fl(2)d单等位基因的敲除显著降低小菜蛾的生殖力和育性

【目的】RNA甲基化是基因转录后水平表观修饰的主要形式,参与了众多重要的细胞学过程。小菜蛾（Plutella xylostella）是危害十字花科蔬菜的重要寡食性害虫,与RNA甲基化相关基因的功能尚未见报道。本研究通过克隆小菜蛾的RNA甲基化蛋白同源基因fl(2)d,鉴定其表达模式,并敲除该基因以探究其生物学功能。【方法】通过小菜蛾基因组网站查找fl(2)d基因序列,PCR扩增其蛋白质编码序列（CDS）;采用实时荧光定量PCR（qRT-PCR）技术,检测小菜蛾不同发育阶段个体以及成虫生殖腺中fl(2)d的相对表达量;运用CRISPR/Cas9结合卵的显微注射技术,对小菜蛾fl(2)d进行编辑;将fl(2)d被编辑过的成虫与野生型成虫杂交,并对其产生的后代进行近交,筛选fl(2)d突变品系;观测并比较突变体与野生型个体遗传特性、生物学参数和表型的差异,明确fl(2)d的功能。【结果】克隆得到长度为912 bp的fl(2)d CDS,fl(2)d在雌蛹、雌成虫和卵中的表达量较高,雄成虫和雄蛹的表达量较低,幼虫期的表达量最低,成虫卵巢中表达量显著高于精巢。通过向小菜蛾的卵注射靶向fl(2)d的向导RNA（sgRNA）和Cas9蛋白的混合物,对所产生的阳性后代进行10代的单对近交筛选,获得3种杂合的移码突变品系,分别缺失了4个（Δfl(2)d213-4）、5个（Δfl(2)d213-5）和7个（Δfl(2)d214-7）碱基。在上述品系的筛选过程中,发现了6只缺失4个碱基的纯合突变个体,2只缺失5个碱基的纯合突变个体;缺失4个碱基的纯合个体成功配成了两对,近交未产卵;剩余的2只缺失4个碱基的雄性纯合个体和2只缺失5个碱基的雄性纯合个体分别与同世代的雌性杂合突变个体近交后仍未产卵。说明fl(2)d纯合突变的个体存活率极低,且可能无法产生后代。通过分析后代基因型的分离比,发现杂合突变个体近交以及杂合突变个体与野生型个体杂交产生的后代中,杂合突变个体与野生型个体的比例分别略小于2和1,说明fl(2)d杂合突变会影响小菜蛾正常的生长发育,并导致部分个体死亡。杂合突变体后代中含有突变的雌雄个体比例接近1﹕1（P<0.05）,推测小菜蛾fl(2)d可能与性别决定无关。只要是有突变品系小菜蛾所参与的交配,雌成虫产卵量和卵的孵化率均显著低于野生型（P<0.01）,所产的卵多数发育异常,表现为失水皱缩、不能正常孵化。通过对成虫的生殖腺进行解剖,发现在野生型雌成虫与突变体雄成虫交配后,卵巢内卵的附着量较未交配的个体明显减少;未交配的突变体雌成虫卵巢内卵的附着量亦少于野生型,而突变体雄成虫的精巢未见明显异常。部分能够孵化的杂合突变个体在整个发育过程会发生不同程度的畸变,导致不能正常完成整个世代;另外一些杂合突变个体未见异常,可以将突变类型遗传给后代。根据上述发现,提出了基于fl(2)d的小菜蛾遗传防控模型。【结论】fl(2)d参与小菜蛾的生殖过程和胚胎发育,突变后显著影响后代种群数量,是开展小菜蛾遗传控制的理想靶标。     

利用双杂合位点标记资料构建芒果遗传图谱

为了建立芒果 (MangiferaindicaL )的分子标记遗传图 ,用 15对AFLP (AmplifiedFragmentLengthPolymorphism)引物组合扩增了芒果品种间杂交组合 (Keitt×Tommy Atkins)的 6 0个F1单株 ,获得了 191个多态性位点。它们的分离表现为双杂合 (Aa×Aa)和测交 (Aa×aa)分离两种类型 ,但前者占了 5 9 7%。为了充分利用双杂合位点分离所提供的遗传信息 ,我们根据群体中任意两个双杂合位点隐性个体出现的数目 ,利用二项式分布概率理论推断它们是否连锁以及它们彼此间的相引或相斥关系。在该芒果群体呈 3:1分离的 81个多态性标记中 ,39个被分为 14组 ,以此为基础构建了 15个连锁群 ;这些连锁群共覆盖了 35 4 1cM的芒果基因组。其中 ,最小与最大遗传距离分别为 3 7cM和 2 8 9cM。此外 ,对 18个 1∶1分离类型的标记 ,直接利用Mapmaker作图软件构建了两个芒果连锁群。本文对所提出的利用双杂合位点构建果树遗传图谱的策略进行了讨论。     

杏杂合位点共显性标记的分离方式及连锁图谱构建

以‘串枝红’ב金太阳’杏的F1代为材料,在分析共显性标记分离方式的基础上利用简单重复序列（SSR）和相关序列扩增多态性（SRAP）标记,构建了两张杏分子连锁图谱。‘串枝红’图谱共14个连锁群,包含37个SSR标记和32个SRAP标记,连锁群总长度为1086.6cM,每连锁群平均长度77.6cM,标记间平均连锁距离为1...     

大白菜InDels标记开发及其在剩余杂合体鉴定中的应用

大白菜(Brassica rapa ssp.pekinensis)参考基因组序列的公布及测序成本的降低为大规模开发插入/缺失(Insertion/Deletion,InDel)标记提供了可能.本研究以性状差异明显的大白菜自交系He102与06-247为亲本,开展了全基因组重测序、标记开发和验证工作.二者测序深度分别为10×和8×,经筛选共获得330 218个InDels差异位点,出现频率为1.2个/kb,其中有11 238个差异位点位于编码区,包括5 184个差异基因,每个基因平均包含2.2个差异位点.分别以He102与06-247测序数据中筛选出的多态性InDels位点为参照,从10条染色体上随机选择933个位点进行了PCR扩增及电泳检测验证.结果表明,测序深度对假阳性率有直接影响,以测序较深的材料中筛选到的差异位点为参照,其验证结果的假阳性率亦较高,反之则较低.本研究中以He102与06-247为参照选择InDels位点验证的平均假阳性率分别是51.9％和22.5％.从933个位点中共筛选出593个在亲本之间实际表现为多态性的位点,这些差异位点中有375个位于编码区,是潜在的功能性标记.利用上述差异位点,检测了He102与06-247衍生的F7代重组自交系群体,明确了F7代各株系在593个位点的纯/杂合状态,为利用剩余杂合株系精细解析和精准定位大白菜耐抽薹、抗病毒、抗干烧心等重要农艺性状奠定了基础.     

Fine Mapping of C(Chromogen for Anthocyanin)Gene in Rice

Seven residual heterozygous lines(RHLs)displaying different genotypic compositions in the genomic region covering probable locations of C (Chromogen for anthocyanin)gene on the short arm of rice chromosome 6 were selected from the progenies of the indica cross Zhenshan 97B/Milyang 46.Seeds were harvested from each of the seven plants,and the resultant F2:3 populations were used for fine mapping of C gene.It was shown in the populations that the apiculus coloration matched to basal leaf sheath coloration in each plant.By relating the coloration performances of the populations with the genotypic compositions of the RHLS,the C locus was located between rice SSR markers RM314 and RM253.By using a total of 1279 F2:3 individuals from two populations showing coloration segregation.the C locus was then located between RM111 and RM253,with genetic distances of 0.7 cM to RM111 and 0.4 cM to RM253.Twenty-two recombinants found in the two populations were assayed with seven more markers located between RM111 and RM253,including six SSR markers and one marker for the C gene candidate,OsC1.The C locus Was delimited to a 59.3-kb region in which OsC1 was located.     

Validating a segment on the short arm of chromosome 6 responsible for genetic variation in the hull silicon content and yield traits of rice

Rice is a typical silicon-accumulating plant and the beneficial effect of silicon on rice has long been recognized. In a previous study using 244 recombinant inbred lines (RILs) of an indica rice cross, Zhenshan 97B/Milyang 46 grown in 2003, four QTLs were detected for hull silicon content. QTL qHUS-6 had the largest effect among these, and the same interval also had significant effects on yield traits in the same population. The primary objective of this study was to validate the QTL effect in this region on HUS and yield traits. The same RIL population and another RIL population of lower heterogeneity were grown in 2004. QTL qHUS-6 was found to have significant additive effects on hull silicon content with a consistent direction in the two populations. From a residual heterozygous line selected from RILs of the same cross, 15 F_2:3 lines that differed only in a 2.15-Mb segment extending from RM587 to RM6119 on the short arm of chromosome 6 were derived. In these lines, qHUS-6 displayed a major effect, so did QTLs for yield traits previously detected in the same region. Two more QTLs for HUS detected in 2003, qHUS-1-1 and qHUS-1-2, also had consistent effects in the Zhenshan 97B/Milyang 46 RIL population in 2004. Thus this study verified three candidate regions for fine mapping HUS QTLs and determining the genetic relationship between silicon content and yield traits in rice.     

应用剩余杂合体衍生的近等基因系分解水稻产量性状QTL

以杂合区间为RM587-RM402的水稻剩余杂合体（RHL）衍生群体为材料,应用SSR标记检测,筛选到杂合区间分别为RM587-RM225、RM204-RM6119和RM6119-RM402的3个单株,进一步检测其F2群体,分别获得母本纯合型材料10株、父本纯合型材料10株和杂合型材料20株。种植这3套近等基因系材料,考查单株产量及其构成因子每株穗数、每穗实粒数和千粒重。经应用目标区间内等位基因效应分析和交迭重组染色体片段代换系分析,分解出3个控制每穗实粒数的QTL和2个控制单株产量的QTL,这些QTL分别位于物理距离为0.66 ~ 2.49 Mb的区间中,全部表现为加性作用为主,增效等位基因除qNFGP6 1来自父本密阳46外,其余均来自母本珍汕97B。提出了构建新型遗传材料,提高水稻QTL精细定位效率的策略。     

BrVIN3.1 互作蛋白BrZAT12 在大白菜春化途径中行使功能初探

BrVIN3.1 在春化过程中通过调控BrFLC1 的表观修饰状态，进而调控其表达控制大白菜花期。本试验以BrVIN3.1 （Bra020445）为诱饵，进行酵母cDNA 文库筛选，从中筛选到它的互作蛋白BrZAT12（Bra006691）；进一步进行酵母双 杂一对一验证，证明BrZAT12 能够与BrVIN3.1 互作。采用低温处理不同花期的大白菜材料，进行表达模式分析，发现 BrZAT12 的表达模式在抽薹性状不同的大白菜材料中存在差异，在易抽薹材料中的表达量明显高于耐抽薹材料，且在耐抽薹 材料中变化幅度较小，同时BrZAT12 的表达模式与BrVIN3.1 相关。推测BrZAT12 可能在大白菜春化调控的开花途径中行使 功能。     

Comparison of homozygous and heterozygous self-compatible seedlings in an almond breeding programme

Homozygous self-compatible almond cultivars have not been reported. It is unclear if they are more inferior than heterozygotes or simply have not yet been detected. To investigate if homozygous individual are generally inferior, the self-compatibility genotype, homozygous or heterozygous, was determined by stylar ribonuclease assay in a population of 241 almond trees obtained by self-fertilisation of self-compatible selections. The resulting zymograms showed that 129 of the seedlings were homozygous and 112 heterozygous. For three years the differences observed between these two classes of self-compatible individuals were analysed with respect to 16 agronomic characteristics. In general, there were no important differences between the two classes. Both showed a low degree of productivity, probably as a result of their inbred origin. Some selected homozygous individuals were used in crosses, which were planned so as to ensure the self-compatibility of 100% of the descendants and to eliminate the laborious task of testing the seedlings for self-compatibility. This revised version was published online in August 2006 with corrections to the Cover Date.     

玉米基因雄性不育双杂合保持系的选育研究

本研究从1982年开始,选用玉米ms1、ms2和ms10三个雄性核不育基因以及易位断点与相应ms基因紧密连锁的T4-6b、T9-10a、T4-10f和T6-10（5519）四个易位系作为基础材料，采用细胞遗传技术育成若干个骨干自交系的基因雄性不育双杂合保持系。至今，已选育出黄早四和Mo17背景的ms2基因杂合保持系。保持系与ms2基因不育系杂交，子代