摘要
栽培燕麦(Avena sativa L.)隶属禾本科燕麦属一年生草本植物,是一种世界性栽培的优质作物,粮饲兼用,兼有食疗功能。全球燕麦品种资源丰富,国外主要栽培带稃型燕麦,国内主要栽培裸粒燕麦。燕麦是一种健康食品,其蛋白含量几乎等同于大豆。世界卫生组织研究表明,燕麦蛋白含量与肉类、牛奶和鸡蛋相当。
燕麦有着重要的商业价值,对燕麦基因组起源进行深入了解可以为育种提供相关背景知识,从而有助于改良燕麦品质；对春化和光周期影响燕麦抽穗期进行调查有助于提高燕麦产量。目前关于燕麦D染色体组组份分析的报道几乎没有,而D染色体组的起源也未知,同时春冬型燕麦产生的原因尚不确定。本文通过对燕麦最小染色体18D质粒文库进行测序,根据序列组成设计FISH探针,从而特异标记燕麦属植物染色体；对燕麦IOI (Integrated Oat Improvement)居群进行光照和温度控制,统计其抽穗期,并对抽穗期差异很大的植株进行春化和光周期基因片段克隆测序,以及对这些植株的籽粒进行储藏蛋白遗传多样性分析。为进一步了解燕麦D染色体组起源和深入了解春冬型燕麦产生的原因提供参考。其主要结果如下：
(1)对栽培燕麦最小染色体18D的基因组文库进行测序,得到来自314个克隆的DNA序列,组配成了99个完全一致或基本一致的片段重叠群(contigs)序列。用Censor检测在RepBase中被描述过的已知重复序列。8个重复类型散布在50个片段重叠群里。多数重复类型属于7种转座子和还原转座子类型。对重复片段进行比对分析,发现22个片段重叠群里的24个区域与其他物种存在基因同源,47个区域与燕麦及其近缘种基因组序列匹配。这些结果展示了燕麦基因组序列转座子基因片段的类型和密度,也为鉴别燕麦特异或单一序列片段提供可能。结果表明通过分离染色体和简化复杂基因组,鉴别燕麦特异染色体编码区的成功率较低。
(2)通过探针A336和pAml标记A. sativa (AACCDD)根尖中期细胞染色体,结果显示：a)探针A336分布在燕麦染色体的着丝粒；b)探针A336对A染色体组和D染色体组有偏好性,不优先分布在C染色体组着丝粒；c)随探针A336浓度增大,所有染色体着丝粒可能都被标记。结果表明了探针A336可以用于标记燕麦染色体着丝粒。
(3)通过探针A250、A305和A436标记A.canariensis(AcAc)根尖中期细胞染色体,结果显示：探针A250、A305和A436均对A.canariensis的所有Ac染色体识别,表明了18D染色体与Ac染色体组关系极为亲密。
(4)通过探针pITS和pAm1标记A.sativa(AACCDD)和A.canariensis(AcAc)根尖中期细胞染色体,结果显示：a)探针pAm1几乎在A.sativa所有C染色体上出现整条信号；b)探针pITS在A.sativa A染色体或D染色体端部出现6个信号；c)探针pITS在A.canariensis4条染色体端部出现信号。结合已知信息：探针pITS会在2条A染色体端部和4条D染色体端部出现信号,结果表明pITS在Ac染色体上的信号和在D染色体的上的信号分布一致。
(5)通过探针A3-19、pAs120a和pAm1标记A.sativa(AACCDD)、A. maroccana (AACC)、A.vaviloviana(AABB)、A.longiglumis(A1A1)和A. canariensis(AcAc)根尖中期细胞染色体,结果显示：a)探针A3-19分布在染色体端部和近端部；b)探针A3-19在A1染色体组有2个端部和2个近端部信号分布；c)探针A3-19在Ac染色体组有2个近端部信号分布；d)探针A3-19在B染色体组也有2个端部和2个近端部信号分布；e)探针A3-19在D染色体组有2个近端部信号分布。结果表明探针A3-19在Ac染色体组上信号分布与在A1染色体组上信号分布不一致,但是却与在D染色体组信号分布一致；探针A3-19在A染色体组和B染色体组上有一致的信号分布。
(6)燕麦IOI居群植株在日长和温度的不同处理条件下,抽穗与否以及抽穗时间存在很大差异。与低温需求相比,日长对燕麦抽穗期的影响更大。根据植株抽穗期的差异,将供试材料初步分为了冬春两型。结合地理起源,结果进一步显示：在地中海区域,燕麦植株春化反应存在多样性,而春化作用不敏感机制可能发生在地中海北岸。
(7)春化和光周期基因的6个片段用于分析燕麦IOI居群冬春型植株间的遗传差异。TCS单倍型节约网状分析把6个片段序列分成Spring(S)和Winter(W)两个独立的组。春化基因片段(VRN1-6、VRN1-19)和光周期基因片段(VRN3-23、 VRN3-24)的S组包含了Dancer_CDC、Kanota、Ogle、Starter.Buffalo和Heinrich;其W组包含了98-28Cn3、Kingfisher、Fleuron、Wistar、Bage1419/36和Landhafer.而春化基因片段VRN1-1的Kanota却分布在其W组里,春化基因片段VRN1-18的Kanota、Ogle也是分布在其W组。结果揭示了春化和光周期基因在冬春型燕麦植株之间存在一定程度的遗传变异,为了解其他温带谷物春化和光周期基因序列变异提供参考。
(8) SDS-PAGE用于分析冬春型燕麦IOI居群植株球蛋白、谷蛋白和醇溶蛋白的遗传多样性。每份材料的蛋白条带约为7条。球蛋白主要集中在50KD区域,谷蛋白大量分布在26-33KD之间,而醇溶蛋白在小于60KD区域散布。球蛋白条带多态性相对较低,谷蛋白和醇溶蛋白条带多态性高,醇溶蛋白条带最为分散。各份材料之间条带分布具有一定的差异。在春型植株和冬型植株间,醇溶蛋白条带分布相对一致,谷蛋白条带分布一致性较低。结果表明储藏蛋白多样性分布不能很好地揭示春型燕麦和冬型燕麦之间的差异。
综上,探针A250、A305、A436标记结果表明Ac染色体组与D染色体组关系密切；探针pITS和A3-19标记结果表明Ac染色体组的信号分布与D染色体组信号分布一致。因而,栽培燕麦的D染色体组可能来自含有Ac染色体组的二倍体物种Acanariensis。春化和光周期对燕麦IOI居群春冬两型植株抽穗期影响很大；春化和光周期控制基因在春冬两型植株间存在一定遗传变异；储藏蛋白遗传多样性不能很好的揭示春冬两型植株间差异。这些结果表明了春化和光周期对燕麦春冬两型植株影响确实存在一定程度的遗传差异,可能是因为候选基因片段在供试材料中分化所致。
Hexaploid oat (Avena sativa L.) is an annual herbaceous plant in the Poaceae, which is cultivated worldwide for food and fodder, as well as for specialty uses in diet therapy and nutrition.Genetic resources of oat are abundant and variable. Oats that thresh free from their hulls (naked oats) are cultivated widely in China, while hulled oats are most commonly grown in the other countries. Oats are generally considered as a healthy food because they are high in fibre, beneficial oils, and good-quality protein. Oat protein is nearly equivalent in quality to soy protein, which World Health Organization research has shown to be nutritionally equivalent to meat, milk, and egg protein.
Tools for genetic improvement of oat are being developed. However, genomic analysis of oat is challenging due to its large, repetitive hexaploid genome. Studies reported of this thesis were conducted to better understand the cytogenetic organization of the oat genome, and to provide the oat research community with new tools for characterizing genetic variability in oat, and the application of genomic tools to discover genetic factors affecting flowering time, an important adaptive characteristic of oat. In this study, we have sequenced and analysed18D-specific library from hexaploid oat(Avena sativa L.), then chose some special sequences as FISH probes to detect species in Avena; we also investigated response to vernalization and photoperiod in a population of384diverse oat varieties (Integrated Oat Improvement, IOI). We then cloned and sequenced the fragments of a candidate gene and analysed the genetic diversity of storage proteins in oat. The main results are as follows:
(1) We have sequenced, assembled, and characterized a set of complexity-reduced genomic clones derived from a chromosome18D-specific library from hexaploid oat. Sequences from314clones were assembled into99contigs of identical or nearly identical sequence. The Censor tool was used to identify similarity to known and characterized repeat sequences in RepBase. Eight repeat classes were scattered throughout50contigs, with most repeats belonging to seven transposon and retrotransposon classes. After accounting for known repeats, additional matches to orthologous genes from other species were identified in24regions of22contigs, and an additional47regions matched genomic sequences from oat and other related species. These results provide information about the types and density of transposable elements in the oat genome, as well as the potential for identifying unique or chromosome-specific sequence elements in oat. Overall, these results predict a low success rate in identifying chromosome-specific coding regions in oat through chromosome isolation and genome complexity reduction.
(2) The probes of A336and pAml were used to label chromosomes in mitotic metaphase plates of A. sativa (AACCDD). The results are as follows:a) chromosomes in A. sativa were labelled in the centromere by probe A336. b) A genome and D genome are prior to be labelled by probe A336. c) all chromosomes would be labelled with the high concentration of probe. These results suggested probe A336may be used to label centromeres in A. sativa.
(3) The probes of A250, A305, and A436were used to label chromosomes in mitotic metaphase plates of A. canariensis (AcAc). As a result, all chromosomes in A. canariensis probes were labelled separately by three probes A250, A305, and A436. This result indicated there might be a close relationship bettween18D chromosome and Ac genome.
(4) The probes of pITS and pAml were used to label chromosomes in mitotic metaphase plates of A. sativa (AACCDD) and A. canariensis (AcAc). The results are as follows:a) chromosomes in C genome from A. sativa were labelled by probe pAm1. b) six telomeres in A/D genome from A. sativa were labelled by probe pITS. c) four telomeres in A. canariensis (Ac) were labelled by probe pITS. Together with previous results that two telomeres in A genome and four in D genome from A. sativa were labelled by probe pITS, we could infer there might be a similar distribution of pITS bettween Ac genome and D genome.
(5) The probes of A3-19, pAs120a and pAml were used to label chromosomes in mitotic metaphase plates of A. sativa (AACCDD), A. maroccana (AACC), A. vaviloviana (AABB), A. longiglumis (A1A1), and A. canariensis (AcAc). The main results are as follows:a) all signals labelled by A3-19are detected in telomeres or subtelometres. b) two telomeres and two subtelomeres in A genome were labelled by probe A3-19. c) similarly, two telomeres and two subtelomeres in B genome were labelled by probe A3-19. d) two subtelomeres in Ac genome were labelled by probe A3-19. e) two subtelomeres in D genome were labelled by probe A3-19. These results suggested the follows:a) distribution of A3-19on Ac genome is different to A1genome, but similar to D genome. b) A genome is close to B genome.
(6) The IOI population was at different controlled temperatures and light regimes to determine the time to flowering under each condition. It was found that day-length had a greater influence on heading date, and that most oat varieties did not flower under short- day conditions. Based on the heading date, winter types and spring types were identified. Taking into account of geographical origin, the results further demonstrated that vernalization insensitive forms might occur in the north Mediterranean region and the adjacent northern territories.
(7) Six fragments of vernalization and photoperiod candidate genes were used to analyse genetic diversity between winter type and spring type. The fragment sequences were clustered into spring and winter groups by TCS network analysis. Dancer_CDC, Kanota, Ogle, Starter, Buffalo, and Heinrich were contained in spring group of vernalization gene fragments (VRN1-6, VRN1-19) and photoperiod gene fragments (VRN3-23, VRN3-24), while the Kingfisher, Fleuron, Wistar, Bage1419/36, and Landhafer were include in winter group. However, the winter group also included Kanota VRN1-1, as well as Kanota and Ogle VRN1-18. These results indicated that there should be some genetic difference between winter type and spring type in oat population. These sequences provided a guide to explore the vernalization and photoperiod genes in other temperate cereals.
(8) SDS-PAGE was used to analyse the genetic diversity of globulin, glutelin, prolamin between winter type and spring type in IOI population. More than seven protein bands were found in each accession. Globulin was focused on the region of50KD, whereas glutelin was manily distributed in the region between26KD and33KD. However, prolamin was scattered in the region less than60KD. There is a low polymorphism for globulin, and relatively high in glutelin, prolamin bands are most dispersed. Each accession displayed a variable distribution of bands. However, there was a homogeneous distribution of prolamin bands among spring types, separately. These results indicated the difference of spring type and winter type cannot be clarified well by their seeds storage proteins diversity.
As an overall summary:probes of A250, A305, and A436suggested there might be a close relationship bettween18D chromosome and Ac genome; probe of pITS and A3-19showed there might be a similar distribution of pITS bettween Ac genome and D genome. These results indicated Ac genome is close to D genome, and A. canariensis (AcAc) could be the D genome donor of Avena sativa. Vernalization and photoperiod had a great influence on heading date of spring type oat and winter type in the IOI population; there should be some genetic difference between winter type and spring type in oat population; there is a relative abundance of genetic diversity of protein between winter type and spring type in IOI population but winter type and spring type cannot be clarified well by their storage protein diversity. These results indicated there are differences in vernalization and photoperiod response that are possibly attributable to specific candidate genes segregating in the IOI population.
引文
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