【作者】 王炯；

【导师】 洪棋斌；

【作者基本信息】 西南大学 ， 细胞生物学， 2017， 硕士

【摘要】 柑橘不仅是世界上第一大水果,也是我国重要的果树之一。作为一种常绿植物,柑橘的光合效率明显低于多数C3木本植物,而光合作用是形成植物生物产量的根基,不仅影响树体生长,也对果实的产量和品质造成影响。光合效率是一种数量性状,影响因素众多,目前在柑橘中的遗传研究报道较少。测试杂交群体的重要光合作用相关性状,研究其遗传关系,同时开发新型标记构建柑橘遗传连锁图谱,挖掘与柑橘光合作用相关的QTLs(Quantitative Trait Loci)及紧密连锁的分子标记目前尚未见报道。相关研究对柑橘光合效率的研究和改良具有重要的理论和实践价值,将有助于提高柑橘高光合效率育种的水平。本研究利用梨橙2号×晚蜜2号所得的80株F1代材料为作图群体,开发COS(Conserved Ortholog Sequence)引物和SSR(Simple Sequence Repeat)引物,并利用已公布的其它SSR引物,构建了新的柑橘遗传连锁图谱。同时,采用LI-6400XT便携式光合仪和SPAD502叶绿素仪,测定和分析了亲本和作图群体的光合作用相关性状。最后结合所构建的含所有标记的遗传图谱对柑橘的光合作用相关性状进行QTL定位分析。主要结果如下:1.引物设计。根据测序克里迈丁和甜橙基因组信息,从Phytozome网站下载了1184条测序克里迈丁Conserved ortholog sequences,在NCBI(National Center for Biotechnology Information)中与测序甜橙序列blast比对后,筛选获得在启动子区或编码区内克里迈丁和甜橙具有一个或多个Gaps的保守直系同源序列275条,并从这些序列成功设计出261对COS引物。运用SSRIT(Simple Sequence Repeat Identification Tool)等工具搜索测序克里迈丁COS及临近序列中的SSR序列,设计出与特定COS序列相关的SSR引物65对,命名为CSSRC。2.引物筛选和群体扩增。首先采用亲本及6个F1代个体,对326对新开发合成的COS、CSSRC引物进行了引物筛选,从中筛选出198对谱带清晰、稳定性好、具有多态性的引物;COS引物164对,多态性比率约63%,CSSRC引物34对,多态性比率约52%。以筛选出的多态性引物及41对已公布的在该群体具多态性的SSR引物对全部样品进行扩增分析。3.遗传连锁图谱构建。运用Join Map?4.0软件,分别对COS标记以及COS标记合并其它SSR标记进行遗传连锁分析,得到两张新柑橘遗传连锁图谱:仅含有COS标记的图谱,总计165个标记,10个连锁群,总长度为816.1 c M,标记间平均距离约为4.9 cM;含COS标记和其它SSR标记的图谱,总计240个标记,9个连锁群,总长度为1186.4 c M,标记间平均距离约为4.9 c M。4.亲本及作图群体光合作用相关性状测定和分析。(1)净光合速率(net Photosynthetic rate,Pn)。群体平均值为6.49μmol/m~2·s,变异范围为2.27~9.92μmol/m~2·s,分布接近正态分布;双亲的Pn分别为6.45μmol/m~2·s和6.68μmol/m~2·s,亲中值为6.57μmol/m~2·s;群体平均值与亲中值非常接近,但后代Pn变异大,出现明显高于或低于亲本的个体,杂交育种改良空间比较大。(2)蒸腾速率(Transpiration rate,Tr)。群体平均值为2.00 mmol/m~2·s,变异范围为0.80~3.76 mmol/m~2·s,分布接近正态分布;双亲的Tr分别为1.74mmol/m~2·s和1.80 mmol/m~2·s,亲中值为1.77 mmol/m~2·s;群体平均值较亲中值更高,后代Tr变异大,出现明显高于或低于亲本的个体,杂交育种改良空间比较大。(3)胞间CO2浓度(Intercellular CO2 concentration,Ci)。群体平均值为202.07μmol/mol,变异范围为119.74~272.74μmol/mol,分布接近正态分布;双亲的Ci分别为249.39μmol/mol和255.04μmol/mol,亲中值为252.22μmol/mol;群体平均值较亲中值更低,但后代Ci出现了明显高于或低于亲本的个体,杂交改良有一定空间。(4)气孔导度(Conductance to H2O,Cond)。群体平均值为0.08 mol/m~2·s,变异范围为0.03~0.15 mol/m~2·s,分布接近正态分布;双亲的Cond分别为0.12mol/m~2·s和0.13 mol/m~2·s,亲中值为0.13 mol/m~2·s;群体平均值较亲中值更低,后代Cond出现明显低于亲本的个体,高于亲本的个体较少,表明通过杂交育种提升气孔导度的空间有限。(5)叶绿素含量SPAD值。群体平均值为71.55,变异范围为58.85~79.28,分布接近正态分布;双亲的叶绿素含量SPAD值分别为77.42和79.37,亲中值为78.40;群体平均值较亲中值更低,但后代SPAD值出现了明显低于亲本的个体,未出现高于SPAD较高亲本的个体,表明杂交培育叶绿素含量显著提升的后代的难度比较大。5.光合作用相关性状的相关性分析。运用SPSS软件进行相关性分析,结果显示:(1)Pn分别以相关系数0.639和0.762与Tr、Cond呈极显著正相关,以相关系数0.251与叶绿素含量SPAD值呈显著正相关,以相关系数0.105与Ci呈正相关。表明Pn可能受到Tr、Cond和叶绿素含量的显著影响。(2)Tr分别以相关系数0.552和0.742与Ci、Cond呈极显著正相关,以相关系数0.210与叶绿素含量SPAD值呈正相关。表明Tr可能与Ci和Cond有非常密切的关系。(3)Ci以相关系数0.602与Cond呈极显著正相关,以相关系数0.171与叶绿素含量SPAD值呈正相关。表明Ci可能与Cond关系密切。(4)Cond以相关系数0.189与叶绿素含量SPAD值呈正相关。表明叶绿素含量可能并非Cond的主要影响因子。6.光合作用相关性状的QTL分析。运用Map QTL6.0分析软件,采用区间作图法(Interval Mapping,IM)对柑橘光合作用相关性状进行QTL初步检测,再采用多重区间作图法优化,两者检测结果一致,结果如下:(1)在LOD≥3.10的条件下,共检测到5个与柑橘Pn相关的QTLs,其中3个QTLs分布在LW3上,命名为qtl3.2(Pn)、qtl3.3(Pn)和COSC3-35(Pn),贡献率分别为16.6%、17.6%、17.8%;2个QTLs分布在LW6上,命名为qtl6.4(Pn)和TAA1(Pn),贡献率分别为16.4%和16.5%。(2)在LOD≥3.90的条件下,共检测到5个与柑橘Tr相关的QTLs,其中1个QTL位点分布在LW2上,命名为qtl2.1(Tr),贡献率为21.0%;4个QTLs分布在LW3上,命名为qtl3.1(Tr)、qtl3.2(Tr)、qtl3.3(Tr)和COSC3-35(Tr),贡献率分别为21.8%、21.9%、20.5%和20.5%。(3)在LOD≥3.60的条件下,共检测到6个与柑橘Ci相关的QTLs,全部分布在LW2上,命名为qtl2.2(Ci)、qtl2.3(Ci)、qtl2.4(Ci)、qtl2.5(Ci)、qtl2.6(Ci)和COSC2-7(Ci),贡献率分别为19.0%、19.9%、19.7%、19.3%、18.9%和20.1%。(4)在LOD≥2.57的条件下,共检测到4个与柑橘Cond相关的QTLs,其中1个QTL位点分布在LW2上,命名为COSC2-24(Cond),贡献率为13.9%;2个QTLs分布在LW3上,命名为qtl3.3(Cond)和COSC3-35(Cond),贡献率均为13.8%;1个QTL位点分布在LW9上,命名为Lc SSR115(Cond),贡献率为15.5%。(5)在LOD≥2.33的条件下,共检测到5个与柑橘叶绿素含量相关的QTLs,其中4个QTLs分布在LW6上,命名为qtl6.1(chl)、qtl6.2(chl)、qtl6.3(chl)和COSC6-18(chl),贡献率分别为12.8%、13.3%、12.6%和13.2%;1个QTL分布在LW9上,命名为Lc SSR122(chl),贡献率为14.1%。7.光合作用相关QTL所在区域的基因分析。重点对定位的净光合速率QTL所在的区间进行了基因分析,发现在Lc SSR127~COSC3-35区间,含有烟酰胺腺嘌呤二核苷酸(NAD+)转运体、渗透压钾转运体等与光合作用可能直接相关的功能基因,这些功能基因的发现,为开展这些基因的研究以及开展柑橘光合作用的MAS(Marker-assisted selection)研究提供了参考。更多还原

【Abstract】 Citrus is the largest fruit crop in the world,and one of the most important fruit trees in China.Photosynthesis is the foundation of forming biological production of plant,not only can it affect the grouth of citrus,but also it can influence the yield and quality of citrus fruit.However,as an evergreen plant,photosynthesis efficiency of citrus is lower than most of C3 woody plants.Photosynthetic efficiency is a quantitative trait and is affected by many factors.Genetic research of photosynthetic efficiency is less reported in citrus than in other plants.And as far as we know,there are no public reports in citrus photosynthesis researches in such studies as measuring important traits related to photosynthesis in a cross population and probing their genetic relationship,developing new molecular markers to construct linkage map and digging quantitative trait loci(QTLs)and close linked molecular markers of important traits related to photosynthesis.These studies will be important for the research and improvement of citrus photosynthesis efficiency both in theory and in practice,and it also contributes to improving the breeding level of photosynthesis efficiency of citrus.In the present work,new COS(Conserved Ortholog Sequence)and SSR(Simple Sequence Repeat)markers were developed to construct a citrus genetic linkage map.Important photosynthesis-related traits were measured and analysed in a cross population of a local sweet orange cultivar ?Li2’(C.sinensis(L.)Osb.)and a tangor ?Wan2’(C.unshiu(Mark.)Marc.× C.sinensis(L.)Osb.)with 80 F1 progenies by the LI-6400 XT Portable Photosynthesis System and the SPAD-502 Chlorophyll Meter Model.Genetic relationshipes of the traits were studied and photosynthesis-related QTLs were digged.The main results were as follows:1.PCR primer design.According to the published C.sinensis and C.clementina genomic sequencing information,1184 conserved ortholog sequences(COS)of C.clementina genome were downloaded on the phytozome.net.275 sequences with one or more Gaps in the promoter regions or coding regions of clementine and sweet orange were obtained.261 COS primers were successfully designed.Moreover,65 CSSRC primers related to COS were designed according to the search results of specific areas of clementine genome.2.PCR primer screening and amplification.All primers including 326 newly developed COS and CSSRC primers were tested,and 198 showed polymorphism among two parental genotypes and six progenies of their F1 population.164 COS of 198 primers were selected with a polymorphic rate of 63%,together with 34 CSSRC primers with a polymorphic rate of 52%.Later,amplification analysis on all samples was proceeded using selected primers and 41 previously published SSR primers.3.Genetic linkage map construction.Two citrus genetic linkage map were constructed by combining the segregating data.One map integrated only 165 COS markers with ten linkage groups and covered 816.1 c M of citrus genome with the mean of 4.9 c M between adjacent loci.The other map included 240 markers contained all COS markers and other SSR markers.It had nine linkage groups spanning 1186.4 c M,and the average genetic distance between markers was 4.9 c M.4.Measure and analysis of photosynthesis-related traits.(1)Net photosynthetic rate(Pn).The average of the mapping population was 6.49 μmol/m2·s with a variation range from 2.27 to 9.92 μmol/m2·s,and the frequency distribution of Pn was a normal distribution.Pn of the parents were 6.45 μmol/m2·s and 6.68 μmol/m2·s respectively,and the mid-parents value was 6.57 μmol/m2·s;The population mean and the mid-parents value were very close,but the Pn of some offsprings has a great variation with obviously higher or lower than the parents,so the improvement space of the cross breeding is larger.(2)Transpiration rate(Tr).The average of the mapping population was 2.00 mmol/m2·s with a variation range from 0.80 to 3.76 mmol/m2·s,and the frequency distribution of Tr was a normal distribution.Tr of the parents were 1.74 mmol/m2·s and 1.80 mmol/m2·s respectively,and the mid-parents value was 1.77 mmol/m2·s.The population mean was higher than the mid-parents value,but the Tr of some offsprings has a great variation with obviously higher or lower than the parents,so the improvement space of the cross breeding is larger.(3)Intercellular CO2 concentration(Ci).The average of the mapping population was 202.07 μmol/mol with a variation range from 119.74 to 272.74 μmol/mol,and the frequency distribution of Ci was a normal distribution.Ci of the parents were 249.39 μmol/mol and 255.04 μmol/mol respectively,and the mid-parents value was 252.22 μmol/mol,The population mean was lower than the mid-parents value,but the Ci of some offsprings were obviously higher or lower than the parents,so it has a certain space of the cross breeding to improve.(4)Conductance to H2O(Cond).The average of the mapping population was 0.08 mol/m2·s with a variation range from 0.03 to 0.15 mol/m2·s,and the frequency distribution of Cond was a normal distribution.Cond of the parents were 0.12 mol/m2·s and 0.13 mol/m2·s respectively,and the mid-parents value was 0.13 mol/m2·s.The population mean was lower than the mid-parents value.the Cond of some offsprings were obviously lower than the parents,but the offsprings higher than the parents were less.It showed that using cross breeding to improve Cond is limited.(5)Chlorophyll concentration SPAD value.The average of the mapping population was 71.55 with a variation range from 58.85 to 79.28,and the frequency distribution of SPAD value was a normal distribution.SPAD value of the parents were 77.42 and 79.37 respectively,and the mid-parents value was 78.40,The population mean was lower than the mid-parents value,Although the SPAD value of some offsprings were obviously lower than the parents,the offsprings higher than the parents were less.It showed that it is difficult to obviously improve chlorophyll concentration using cross breeding.5.Correlation analysis.Correlation of photosynthesis-related traits was analysed using a SPSS statistics software.The results appeared as shown below:(1)The Pn showed highly significant positive correlation respectively to Tr and Cond with correlation coefficient of 0.639 and 0.762,and it was significant positive correlation to SPAD value with correlation coefficient of 0.251,it was positively correlated to Ci with correlation coefficient of 0.105.It showed that Pn could significantly affected by Tr,Cond and chlorophyll concentration.(2)The Tr showed very significant positive correlation respectively to Ci and Cond with correlation coefficient of 0.552 and 0.742,and it was positively correlated to SPAD value with correlation coefficient of 0.210,The results showed that Tr,Ci and Cond have a very close relationship.(3)The Ci showed very significant positive correlation to Cond with correlation coefficient of 0.602,and it was positively correlated to SPAD with correlation coefficient of 0.171.It showed that Ci and Cond have a highly close relationship.(4)The Cond showed positively correlated to SPAD value with correlation coefficient of 0.189,it showed that chlorophyll concentration may wasn’t main influential factor for Cond.6.QTL analysis.Based on the second citrus genetic linkage map and photosynthesis traits data,QTL of Pn,Tr,Ci,Cond and SPAD of chlorophyll concentration were indentified by using Map QTL 6.0.The main results were as follows:(1)On the basis of LOD≥3.10,five QTLs of Pn,respectively named as qtl3.2(Pn),qtl3.3(Pn),COSC3-35(Pn),qtl6.4(Pn)and TAA1(Pn),were mapped on the linkage group LW3 and LW6 and they explained 16.6%,17.6%,17.8%,16.4% and 16.5% of phenotypic variation respectively.(2)Based on LOD≥3.90,five QTLs of Tr,respectively named as qtl2.1(Tr),qtl3.1(Tr),qtl3.2(Tr),qtl3.3(Tr)and COSC3-35(Tr),were located on the linkage group LW2 and LW3,and they explained 21.0%,21.8%,21.9%,20.5% and 20.5% of phenotypic variation respectively.(3)On the basis of LOD≥3.60,six QTLs of Ci,respectively named as qtl2.2(Ci),qtl2.3(Ci),qtl2.4(Ci),qtl2.5(Ci),qtl2.6(Ci)and COSC2-7(Ci),were located on the linkage group LW2,and they explained 19.0%,19.9%,19.7%,19.3%,18.9% and 20.1% of phenotypic variation respectively.(4)Based on LOD ≥ 2.57,four QTLs of Cond,respectively named as COSC2-24(Cond),qtl3.3(Cond),COSC3-35(Cond)and Lc SSR115(Cond),were located on the linkage group LW2,LW3 and LW9,and they explained 13.9%,13.8%,13.8% and 15.5% of phenotypic variation respectively.(5)On the basis of LOD ≥ 2.33,five QTLs of chlorophyll concentration,respectively named as qtl6.1(chl)、qtl6.2(chl)、qtl6.3(chl),COSC6-18(chl)and Lc SSR122(chl),were located on the linkage group LW6 and LW9,and they explained 12.8%,13.3%,12.6%,13.2% and 14.1% of phenotypic variation respectively.7.Genetic analysis.The genomic region of QTL of the net photosynthetic rate was analysed,and some functional genes directly related to photosynthesis were found,such as chlorophyll nicotinamide adenine dinucleotide(NAD+)transporter 1 and osmotic stress potassium transporter,were found in Lc SSR127~COSC3-35.it will provide reference for these genes and citrus photosynthesis marker-assisted selection breeding research.更多还原