柑橘纯合种质创制及早金甜橙DH系代谢与转录组分析

发布时间：2018-01-02 05:34

本文关键词：柑橘纯合种质创制及早金甜橙DH系代谢与转录组分析　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：纯系材料在植物育种及基因组学研究中发挥着重要作用。然而,由于柑橘具有高度杂合性、童期长、树体庞大、珠心胚和某些品种自交不亲和等特性,采用常规育种途径在柑橘上很难获得纯合种质。诱导配子体胚胎发生,包括花药培养、小孢子离体培养、诱导孤雌生殖、离体子房培养等,可以直接获得完全的纯系材料,省时省力。自上世纪70年代,柑橘通过诱导配子体胚胎发生已获得一些纯合材料,然而这些纯系大都具有相似的遗传背景,且都存在再生困难的问题,使得柑橘纯系种质资源种类及数量无法达到应用的要求,限制了柑橘纯系材料的应用。本文通过花药培养和诱导孤雌生殖的方法,创造柑橘纯系种质,以拓展柑橘纯合种质资源类型及数量;对早金甜橙及其DH系进行芳香烃代谢及转录组差异分析,评价其单倍化过程中代谢及基因表达变化。主要结果如下:1、花药培养诱导柑橘纯系。通过四种诱导培养基对15个柑橘品种进行花药培养诱导,并对诱导获得的全部胚状体及愈伤组织进行倍性及分子标记鉴定,获得了早金甜橙(Citrus sinensis Osbeck)单倍体系15个,纯合二倍体系10个(其中2个DH系已再生植株,为甜橙纯合植株诱导成功的首次报道),同源四倍体系2个;红夏橙(C.sinensis Osbeck)单倍体愈伤组织系1个;暗柳橙(C.sinensis Osbeck)单倍体愈伤组织系2个;鸡尾葡萄柚(C.paradis Macf.)单倍体愈伤组织系1个(为葡萄柚纯系诱导成功的首次报道)。利用均匀分布于柑橘9条染色体的31对SSR引物对早金甜橙16个纯系的遗传组成进行深入分析,证实这些纯系的纯合性为100%,并且起源于不同基因型的小孢子;通过对其带型统计进行小孢子等位基因分离规律分析,表明在29个双带位点中,有16个位点的等位基因分离遵循1:1的分离规律。2、诱导孤雌生殖创制柑橘纯系。利用γ射线辐射的枳(Poncirus trifoliata Raf.)和铜城72-1锦橙(C.sinensis Osbeck)不同辐射剂量的花粉对HB柚(C.grandis Osbeck)、高班柚(C.grandis Osbeck)、凤凰柚(C.grandis Osbeck)、华农红柚(C.grandis Osbeck)、秋辉橘橙([C.reticulata Blanco×(C.paradis Macf.×C.reticulata Blanco)]×(C.reticulata Blanco×C.sinensis Osbeck))以及尤力克柠檬(C.limon Burm.f.)6个柑橘单胚或者单多胚混合品种授粉诱导孤雌生殖。花粉活力检测表明辐射对花粉活力没有影响;授粉后对花粉管行为进行观察发现,辐射后的花粉管可以正常萌发伸长至胚珠。经胚挽救,对获得的植株进行倍性及分子标记鉴定,确定获得HB柚单倍体4株,同时由HB柚×255Gy枳授粉组合获得三倍体1株、由华农红柚×500Gy枳授粉组合获得三倍体1株。核型分析和分子标记确定了HB柚单倍体来源于孤雌生殖。3、三倍体花粉诱导孤雌生殖。利用三倍体葡萄柚Melgold(C.paradis Macf.)与Oroblanco(C.paradis Macf.)的花粉对HB柚、高班柚、凤凰柚、华农红柚、秋辉及沙田柚(C.grandis Osbeck)6个柑橘单胚品种授粉诱导孤雌生殖,通过胚挽救并对获得的植株进行倍性鉴定,确定由HB柚×Melgold授粉组合获得三倍体植株7株,由高班柚×Melgold授粉组合获得三倍体1株,由秋辉×Oroblanco三倍体组合获得四倍体1株。4、早金甜橙及其DH系芳香烃代谢及转录组差异分析。对早金甜橙及其DH系叶片进行形态学分析,其叶面积、翼叶面积、叶面积与翼叶面积比及叶形指数均存在显著性差异。通过GC-MS对芳香烃代谢产物进行鉴定分析,共检测到45种芳香烃类化合物,其中42种物质含量存在显著性差异;在DH系,其总含量高于早金甜橙,含量上调积累的有12种,含量下调积累的有30种,其中20种物质未检测到。通过RNA-seq对基因表达差异进行分析,以FDR≤0.001且表达量≥2倍为筛选标准,共筛选到差异表达基因1863个,占总基因量的5.77%;其中DH系上调表达基因506个(占总差异基因的27.16%),下调表达基因1357个(占总差异基因的72.84%)。这些差异基因主要参与防御、逆境及刺激响应、代谢及细胞死亡等生物过程,其中上调表达差异基因还参与芳香化合物合成过程,这可能是引起DH系芳香烃代谢差异的主要原因。
[Abstract]:Pure lines play an important role in plant breeding and genomics research. However, due to the citrus highly heterozygous, long juvenile period, large tree, nucellar embryo and some varieties of self incompatibility and other characteristics, using conventional breeding in citrus. It is difficult to obtain a homozygous gametophyte embryo induction. Happen, including anther culture, isolated microspore culture, parthenogenesis, ovary culture in vitro, can directly obtain pure lines, complete time-saving. Since the last century in 70s, through the induction of embryogenesis in Citrus gametophyte has obtained some homozygous materials, however, most of these homozygous lines with similar genetic background, and there is regeneration difficult problem, the pure citrus Germplasm resources types and quantity cannot meet the application requirements, limit the application of Citrus pure lines. This paper through anther culture and induction of parthenogenesis, Pure creation of Citrus germplasm, to develop citrus germplasm resources of homozygous type and quantity of earlygold sweet orange; and its DH system to analyze the differences of aromatic hydrocarbon metabolism and transcriptome, metabolism and gene expression to evaluate its haploid process. The main results are as follows: 1. The induction of Citrus pure induction medium for anther culture. The 15 varieties of Citrus anther culture induced by four, and ploidy and molecular marker identification of embryoid and callus induction of the earlygold sweet orange (Citrus sinensis Osbeck) system of 15 haploid, 10 homozygous diploid lines (DH lines have been reported for the first time in 2 regeneration plants. For the orange homozygous plants successfully induced), which is four times the 2 homologous system; red Valencia Orange (C.sinensis Osbeck) haploid callus line 1; anliucheng (C.sinensis Osbeck) haploid callus line 2; chicken tail grapefruit (C.paradis Macf.) Haploid callus line 1 (for the first time reported for grapefruit pure lines successfully induced). Using 31 pairs of SSR primers were evenly distributed on 9 chromosomes of Citrus genetic earlygold sweet orange 16 pure lines composed of in-depth analysis, confirmed the homozygosity of these homozygous lines was 100%, and the microspore originated from different genotypes; through the band type statistics allele separation law of microspore analysis, show that in 29 pairs of loci, 16 alleles from.2 follow the separation law of 1:1, parthenogenesis create citrus pure lines. Using gamma ray radiation (Poncirus trifoliata Raf.) and Poncirus Tongcheng 72-1 Jincheng orange (C.sinensis Osbeck) with different radiation doses of pollen (C.grandis Osbeck), HB grapefruit pomelo (C.grandis Osbeck), high (C.grandis Osbeck), Phoenix pummelo (C.grandis Osbeck) Huanong red grapefruit, orange orange ([C.reticulata Blanco Qiu Hui * (C.paradis Macf. * C.reticulata * Blanco)] (C.reticulata) Blanco * C.sinensis Osbeck) and Eureka Lemon (C.limon Burm.f.) 6 citrus single embryo or single hybrid varieties of Polyembryonic pollination induced parthenogenesis. Pollen viability test showed that radiation had no effect on pollen viability; after pollination of pollen tube behavior were observed, the radiation tube normal germination extends to the ovule. Through embryo rescue, ploidy and molecular marker identification of plants, obtained 4 strains of HB pummelo haploid, and by HB pummelo * 255Gy trifoliate pollination combination to obtain triploid 1 strains by Huanong red grapefruit 500Gy * trifoliate pollination combination to obtain triploid 1 strains. Karyotype analysis and molecular marker determination HB grapefruit originated from the parthenogenesis haploid.3, parthenogenesis induced triploid pollen. The triploid of Melgold (C.paradis Macf.) and grapefruit Oroblanco (C.paradis Macf.) pollens of HB pummelo, Phoenix High pomelo, pomelo, Huanong red grapefruit, autumn Hui (C.grandis Osbeck) Shatinyu and 6 citrus Monogerm varieties pollination induced parthenogenesis, through embryo rescue and ploidy identification of the plant, determined by the combination of HB pummelo * Melgold pollination to obtain triploid 7 strains by the combination of high class pummelo * Melgold pollination obtain triploid tetraploid 1 strains, 1 strains of.4 by the combination of autumn Hui * Oroblanco triploid, difference analysis of aromatic hydrocarbon metabolism and transcription group earlygold sweet orange and DH. Morphological analysis of earlygold sweet orange and DH leaves, leaf area, wing leaf area, leaf area and leaf area ratio and leaf index were wing there was a significant difference. The GC-MS for aromatic metabolites were identified and analyzed, 45 kinds of aromatic compounds were detected, of which there is a significant difference between the 42 kinds of substances; in DH, the total content is higher than that of earlygold sweet orange, raised the content of accumulation There are 12 kinds of content, reduced accumulation of 30 species, including 20 kinds of substances were not detected. Through the analysis of the differences on the expression of RNA-seq gene in FDR is less than or equal to 0.001 and the expression amount is greater than or equal to 2 times the screening criteria were screened 1863 differentially expressed genes, the total amount of gene 5.77%; wherein DH is raised the expression of 506 genes (total differential gene 27.16%), down regulate the expression of 1357 genes (total 72.84% differentially expressed genes). These genes were mainly involved in defense, stress and stimulation response, metabolism and cell death and other biological processes, which up-regulated differentially expressed gene is also involved in the synthesis process of aromatic compounds, which may be mainly caused by the the reason of DH aromatic hydrocarbon metabolism differences.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S666

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