三倍体百合种子胚乳高度发育现象的细胞及分子机制研究

It was observed that the endosperm of lily distant or interploid crosses (3x x 2x/4x) developed well and the crosses were usually successful when it contained ≥ 5 same genomes; otherwise, it hardly developed or aborted and the crosses were usually unsuccessful. Theoretic analysis indicates that this phenomenon related with lily tetra-sporic embryo sac, because it is deduced that “each embryo sac of triploid lilies usually contains an aueuploid egg cell and a hexaploid secondary nucleus regardless of abnormal meiosis”, based on normal megasprogenesis. In order to unveil its mechanism at cellular and molecular level, different interploid crosses will be selected and fertilized with artificial method based on the difference of their endosperm development. After fertilization, the ovules will be analyzed with anatomic method to check the extent of endosperm development. Based on the extent of endosperm development, the ovules and leaves at different stages after fertilization will be sampled. The samples will be tested for their phytohormone and starch synthase RNA will be isolated from ovules and leaf respectively, then cDNA will be prepared. The cDNA of different crosses will be sequenced. By analysis of the above data, it would be possible to find some specific genes related with endosperm development in Lilium. By comparing the genes with the identified genes expressed in endosperm of Arabidopsis, it will provide some cytological and molecular evidences to unveil the mechanism of highly developed endosperm of triploid Lilium.

通过百合远缘杂交和倍性间杂交观察到，当杂交组合的胚乳中含有5个或5个以上相同的基因组时，胚乳发育较好，杂交容易成功；否则胚乳不发育或很难发育，杂交也难以成功。分析表明这与百合产生四孢八核胚囊有关，因为根据其大孢子形成过程得到推论“三倍体的百合因减数分裂异常产生的卵细胞为非整倍体，但极核永远为六倍体”。为了从细胞水平和分子水平上揭示这种现象的机制，本项目拟根据胚乳发育的差异首先人工授粉进行不同类型组合的杂交，利用组织切片确定胚乳发育程度，用GISH和FISH技术分析胚乳的基因组构成，根据胚乳发育的程度对不同发育时期的胚珠和叶片取样，分析激素和 淀粉合成酶水平，提出mRNA后，制备cDNA，并对不同组合的cDNA测序，通过分析，发现与百合胚乳发育相关的特异基因，从而为揭示三倍体百合胚乳高度发育机制提供细胞和分子方面的证据。

三倍体百合不仅是百合育种的重要方向，而且三倍体百合尽管雄性不育但可以作母本与适宜的二倍体或四倍体杂交获得非整倍体后代，其原因从理论分析其胚囊类型的特殊性获得了解释，但尚缺乏细胞和分子方面的证据。本项目主要进行了百合的三倍体和二倍体或四倍体杂交，从分子水平上分析了亲和性杂交组合和不亲和性杂交组合转录组和代谢组的差异，初步解释了三倍体百合胚乳发育的分子机制；用FISH技术分析了三倍体百合胚乳的倍性和基因组构成，结果表明胚乳的基因组构成与理论分析基本一致，即：百合异常减数分裂胚囊中极核的倍性总是体细胞的两倍；用GISH技术分析三倍体和二倍体杂交后代基因组构成，结果表明其后代皆为非整倍体；用有性多倍体化和倍性间杂交获得三倍体百合品种并进行了国际登录；用GISH和FISH技术分析了百合组间杂交后代，表明已获得了包括龙牙百合与兰州百合在内的多个类群远缘杂交的后代，且用LA x AAAA杂交获得了奇异源四倍体（LAAA）的百合。这些结果对于避免百合及其它多年生花卉育种的盲目性有较高的参考价值。一些相关结果已发表在《Breeding Science》《Scientia Horticulturae》《Acta Horticulturae》等园艺学的主流期刊上，还有些结果整在整理中或投稿中。