were analyzed. Daqingke was defined as the hybrids whose plant height was over 135 cm and heading date was over 121d in this study. The results were as follows:There is one Daqingke named 9522A/Zijianxian-1 among 7 hybrids which were based on 9522A as female parent and there are 8 Daqingke which included all 8 hybrids based on LiuqianxinA as female parent among 38 hybrids which were based on Japonica sterile lines as female parents and Indica as male parents.There were one Daqingke namedⅡ-32A/C57 among 4 hybrids which were based onⅡ-32A as female parent among 17 hybrids which were based on Indica sterile lines as female parents and Japonica as male parents. Others of 17 hybrids were non-Daqingke. Five SSR markers including RM9, RM283, RM429, RM515 and RM483 had significant positive correlation with plant height. RM9, RM283, RM429 and RM515 had extramarked positive correlation with plant height. Ten SSR markers including RM9, RM44, RM206, RM152, RM276, RM228, RM515, RM211, RM432 and RM454 had significant positive correlation with heading date.8 primers including RM9, RM44, RM206, RM152, RM515, RM211, RM432 and RM454 had extramarked positive correlation with heading date. Hybrids between subspecies of Indica and Japonica could be identified according to existence of both Indica special marker and Japonica special markers. Daqingke among subspecies hybrids based on 9522A as female parent could be identified by two primers of RM50 and RMll(or RM25、RM152、RM228、RM251、RM252、RM286,RM302、RM415). Daqingke among subspecies hybrids based onⅡ-32A as female parent could be identified by RM9 or RM218. All of subspecies hybrids based on LiuqianxinA were Daqingke. They could be identified by one of six primers such as RM9、RM152、RM279、RM413、RM415 and RM429 in order to distinguish LiuqianxinA and other Japonica sterile lines.New hybrid combination 3726A/Minghui 63 which had high seed setting rate (87.1%), moderate plant height (122 cm) and suitable heading date (109 d) was discovered by investigating three major agronomic traits of 55 subspecies hybrid rice in this study. It might be used in production after further verification.更多还原