利用SSR荧光标记对野慈姑异交率的估测

对自交亲和植物交配系统进行估测,有利于了解植物的繁殖状态、自异交进化的轨迹和特定种群的自然历史或选择压力。自交亲和的野慈姑有性繁殖和克隆繁殖并存,且其花序内和无性系分株间存在雌雄花同时开放的现象即给自交带来了机会。本研究利用SSR标记估测野慈姑自然种群的异交水平,并比较分析异交率估测中荧光定量法的准确性和优势,探讨野慈姑不同自然种群微卫星位点的多态性。结果表明,从28对SSR引物中筛选出3对多态性较高的引物,其多种群水平的位点数分别是5、6、6;对1个自然种群的6棵植株共计31个果实的异交率进行估测,其整体异交率为92.87%±2.5%,揭示了野慈姑自然种群的交配系统以异交为主,且无性繁殖对后代的贡献有限;单引物水平下,荧光定量技术检测出的多态性位点数和杂合率均高于NativePAGE成像电泳,且其对异交率估测的结果也更准确;另外,从检测效率实验耗时和实验成本等方面综合分析,本研究推荐使用荧光定量技术。     

泽苔草交配系统的RAPD分析

利用RAPD显性标记位点对泽苔草(Caldesia parnassifolia)湖南茶陵湖里居群进行了交配系统研究。采用10个家系共240个子代样品测定了该居群花粉库基因频率、固定指数(F)、多位点异交率(tm)和单位点异交率(ts)。结果显示该居群异交率处于较高的水平(tm=1.008),多位点异交率与单位点异交率差异不显著(tm-ts=-0.021),固定指数(F)的估计值小于0(F=-0.200),表明该居群双亲近交不明显,而以异交为主。这可能是受居群密度、传粉者的活动能力等因素影响的结果。     

用微卫星DNA标记对我国杂交水稻主要恢复系遗传差异的检测分析

选用分布于水稻（Oryza sativaL.)12条染色体上的25对SSR（Simple sequence repeats)引物,分析了生产中广泛应用的35个杂交 水稻恢复系,在35个杂交水稻恢复系材料间共检测出65个等位基因（alleles),平均每对SSR引物可检测到2.6个等位基因,PIC（Polymorphism index content）值的变动范围为0.206-0.682,平均值为0.414。聚类分析表明,我国杂交 水稻恢复系资源比较丰富,但其遗传差异较小,遗传背景单一,从而在很大程度上限制了我国水稻杂种优势的利用。     

用微卫星DNA标记检测中国主要杂交水稻亲本的遗传差异

选用分布于水稻(OryzasativaL.)12条染色体上的20对SSR(Simplesequencerepeats)引物,分析了具有多种质源和较大应用面积的24个水稻胞质雄性不育系、1个光(温)敏核不育系、3个保持系和5个生产中应用较广的恢复系。在以上33份杂交水稻亲本材料间共检测出102个等位基因(alleles),平均每对SSR引物可检测到5.1个等位基因。PIC(polymorphicindexcontent)值的变动范围为0.274～0.773,平均PIC值为0.554。从56对SSR引物中筛选出5对引物,能够有效地区分所有供试的水稻雄性不育系和恢复系。杂交水稻亲本的聚类分析表明:(1)我国水稻雄性不育系遗传变异丰富,但生产中主要应用的水稻雄性不育系遗传背景比较单一。(2)生产中应用面积较大的水稻雄性不育系遗传变异较恢复系差。(3)生产中主要应用的水稻雄性不育系与恢复系分别聚类于不同的类群,且遗传关系较远。     

利用重测序技术开发高粱多态性SSR分子标记

SSR分子标记由于具有成本低廉、容易操作等特性,使其在分子标记辅助育种中广泛应用。目前高粱SSR标记多基于测序已经完成美国高粱品种BTX623基因组开发,在应用中筛选多态性标记的效率低。对不育系和恢复系组成的26个高粱材料进行了重测序,然后进行生物信息学分析,最终开发了在26份材料中至少含有2种多态型的SSR标记24 441个。这些SSR标记在26份材料中表现出的基因型多态类型在2-7种之间,2种的有16 694个,7种的仅有77个。另外本研究还进行了筛选单拷贝基因处的多态性SSR分析,共筛选到6 733个。随机挑选均匀覆盖10条染色体的单拷贝基因处SSR标记50对,利用辽宁高粱杂交种辽糯3号进行测试,其中49对能扩增出产物,成功率高达98%。其后利用2个品种和74份微核心种质资源测试表明,50对SSR标记在2个品种中有18对表现出多态性,挑选了一对引物在74份微核心种质中可见8种多态型。本研究表明利用不育系和恢复系材料进行重测序能有效开发多态性高的SSR标记。     

用遗传标记监测不同环境和经营措施下的加勒比松森林群体遗传动态

对古巴加勒比松的6个群体（包括天然林、采伐林、母树林和种子园）进行了同功酶分析,根据5个酶系统8个位点的同功酶数据,对各群 交配系统以及群体遗传变异和结构进行了分析,天然林、种子园和母树林的多位点异交率和绝大多数单位点异交率都和完全异交无显著差异,过渡采伐的松树岛群体多位点异交率显著小于完全异交,而只有一半单位点异交率显著小于完全异交,而且该群体单位点平均异交率和多位点异交率均低于其它3个群体的估计值。采伐群体中同功酶变异和基因多样性与天然林群体JAG的相似,但低于其它群体,其近交系数较大,但小于天然林MAN和中国栽培群体的近交系数。中国引种栽培群体无论是同功酶变异还是基因多样性都显著高于古巴群体,与所有古巴群体的遗传距离都显著大于古巴群体之间的遗传距离。结果表明过度采伐导致群体自交程度增加,营建种子园可有效减少近交。自然分布区以外的引种栽培群体遗传变化量大,无论遗传变异和基因多样性都比参试其它群体大。     

EST-SSR标记在水曲柳雌雄鉴定中的应用

选用黑龙江省青山林木种子园成年水曲柳200棵树为实验材料,在来自NCBI中的5 423条白蜡属EST序列和该实验室测得的水曲柳转录组的179 502条序列中查找SSR位点并设计引物,通过PCR扩增筛选有效扩增和有差异片段的引物,用于水曲柳雌雄的鉴别。结果显示:(1)白蜡属EST和水曲柳转录组中的5 423条和179 502条序列中,分别查找到9 488个和3 557个SSR位点,SSR出现的频率分别是174.96%和1.98%,SSR的类型都是以二核苷酸为主。(2)232对引物中有208对引物获得有效扩增,74对引物在雌雄DNA中有差异,27对扩增片段符合预定大小片段。(3)经过3轮筛选,选出19号和56号引物用于200棵水曲柳中,雌雄差异率分别是64.5%和89.5%。研究表明,利用EST-SSR分子标记对水曲柳性别进行鉴定是一种有效可行的方法。     

56个杂交水稻骨干亲本SSR指纹图谱的构建及遗传相似性分析

以中国56个杂交水稻骨干亲本为研究材料,包括水稻雄性不育系和恢复系。从国标中公布的48对水稻SSR引物中筛选出14对稳定性好、多态性高、杂带少且在染色体上分布均匀的引物作为核心引物,建立56个杂交水稻骨干亲本的SSR指纹图谱,结果表明：14对SSR引物在56份材料中共扩增出48个多态性片段,平均每对引物可以检测3．43个等位基因。聚类分析得出56个品种间的遗传相似性系数在0．63～0．98之间,基本上反映了不同品种间的亲缘关系。     

小慈姑的开花状态、传粉机制与交配系统

在自然种群中对沼生植物小慈姑（Sagittaria potamogetifolia Merr.）的开花状态和传粉过程作了观测,并用同工酶遗传标记法定量估测了其3个种群中5个样本的异交率。该种为中心媒传粉为主的虫／风 媒兼性传粉机制,访花昆虫的飞行距离多在0-2m范围内,并与花序密度呈不显著的正相关。异交率（－／^／t）为50.0％－92.8％,表明该种为异交为主的混合交配系统（ixed mating system）。对雌雄同株的小慈姑而言,异交率与各种群花期植株密度无关,异交水平取决于种群中开花花序的两个或两个以上的植株所占比例。     

利用SSR标记分析海南普通野生稻的遗传多样性

选用平均分布于水稻基因组的28对SSR引物,对海南不同纬度5个普通野生稻居群的163份材料进行遗传多样性和遗传结构研究。结果表明:(1)海南普通野生稻具有较高的遗传多样性,28个位点共检测到227个等位变异,平均等位变异数A=8.1071,有效等位变异数Ae=4.4190,平均期望杂合度He=0.4004,实际观察杂合度Ho=0.7062,香农指数I=1.6048;(2)居群的遗传分化系数较大,总的遗传变异中有46.40%存在于居群间(Fst=0.4640);(3)居群内杂合体较高(F is=-0.7069),根据固定指数(F=0.0588)计算出的异交率t=0.8889,说明海南普通野生稻的繁育系统属于一种较高的异交混合交配类型。     

SSR标记对不同黄籽甘蓝型油菜亲本材料的遗传分析

杂交育种中,亲本选配是育种成败的关键。本研究以重庆市油菜工程技术研究中心提供的180份甘蓝型黄子油菜亲本种质为材料,应用分布于不同连锁群的60对SSR标记进行了分析,共检测出308个标记位点,每对引物在不同亲本材料之间的等位基因数在1～11个之间,平均位点为5.1个。其中多态性位点207个,多态率达67.2%。对SSR扩增结果进行UPGMA分析,在遗传距离0.566处,180个品种(系)分为3个类群,聚类结果与种质来源比较一致,本研究为甘蓝型油菜黄子杂交育种和优势组合的选配提供了理论依据。     

Fine mapping and candidate gene analysis of the nuclear restorer gene Rfp for pol CMS in rapeseed (Brassica napus L.)

The Polima (pol) system of cytoplasmic male sterility (CMS) in rapeseed is widely used in China for commercial hybrid seed production. Genetic studies have shown that its fertility restorer gene (Rfp) is monogenic dominant. For fine mapping of the Rfp gene, a near isogenic line comprising 3,662 individuals of BC(14)F(1) generation segregating for the Rfp gene was created. Based on the sequences of two SCAR markers, SCAP0612ST and SCAP0612EM2, developed by Zhao et al. (Genes Genom 30(3):191-196, 2008) and the synteny region of Brassica napus and other Brassica species, 13 markers strongly linked with the Rfp gene were identified. By integrating three of these markers to the published linkage map, the Rfp gene was mapped on linkage group N9 of B. napus. Using these markers, the Rfp locus was narrowed down to a 29.2-kb genomic region of Brassica rapa. Seven open reading frames (ORFs) were predicted in the target region, of these, ORF2, encoding a PPR protein, was the most likely candidate gene of Rfp. These results lay a solid foundation for map-based cloning of the Rfp gene and will be helpful for marker-assisted selection of elite CMS restorer lines.     

甘蓝型油菜千粒重性状的QTL初步定位研究

千粒重是油菜重要的产量相关性状之一,构建油菜遗传连锁图谱是研究其产量性状基因的前提。本研究利用小孢子培养技术,选育出了甘蓝型油菜大粒品系(G-42)和小粒品系(7-9)的纯合DH系DH-G-42和DH-7-9,其千粒重分别为6.24 g和2.42 g,二者比值达2.58。以DH-G-42为母本、DH-7-9为父本,构建了含190个单株的F2遗传作图群体,利用SSR和SRAP标记技术绘制遗传连锁图谱,该图谱共包含20个连锁群,涉及128个SSR标记和100个SRAP标记,图谱总长1546.6cM,标记间平均图距为6.78cM。本研究共检测到3个与千粒重性状相关的QTL,分别位于A9和C1连锁群,其中qSW-A9-1和qSW-A9-2贡献率分别达到10.98%和27.45%,均可视为控制粒重的主效QTL。本研究为后续进行油菜千粒重性状QTL的精细定位分析、分子标记辅助选择育种及新基因的克隆等奠定了基础。     

Analysis of B-genome chromosome introgression in interspecific hybrids of Brassica napus × B. carinata

Brassica carinata, an allotetraploid with B and C genomes, has a number of traits that would be valuable to introgress into B. napus. Interspecific hybrids were created between B. carinata (BBCC) and B. napus (AACC), using an advanced backcross approach to identify and introgress traits of agronomic interest from the B. carinata genome and to study the genetic changes that occur during the introgression process. We mapped the B and C genomes of B. carinata with SSR markers and observed their introgression into B. napus through a number of backcross generations, focusing on a BC(3) and BC(3)S(1) sibling family. There was close colinearity between the C genomes of B. carinata and B. napus and we provide evidence that B. carinata C chromosomes pair and recombine normally with those of B. napus, suggesting that similar to other Brassica allotetraploids no major chromosomal rearrangements have taken place since the formation of B. carinata. There was no evidence of introgression of the B chromosomes into the A or C chromosomes of B. napus; instead they were inherited as whole linkage groups with the occasional loss of terminal segments and several of the B-genome chromosomes were retained across generations. Several BC(3)S(1) families were analyzed using SSR markers, genomic in situ hybridization (GISH) assays, and chromosome counts to study the inheritance of the B-genome chromosome(s) and their association with morphological traits. Our work provides an analysis of the behavior of chromosomes in an interspecific cross and reinforces the challenges of introgressing novel traits into crop plants.     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.     

Regional association analysis delineates a sequenced chromosome region influencing antinutritive seed meal compounds in oilseed rape

This study describes the use of regional association analyses to delineate a sequenced region of a Brassica napus chromosome with a significant effect on antinutritive seed meal compounds in oilseed rape. A major quantitative trait locus (QTL) influencing seed colour, fibre content, and phenolic compounds was mapped to the same position on B. napus chromosome A9 in biparental mapping populations from two different yellow-seeded × black-seeded B. napus crosses. Sequences of markers spanning the QTL region identified synteny to a sequence contig from the corresponding chromosome A9 in Brassica rapa. Remapping of sequence-derived markers originating from the B. rapa sequence contig confirmed their position within the QTL. One of these markers also mapped to a seed colour and fibre QTL on the same chromosome in a black-seeded × black-seeded B. napus cross. Consequently, regional association analysis was performed in a genetically diverse panel of dark-seeded, winter-type oilseed rape accessions. For this we used closely spaced simple sequence repeat (SSR) markers spanning the sequence contig covering the QTL region. Correction for population structure was performed using a set of genome-wide SSR markers. The identification of QTL-derived markers with significant associations to seed colour, fibre content, and phenolic compounds in the association panel enabled the identification of positional and functional candidate genes for B. napus seed meal quality within a small segment of the B. rapa genome sequence.     

Marker-assisted selection of restored male-fertile Brassica napus plants using a set of dominant RAPD markers

Bulked segregant analysis was used to identify RAPD markers in oilseed rape (Brassica napus L.) that were linked to a male fertility restorer gene for Ogura cytoplasmic male sterility. After screening for polymorphisms using 960 primers, 14 RAPD markers were mapped to a 25 cM region including the restorer locus, a mapping population of 242 F₂ individuals being employed. The map was used to select 11 markers that were investigated for polymorphisms between the restorer donor line and 46 recipient lines. A set of four RAPD markers, one in coupling phase with the restorer allele and three with the non-restorer allele, which were informative in all 46 combinations, were used in marker assisted selection of plants homozygous for the restorer allele. A total of 906 homozygous restored plants were found among the 4605 BC₁F₂ plants analysed. Phenotypic data of a subset of the classified plants was compared with the RAPD data and the expected number of recombinants was calculated from the map data. A close correspondence between the expected and observed numbers of plants with a deviating phenotype was found. Thus, use of a set of dominant RAPD markers provides a way obtaining reliable data for marker-assisted selection.     

Yield reduction in Brassica napus, B. rapa, B. juncea, and Sinapis alba caused by flea beetle (Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae)) infestation in northern Idaho

Phyllotreta cruciferae is an important insect pest of spring-planted Brassica crops, especially during the seedling stage. To determine the effect of early season P. cruciferae infestation on seed yield, 10 genotypes from each of two canola species (Brassica napus L. and Brassica rapa L.) and two mustard species (Brassica juncea L. and Sinapis alba L.) were grown in 2 yr under three different P. cruciferae treatments: (1) no insecticide control; (2) foliar applications of endosulfan; and (3) carbofuran with seed at planting plus foliar application of carbaryl. Averaged over 10 genotypes, B. rapa showed most visible P. cruciferae injury and showed greatest yield reduction without insecticide application. Mustard species (S. alba and B. juncea) showed least visible injury and higher yield without insecticide compared with canola species (B. napus and B. rapa). Indeed, average seed yield of S. alba without insecticide was higher than either B. napus or B. rapa with most effective P. cruciferae control. Significant variation occurred within each species. A number of lines from B. napus, B. juncea, anid S. alba showed less feeding injury and yield reduction as a result of P. cruciferae infestation compared with other lines from the same species examined, thus having potential genetic background for developing resistant cultivars.     

Alloplasmic male sterile Brassica napus with Enarthrocarpus lyratus cytoplasm: introgression and molecular mapping of an E. lyratus chromosome segment carrying a fertility restoring gene.

A cytoplasmic male sterility (CMS) system for Brassica napus (2n = 38; AACC) was developed by backcross substitution of its nucleus into the cytoplasm of a wild crucifer, Enarthrocarpus lyratus. Male sterility was complete, stable, and expressed in small flowers with rudimentary anthers. Since the B. napus germplasm lines were complete or partial maintainers of male sterility, the required fertility restorer gene (Rfl) was introgressed from the cytoplasm donor species. Inheritance studies carried out on F1 and F2 populations derived from hybridizing cytoplasmic male sterile and male fertile near-isogenic (PNILs) lines of B. napus 'Westar', revealed a monogenic dominant control for fertility restoration. Bulked segregant analysis with 215 RAPD primers helped in the identification of putative primers associated with fertility restoration. Co-segregation analysis of eight such primers with Rfl gene revealed two markers, OPK 15700 and OPZ 061300, which flank the Rfl locus on either side at a distance of 8.2 and 2.5 cM, respectively. These DNA markers will be useful in marker-assisted selection for improving the commercial potential of this newly developed CMS-fertility-restorer system for hybrid seed production programs in rapeseed.