东乡野生稻耐冷渐渗系的细胞学观察及SSR分析

以前期鉴定筛选的2个东乡野生稻强耐冷渐渗系(IL5243和IL5335)为试材,研究其减数分裂时期的染色体行为特征及外源基因的渗入分子证据。结果表明:(1)IL5243和IL5335中正常减数分裂的花粉母细胞分别达89.93%和90.22%,最终形成正常的成熟花粉粒,花粉离体萌发率分别为(83.03±2.82)%和(81.96±1.73)%,与受体亲本无显著性差异。(2)在减数分裂I中,2个耐冷渐渗系均观察到低频率异常染色体行为,如单价体、"8"字型二价体、多价体,以及后期I有少数花粉母细胞(3.95%～5.15%)存在落后染色体等,表明其染色体组之间发生了交换和重组;在粗线期,2个强耐冷渐渗系均观察到较高频率(IL5243和IL5335分别为27.0%和38.9%)的双核仁,而其双亲都是单核仁。(3)SSR标记和Structure分析进一步证实了栽培稻和野生稻染色体组间发生了交换重组,东乡野生稻部分DNA片段已渗入到强耐冷渐渗系中,这为水稻耐冷基因的挖掘与利用奠定了重要基础。     

东乡野生稻耐冷渐渗系DNA甲基化水平和模式变化研究

根据水稻全基因组和特定位点的CpG岛序列设计引物,采用McrBC酶酶切DNA,以东乡野生稻耐冷渐渗系(IL5335和IL5423)及其双亲为试材,研究其基因组和特定位点的DNA甲基化水平和模式变化特征,探讨甲基化变异对野生优异基因渐渗的影响。结果显示:(1)在覆盖全基因组的83个CpG岛中,IL5335和IL5423的基因组甲基化频率分别为46.6%和53.8%,低于受体亲本协青早B的62.6%;大部分(75.9%~80.7%)受体亲本的甲基化模式在两耐冷渐渗中能稳定遗传,另外一些位点发生了甲基化模式的改变,主要表现为脱甲基化(13.3%~18.1%)和过甲基化(4.4%~6.0%)。(2)在耐冷QTLs区间,两耐冷渐渗系的甲基化水平为13.3%~26.7%,远低于受体亲本的61.4%;它们在该区域的甲基化模式变异主要为脱甲基化(33.3%~40.0%),高于全基因组的平均变异率。(3)分析逆转座子Houba和Osr14区域的51个CpG岛发现,耐冷渐渗系在该区域具有较高频率的甲基化修饰和较低的甲基化模式变异。研究表明,种间杂交渐渗诱发了受体亲本广泛的甲基化水平和模式变异,为野生优异基因的有效利用提供了新的机遇和挑战。     

东乡野生稻及其渐渗系应答低磷胁迫的基因差异表达分析

以东乡野生稻耐低磷渐渗系IL171及其双亲(栽培稻‘协青早B’和东乡野生稻)为试材,采用cDNA-AFLP技术分析其幼苗期应答低磷胁迫的差异表达谱特征,并采用实时荧光定量PCR分析验证差异表达基因的表达特性,为探究东乡野生稻耐低磷胁迫的分子机制、发掘耐低磷相关的基因奠定基础。结果显示:(1)基于17对扩增效果较好的cDNA-AFLP引物分析发现,不同胁迫时间的参试材料与其对照组(正常磷水平)相比,具有很多(20~159个)上调或下调表达的差异片段。(2)与‘协青早B’相比,IL171中特异性上调的差异带有36条,下调表达61条,而东乡野生稻中分别有79条和136条;IL171与东乡野生稻共有的上调差异带为13条,下调差异带有15条。(3)回收纯化其中60条特异性差异表达条带,最终克隆测序获得50个差异表达基因片段TDFs;通过Blast比对和功能分析,可将TDFs分为8类,包括能量与代谢、基因表达调控、信号转导和转录因子等。(4)实时荧光定量PCR验证其中6个TDFs发现,各差异片段的荧光定量PCR表达模式与cDNA-AFLP结果一致,表明该实验的cDNAAFLP差异表达结果可靠,东乡野生稻的部分耐低磷相关基因已成功导入到渐渗系中,是发掘利用东乡野生稻耐低磷相关基因、探究其耐低磷分子机制的重要资源。     

异源DNA导入水稻诱发活跃反转子Tosl7发生可遗传DNA甲基化变异

用水稻(Oryza sativa L.)内源反转座子Tosl7为探针,经Southern杂交在5种含有野生稻(Zizania latifolia Griseb．)(菰)DNA片段的水稻渐渗杂交系中检测到了可遗传DNA甲基化变异。在分析的4种甲基化敏感限制性内切酶中,每种酶切都发生了亲本杂交片段的消失和新片段的出现。发生甲基化变异的位点包括对称和不对称的胞嘧啶碱基,也包括腺嘌呤碱基。序列分析表明,与水稻亲本比较,所研究的5种渐渗杂交系在Tosl7的2个重要区域(5′—LTR和RT)均未发生序列变异。但甲基化敏感—序列特异性PCR分析证实,每种渐渗杂交系在这2个区域内均发生了广泛的DNA甲基化变异。而且,在2种渐渗杂交系中发现5′—LTR和RT区域的甲基化变异存在协同性。甲基化变异可稳定遗传给后代。因为已有的研究表明,在这5种渐渗杂交系中异源DNA导人均导致了Tosl7的激活和转座,因此可以推测DNA甲基化在调控Tosl7活性中可能具有一定作用。但反转座子激活和甲基化变异之间的确切关系尚有待进一步研究。     

冬小麦小RNA高通量测序及生物信息学分析

东农冬麦1号是我国首例可在黑龙江高寒地区种植的强抗寒冬小麦(Triticum aestivum)品种。鉴于分蘖节的耐寒程度直接影响植株的安全越冬, 且MicroRNAs是一类生物体内普遍存在的非编码内源小分子RNA, 在植物的生长发育和适应胁迫等过程中具有重要作用, 该文基于HiSeq深度测序原理, 对5°C和–10°C胁迫下的东农冬麦1号分蘖节进行测序, 并开展生物信息学分析。结果表明, 在5°C文库中有2 229 955条特有小分子RNA序列, 并检测到35条已知miRNA, 属于30个miRNA家族, 其表达丰度介于1–173 810之间; 在–10°C文库中有3 721 449条特有小分子RNA序列, 并发现29条已知miRNA, 属于24个miRNA家族, 其表达丰度为1–105 868。靶基因预测结果表明, 5°C文库的30个miRNA家族中共预测到53个靶基因。功能分析结果显示, 这些基因主要参与转录调节、新陈代谢、胁迫响应和信号转导等过程。已知小麦miRNA的类型和表达丰度在5°C和–10°C两个文库中均有较大差异。     

异源DNA导入水稻诱发活跃反转子Tos17发生可遗传DNA甲基化变异

用水稻(Oryza sativa L.)内源反转座子Tos17为探针,经Southern杂交在5种含有野生稻(Zizania latifolia Griseb)(菰)DNA片段的水稻渐渗杂交系中检测到了可遗传DNA甲基化变异.在分析的4种甲基化敏感限制性内切酶中,每种酶切都发生了亲本杂交片段的消失和新片段的出现.发生甲基化变异的位点包括对称和不对称的胞嘧啶碱基,也包括腺嘌呤碱基.序列分析表明,与水稻亲本比较,所研究的5种渐渗杂交系在Tos17的2个重要区域(5'-LTR和RT)均未发生序列变异.但甲基化敏感-序列特异性PCR分析证实,每种渐渗杂交系在这2个区域内均发生了广泛的DNA甲基化变异.而且,在2种渐渗杂交系中发现5'-LTR和RT区域的甲基化变异存在协同性.甲基化变异可稳定遗传给后代.因为已有的研究表明,在这5种渐渗杂交系中异源DNA导入均导致了Tos17的激活和转座,因此可以推测DNA甲基化在调控Tos17活性中可能具有一定作用.但反转座子激活和甲基化变异之间的确切关系尚有待进一步研究.     

异源DNA导入水稻诱发活跃反转子Tos17发生可遗传DNA甲基化变异(英文)

用水稻(OryzasativaL.)内源反转座子Tos17为探针,经Southern杂交在5种含有野生稻(ZizanialatifoliaGriseb.)(菰)DNA片段的水稻渐渗杂交系中检测到了可遗传DNA甲基化变异。在分析的4种甲基化敏感限制性内切酶中,每种酶切都发生了亲本杂交片段的消失和新片段的出现。发生甲基化变异的位点包括对称和不对称的胞嘧啶碱基,也包括腺嘌呤碱基。序列分析表明,与水稻亲本比较,所研究的5种渐渗杂交系在Tos17的2个重要区域(5'-LTR和RT)均未发生序列变异。但甲基化敏感-序列特异性PCR分析证实,每种渐渗杂交系在这2个区域内均发生了广泛的DNA甲基化变异。而且,在2种渐渗杂交系中发现5'-LTR和RT区域的甲基化变异存在协同性。甲基化变异可稳定遗传给后代。因为已有的研究表明,在这5种渐渗杂交系中异源DNA导入均导致了Tos17的激活和转座,因此可以推测DNA甲基化在调控Tos17活性中可能具有一定作用。但反转座子激活和甲基化变异之间的确切关系尚有待进一步研究。     

基于单片段代换系的水稻芽期耐冷QTL定位和聚合北大核心CSCD

芽期耐冷性是华南双季稻地区水稻育种的一个重要目标。虽然水稻芽期耐冷QTL的标记定位已取得了一定的进展,但是这些QTL/基因尚未在水稻育种中得到有效的应用。定位稳定表达的芽期耐冷QTL,开展QTL聚合育种是水稻芽期耐冷性育种取得突破的关键。在本研究中,利用以粳稻IR65598-112-2为供体,籼稻华粳籼74为受体构建的单片段代换系(SSSL)开展芽期耐冷QTL定位,并进行聚合育种。通过评价SSSL与受体华粳籼74的芽期耐冷性差异,定位了2个稳定的芽期耐冷QTLs(qCTBB-3和qCTBB-12)。试验表明,分别携带有耐冷QTL qCTBB-3和qCTBB-12的SSSL在冷处理后都比华粳籼74表现出更高的幼苗成活率。通过代换作图,发现在qCTBB-3区间存在2个紧密连锁的耐冷QTLs(qCTBB-3a和qCTBB-3b)。利用本研究携带qCTBB-3a/qCTBB-3b的单片段代换系和前期研究鉴定出的芽期耐冷QTL qCTBB-6的单片段代换系为亲本进行杂交,通过分子标记辅助选择,获得了2份含有这3个QTL的聚合系。耐冷性评价表明,来源于两个供体/亲本的QTL不存在显著的上位性效应,聚合系的芽期耐冷性较亲本显著增强。可见,通过聚合芽期耐冷QTLs qCTBB-3a/qCTBB-3b和qCTBB-6能显著提高水稻芽期的耐冷性,获得的QTL及三耐冷QTL聚合系为水稻芽期耐冷性分子育种提供了优良的基因资源和亲本材料。     

棉花种间杂交渐渗系SSR及农艺性状分析

利用87对多态性SSR和EST-SSR引物,对57份棉属种间杂交渐渗系及其6个代表性血缘亲本进行了鉴定分析.共检测到540个等位变异,多态性等位变异占98.5%,EST-SSR变异占63.9%,41个SSR标记定位在棉花基因组的22条染色体上.种质间成对相似系数为0.553～0.937,平均为0.748.渐渗系的SSR聚类与种质材料的系谱来源基本吻合,而和农艺性状聚类结果相差很大,前者更能反映其亲缘关系.     

甘蓝型油菜BnTCP7基因的克隆和表达分析

该研究克隆获得了甘蓝型油菜预测转录因子TCP7-like同源编码基因,命名为BnTCP7,与甘蓝型油菜TCP7-like NC_027757基因的核苷酸序列相似性为95.68%。BnTCP7基因开放阅读框全长为648bp,编码215个氨基酸。BnTCP7氨基酸序列与十字花科(Brassicaceae)中其他19条TCP7或者TCP7-like氨基酸序列比对发现,BnTCP7与芸薹属和萝卜属等TCP7同源蛋白具有很强的相似性和保守性,尤其在靠近N端的TCP保守结构域,具有典型的螺旋-环-螺旋结构,且BnTCP7属于TCP家族Ⅰ类,为亲水性不稳定蛋白。通过BnTCP7和拟南芥(Arabidopsis thaliana)AtTCP7(NC_003076)蛋白的二级和三级结构在线预测比对分析,进一步证实BnTCP7具有TCP家族典型结构。进化树分析表明,BnTCP7蛋白与甘蓝型油菜TCP7-like NC_027757蛋白聚在同一分支,两者亲缘关系最近。利用转录组数据对不同时期不同器官BnTCP7基因表达模式分析发现,其表达量呈现一定差异性,其中营养器官中的表达量高于生殖器官。非生物胁迫及激素处理对甘蓝型油菜幼苗植株中BnTCP7基因的表达影响分析发现,BnTCP7基因不仅响应了冷、热、损伤等非生物胁迫,而且参与了ABA和GA3激素的信号转导,表明BnTCP7基因在植物维持正常生长发育和逆境胁迫中可能发挥重要作用。     

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from <Emphasis Type="Italic">Gossypium bickii</Emphasis>

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated ∼2000 genomic and ∼800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.     

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from Gossypium bickii

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated 2000 genomic and 800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.     

东乡野生稻苗期耐冷性的遗传分析

以强耐冷材料龙稻5号(Oryza sativa ssp. japonica)和冷敏感材料协青早B(O. sativa ssp. indica)为对照品种, 以萎蔫率和死苗率为鉴定指标, 利用江西东乡普通野生稻(O. rufipogon)/协青早B//协青早B构建的回交重组自交系(backcross inbred lines, BILs)群体(BC₁F₉)进行苗期耐冷性鉴定和遗传分析。结果表明, 10°C冷处理7天后, 228个BILs群体的平均萎蔫率为67.4%, 平均死苗率为70.8%。将死苗率≤20%的株系, 在更低温度(8°C昼/5°C夜)下处理5天, 结果显示5243和5335为强耐冷材料, 可用于构建东乡野生稻QTL(quantitative trait loci)近等基因系(near-isogenic lines, NILs)。实验结果显示, 群体萎蔫率和死苗率均呈偏态的连续分布, 暗示东乡野生稻苗期耐冷性表现为质量-数量性状遗传特征, 由主基因-多基因控制。     

东乡野生稻苗期耐冷性的遗传分析

以强耐冷材料龙稻5号（Oryza sativa ssp.japonica）和冷敏感材料协青早B（O.sativa ssp.indica）为对照品种,以萎蔫率和死苗率为鉴定指标,利用江西东乡普通野生稻（O.rufipogon）/协青早B//协青早B构建的回交重组自交系（backcross in-bred lines,BILs）群体（BC1F9）进行苗期耐冷性鉴定和遗传分析。结果表明,10°C冷处理7天后,228个BILs群体的平均萎蔫率为67.4%,平均死苗率为70.8%。将死苗率≤20%的株系,在更低温度（8°C昼/5°C夜）下处理5天,结果显示5243和5335为强耐冷材料,可用于构建东乡野生稻QTL（quantitative traitloci）近等基因系（near-isogenic lines,NILs）。实验结果显示,群体萎蔫率和死苗率均呈偏态的连续分布,暗示东乡野生稻苗期耐冷性表现为质量-数量性状遗传特征,由主基因-多基因控制。     

Identification of a Drought Tolerant Introgression Line Derived from Dongxiang Common Wild Rice (O. rufipogon Griff.)

Construction of introgression lines using cultivated rice as recipient and wild rice is a novel approach to explore primitive and broad genetic resources in rice breeding. We recently generated a set of 159 introgression lines via a backcrossing program using an elite Indica cultivar rice Guichao 2 (O. sativa L. ssp. indica) as recipient and a common wild rice Dongxiang accession (O. rufipogon Griff.) as donor. In this study, we have evaluated the previously constructed 159 introgression lines for drought-tolerance. A total of 12 quantitative trait loci (QTLs) related to drought tolerance were mapped. Furthermore, a drought tolerant introgression line, IL23, was identified and characterized. Genotype analysis of IL23 demonstrated that IL23 contained two QTLs associated with drought tolerance, qSDT2-1 and qSDT12-2, which were located on chromosome 2 and 12 within the two introgressed segments derived from the common wild rice, respectively. Physiological characterization, including measurement of water loss, osmotic potential, electrolytical leakage, MDA content, soluble sugars content and the leaf temperature, revealed that IL23 showed the characteristics associated with drought tolerance. Identification and characterization of IL23 would provide a useful basis for isolation of novel genes associated with drought tolerance and for molecular breeding of drought tolerant rice. Furthermore, the results in this study indicated that construction of introgression lines from common wild rice should be an appropriate approach to obtain favorable genetic materials.     

Sequence-Based SSR Marker Development and Their Application in Defining the Introgressions of LA0716 (Solanum pennellii) in the Background of cv. M82 (Solanum lycopersicum)

The introgression lines (ILs) from cv. M82 (Solanum lycopersicum) × LA0716 (S. pennellii) have been proven to be exceptionally useful for genetic analysis and gene cloning. The introgressions were originally defined by RFLP markers at their development. The objectives of this study are to develop polymorphic SSR markers, and to re-define the DNA introgression from LA0716 in the ILs. Tomato sequence data was scanned by software to generate SSR markers. In total, 829 SSRs, which could be robustly amplified by PCR, were developed. Among them, 658 SSRs were dinucleotide repeats, 162 were trinucleotide repeats, and nine were tetranucleotide repeats. The 829 SSRs together with 96 published RFLPs were integrated into the physical linkage map of S. lycopersicum. Introgressions of DNA fragments from LA0716 were re-defined among the 75 ILs using the newly developed SSRs. A specific introgression of DNA fragment from LA0716 was identified in 72 ILs as described previously by RFLP, whereas the specific DNA introgression described previously were not detected in the ILs LA4035, LA4059 and LA4091. The physical location of each investigated DNA introgression was finely determined by SSR mapping. Among the 72 ILs, eight ILs showed a shorter and three ILs (IL3-2, IL12-3 and IL12-3-1) revealed a longer DNA introgression than that framed by RFLPs. Furthermore, 54 previously undefined segments were found in 21 ILs, ranging from 1 to 11 DNA introgressions per IL. Generally, the newly developed SSRs provide additional markers for genetic studies of tomatoes, and the fine definition of DNA introgressions from LA0716 would facilitate the use of the ILs for genetic analysis and gene cloning.