木荷繁殖生物学特性及种子园交配系统研究

发布时间：2018-01-02 01:20

  本文关键词：木荷繁殖生物学特性及种子园交配系统研究　出处：《中国林业科学研究院》2017年博士论文　论文类型：学位论文

  更多相关文章： 木荷 种子园 繁殖生物学 自交不亲和 交配系统 传粉规律

【摘要】：木荷(Schima superba)为我国东部湿润亚热带常绿阔叶林的主要建群树种,其材性优良,抗逆性强,是我国南方各省区中主要的珍贵优质阔叶用材和高效生物防火树种。自2001年开展木荷良种选育以来,已在浙江、福建、江西、广东和重庆等营建了超过100ha的木荷无性系种子园。但对木荷的繁殖生物学特性和种子园的遗传管理缺乏系统研究,难以为种子园的经营管理及良种生产提供理论和实践指导。鉴于此,本文以进入正常开花结实期的木荷种子园为研究对象,对木荷开花综合特征、传粉机制、繁育系统等进行了深入研究,揭示木荷的繁殖生物学特性,为其种子生产、资源利用和杂交育种提供重要的实践指导和科学依据。同时,研究和揭示了种子园无性系花期同步性、遗传交配系统状况及传粉规律,为木荷种子园营建和高效的遗传管理提供了重要的科学理论依据。主要研究结果如下:1.通过定点观察,对木荷花部综合特征及繁育系统的研究结果表明,木荷5~7月份开花,单花期4~5天。花白色,常多朵排成总状花序,平均每个花序着生7朵花。开花当天柱头即具有可授性,且在整个单花期内均保持较高的可授性。花粉失活较快,花粉活性在花朵刚开放时最高(83.78%),至花朵凋谢时花粉活性仅为5.45%。花开后柱头快速伸长,直至略高于花药,形成柱头和花药在空间上的分离。木荷的杂交指数(OCI)值为4,花粉胚珠比(P/O)值约为6686.67,结合控制授粉试验显示,木荷繁育系统以异交为主,不存在无融合生殖,传粉过程需要传粉者。2.木荷为异花授粉植物,其种子生产必须依靠昆虫传粉。通过观察认为,中华蜜蜂(Apis cerana)、白星花金龟(Protaetia brevitarsis)和棉花弧丽金龟(Popillia mutans)是木荷的主要传粉昆虫。中华蜜蜂和棉花弧丽金龟的访花高峰均发生在10:00~11:00,白星花金龟则无明显的访花高峰。3种传粉昆虫体表均携带花粉,白星花金龟的携粉量显著高于棉花弧丽金龟(P0.001)和中华蜜蜂(P0.001),棉花弧丽金龟的携粉量显著高于中华蜜蜂(P0.001)。体表不同部位携粉量不同,3种传粉昆虫主要的携粉部位为胸腹部,同时也是接触花药和柱头最为频繁和力度最大的部位,因此它们的传粉方式均为腹触式传粉。中华蜜蜂每次访花的花粉移出数、柱头花粉沉降数及传粉者效率均低于白星花金龟和棉花弧丽金龟,但不显著(P0.05),而中华蜜蜂在访花频率上占有明显优势,分别为白星花金龟的29倍和棉花弧丽金龟的8.3倍。综合各项特征表明,中华蜜蜂是木荷最有效的传粉者。3.进一步对木荷自交与异交的亲和性进行比较研究。结果显示,自交与异交花粉均能在柱头上正常萌发,生长至花柱基部。异交花粉管能成功进入子房和胚囊,而自交花粉管不能进入,表明木荷为晚期自交不亲和(LSI)植物。自交和异交授粉均能引起子房SOD、POD和CAT活性的增加。自交和异交授粉48 h,氨基酸含量差异最大,其中自交授粉精氨酸(Arg)的含量显著高于异交(P0.001),表明Arg含量的变化可能与木荷晚期自交不亲和有关。授粉后子房生长素(IAA)和玉米素(ZT)呈先升高后下降的趋势。授粉48 h,异交子房IAA和ZT含量均显著高于自交(P0.05),表明子房中高水平的IAA和ZT有利于授粉受精正常进行。4.差异蛋白组学研究结果显示,自交和异交授粉48 h后的子房存在82个差异蛋白点,经质谱和数据库检索成功鉴定出58个蛋白点,其中自交授粉中表达丰度升高的蛋白点有15个,表达丰度降低的有43个。异交授粉后肌动蛋白和蛋白酶体表达量显著大于自交授粉,而过氧化物酶蛋白、蛋白质二硫键异构酶及植物凝集素蛋白的表达量显著低于自交授粉。转录组测序结果显示,自交与异交授粉48 h子房间筛选出509个差异表达基因,其中与信号转导(Ca2+信号、蛋白激酶与蛋白磷酸酶等)、防御(细胞色素P450与MLP31等)、氨基酸代谢(草酰乙酸脱羧酶等)等相关的基因在自交子房中的表达量显著高于异交,促进生长类激素代谢相关基因的表达量显著低于异交。另外,自交子房中还发现2个特异表达的转录因子:MYB和bHLH,两者共同参与调控木荷晚期自交不亲和反应。这些基因在转录组中表达结果与qRT-PCR分析结果一致。综合分析表明,木荷晚期自交不亲和反应发生时伴随着一系列的信号转导与防卫响应,是一个主动抑制自交花粉管在子房中生长的过程。5.连续2年对木荷种子园中19个无性系进行花期、花量调查,分析了各无性系的开花物候特征和花期同步指数。相关分析结果表明,花量与花期长度、座果数存在显著正相关关系,花期长度和座果数也存在显著正相关关系。无性系组合间花期同步指数存在极显著变异(P0.01),2015年和2016年的变化范围分别为0.552~0.857和0.406~0.808,变异系数分别为12.016%~46.476%和15.375%~51.202%。2015年和2016年的平均花期同步指数分别为0.758和0.713,不存在显著变异(P0.05)。年度间花期同步指数相关系数为0.229,表明年度间花期同步指数具有一定的相关性。种子园无性系年度间花期同步指数较为稳定,可根据花期同步指数对无性系进行筛选和优化,同时辅以人工授粉等措施弥补花期同步性差异,以提高种子园种子产量和质量。6.同时,以木荷种子园投产初期自由授粉子代为研究对象,运用13对SSR引物,对种子园内44个亲本及328个子代进行分析。结果显示,子代群体包含亲本群体所有的等位基因。亲本群体遗传多样性水平适中,子代群体遗传多样性较亲本略有降低,观测杂合度略高于亲本,说明子代群体中实际观察到的杂合单株的比例较亲本有所增加,但差异不大。子代群体的F值为-0.143,存在杂合子过剩。多位点交配系统分析结果表明:种子园异交率较高,亲本的近交现象不显著,但有效花粉供体数目较少。单位点和多位点父本相关性的差值(0.012)大于0,表明只有小部分花粉供体是近亲关系。11个家系间异交率差异不明显,少数存在近交现象。11个家系的父本相关性和有效花粉供体数目变化较大,分别为0.210~0.762和1.3~4.8,说明各家系的父本相关性程度不一致,其中31号最高,最低的是48号。整体而言,木荷种子园异交率高,近交现象不明显;无性系之间基因交流相对充分,遗传多样性丰富,子代能保持亲本所具有的较高的遗传多样性。7.13对SSR引物在木荷种子园中检测到亲本和子代群体总的多态性信息含量(PIC)、观测杂合度(Ho)和期望杂合度(He)分别为0.679、0.714和0.725。在95%的可信水平下总共鉴定得到203个全同胞家系,平均每个采种母树与18个父本产生子代。在自由授粉状态下,木荷无性系种子园自交率为1.5%,自交现象很弱,其交配方式以异交为主。父本繁殖贡献率在0.49%~7.77%之间,平均为2.44%。木荷种子园的主要传粉距离集中在0~60 m,检测到的最大传粉距离为120 m,其中0~60 m的传粉距离内产生的子代数量占已确定父本子代群体的80%以上。种子园存在一定的花粉污染,为7.01%。综合分析认为,木荷种子园花粉传播较为均匀,种子园与花粉污染源隔离距离应在60m以上,120 m以上最佳,以防止传粉昆虫的长距离传粉活动造成花粉污染。
[Abstract]:Schimasuperba (Schima superba) the main constructive species in East China moist subtropical evergreen broad-leaved forests, its excellent material properties, strong resistance, is the main precious hardwood species and efficient biological fire provinces in the south of China. Since 2001 to carry out breeding in Schima superba has been in Zhejiang, Fujian, and Jiangxi. Guangdong and Chongqing construction of schimasuperba clonal seed orchard of more than 100HA. But the lack of systematic research on the genetic management of Schima superba reproductive characteristics and seed orchard, to provide theoretical and practical guidance for seed orchard management and seed production. In view of this, this paper to enter the normal flowering and fruiting stage of Schima superba seed orchard for research the object, flowering comprehensive characteristics, pollination mechanism of Schima superba, breeding system conducted in-depth research, reveal the reproductive characteristics of Schima superba, its seed production, utilization and breeding resources to provide heavy To practice guidance and scientific basis. At the same time, with the study of clonal seed orchard of flowering synchrony, genetic mating system and pollination, provides an important theoretical basis for the scientific management of Schima superba seed orchard establishment and genetic efficiency. The main results are as follows: 1. through the fixed-point observation research on wood, Lotus comprehensive department the characteristics and breeding system. The results showed that the flowering of Schima 5~7 month, single flower period of 4~5 days. Many white flowers, often arranged in racemes, inflorescences per 7 flowers. On the day of anthesis. With the receptivity, and maintain high receptivity in the single flower. Rapid inactivation of pollen the highest activity in pollen, just the flowers open (83.78%), to flower pollen activity is only 5.45%. flower stigma after rapid elongation, until slightly higher than anther, stigma and anther separation form in space. The hybrid index (wood bearing The OCI value is 4), pollen ovule ratio (P/O) value is about 6686.67 and the combination of controlled pollination experiments showed that Schima superba dominated by outcrossing breeding system, no apomixis, pollination of pollinator.2. Schima cross pollination, the seed production must be pollinated by insects. It has been observed that the bees (Apis, cerana) brevitarsis (Protaetia brevitarsis) and cotton beetle (Popillia mutans) arc is the main pollinators. The bees and Schima superba cotton arc beetle foraging peaks occurred at 10:00~11:00, white gold star turtle no flower peak.3 pollinators were obvious surface pollen carrying visit. Carrying amount of powder brevitarsis was significantly higher than that of cotton arc beetle (P0.001) and the Chinese honeybee (P0.001) powder, carrying amount of cotton was significantly higher than that of the arc beetle bees (P0.001). Different parts of the body with 3 kinds of different amount of powder, the main pollination insects To the site for carrying powder is also in contact with the chest and abdomen, anther and stigma is the most frequent and the largest part, so the pollination they were abdominal touch pollination. The number of each flower visiting Chinese bee pollen removal, pollen deposition and pollinator efficiency were lower than brevitarsis and cotton popillia beetles, but not significantly (P0.05), and the bee occupies obvious advantage in visitation frequency, were 8.3 times 29 times brevitarsis and cotton. The arc beetle showed that the characteristics of Chinese bee pollination is the most effective.3. schimasuperba further affinity for self and schimasuperba to make a comparative study. The results showed that the outcrossing and selfing pollen can germinate on the stigma, growth to base of style. Outcrossing pollen tube can successfully enter the ovary and embryo sac, and self pollen tube can not enter, show that the advanced self incompatibility schimasuperba (L SI). Plant selfing and outcrossing pollination can cause ovary SOD, increase POD and CAT activity. The self and cross pollinated 48 h, amino acid content of the biggest difference, the self pollination of arginine (Arg) were significantly higher than those of outcrossing (P0.001), indicating that the change of Arg content may be related to late wood bearing self incompatibility. Ovary after pollination, auxin (IAA) and Zeatin (ZT) increased firstly and then decreased. Pollination 48 h, outcrossing ovary IAA and ZT were significantly higher than that of self (P0.05), showed that in high levels of IAA and ZT is conducive to the normal pollination and fertilization by.4. protein study results show that the self and cross pollinated ovary after 48 h there are 82 differential protein spots by mass spectrometry and database retrieval successfully identified 58 proteins, including self pollination in up-regulated 15 proteins, expression decreased abundance was 43. Cross pollination after actin Protein and proteasome expression was significantly greater than that of self pollination, and the peroxidase protein expression of two protein disulfide isomerase and lectin protein was significantly lower than that of self pollination. Transcriptome sequencing results showed that the self pollination and cross pollination of 48 h sub room screened 509 differentially expressed genes, including Ca2+ (signal and signal transduction protein kinase and protein phosphatase, etc.), defense (cytochrome P450 and MLP31), amino acid metabolism (oxaloacetate decarboxylase etc.) and related gene expression in hybrids was significantly higher than that in the self promoting outcrossing, expression of growth hormone metabolism related genes was significantly lower than that of outcrossing. In addition, since the sub room also found 2 specific transcription factors: MYB and bHLH, both of them are involved in the regulation of late Schima superba self incompatibility. These gene expression results with qRT-PCR in comprehensive transcriptome. Analysis shows that the late schimasuperba self incompatibility occurs when accompanied by a series of signal transduction and defense response, is an active in inhibiting the growth of the ovary in the process of self pollen tube.5. for 2 consecutive years of 19 clones in seed orchard of Schima superba flowering, flower quantity survey, flowering phenology and synchronous flowering index analysis of the clones. The results of correlation analysis showed that the amount of flowers and flowering length, there is a significant positive correlation between fruit number, there are significant positive correlation between the length of flowering and fruit number. There is significant variation between clones combined flowering synchrony index (P0.01), the change range of 2015 and 2016 were 0.552~0.857 and 0.406~0.808, the coefficient of variation were 12.016%~46.476% and 15.375%~51.202%.2015 and 2016 average flowering synchrony index were 0.758 and 0.713, there is no significant variation (P0.05). The same year fl. Step index correlation coefficient was 0.229, showed that the annual flowering synchrony index has certain correlation. Clones in seed orchard of inter annual flowering synchronization index is relatively stable, according to the index of synchronous flowering clones were screened and optimized, supplemented by artificial pollination and other measures to compensate for the flowering synchrony differences, in order to improve the seed yield and quality of.6. Park seeds at the same time, to the early stage of production of Schima superba seed orchard open pollinated progeny as the research object, using 13 pairs of SSR primers on the seed orchard of 44 parents and 328 offspring were analyzed. The results showed that the offspring population contains parental populations all alleles. The genetic diversity level of moderate parental populations in progeny population, genetic diversity a parent was slightly reduced, the observed heterozygosity is slightly higher than that of the parents, shows the proportion of heterozygous individuals in progeny populations observed were increased, but the difference is small. The offspring group The F-measure is -0.143, there are heterozygote excess. Multilocus mating system analysis results showed that the seed orchard outcrossing rate is higher, the phenomenon of inbreeding were not significant, but the effective number of pollen donors. Less difference unit point and multilocus paternal correlation (0.012) is greater than 0, showed that only a small portion of the pollen donor.11 is a close relationship between families of outcrossing rates were not significantly different, there are few male parent pollen correlation and effective inbreeding.11 pedigrees of donor number changes greatly, respectively 0.210~0.762 and 1.3~4.8, the degree of correlation between male parent families is not a show, the 31 was highest, the lowest is 48 overall. In terms of Schima seed orchard high outcrossing rate, inbreeding phenomenon is not obvious; gene exchange between clones is relatively full, rich genetic diversity, genetic diversity of parent offspring can keep the higher.7.13 of SSR primers in Schima superba seed orchard To the parents and progeny of the total polymorphism information content detection (PIC), observed heterozygosity (Ho) and expected heterozygosity (He) were 0.679,0.714 and 0.725. in 95% of the total level identified 203 full sib families, every mother tree with 18 male parent to produce progeny. In the free pollination condition, Schima superba clonal seed orchard of selfing rate was 1.5%, the phenomenon of self is very weak, the mating pattern dominated by outcrossing. The male reproductive contribution rate between 0.49%~7.77%, average 2.44%. as the main pollination seed orchard in Schima superba distance 0~60 m, detect the maximum pollination distance is 120 m, which produced 0~60 m in the range of Pollination Progeny ascertained father population of more than 80%. The book of pollen contamination in seed orchards, that is the comprehensive analysis 7.01%., Schima seed orchard pollen spread evenly, seed orchard and flower powder pollution source isolation The distance should be above 60m and above 120 m to prevent pollen contamination from pollinating insects in long distance pollination activities.

【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S792.99
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本文编号：1367060