【作者】 涂玉琴；

【导师】 李再云；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 远缘杂交是进行作物遗传改良的重要途径。作物的近缘野生种中包含有大量的有利性状如对各种生物和非生物的抗耐性。通过远缘杂交(包括有性杂交和体细胞杂交)将这些野生种的有利基因转移到作物中,不仅创造新的物种和改良作物的特性,而且拓宽了栽培作物的遗传基础,增加其在育种程序中的遗传多样性。菘蓝(Isatis indigotica Fort.,2n=14,Ⅱ)属于十字花科菘蓝族二年生草本植物,它不仅是一种染料作物,具有较大的靛蓝生产能力,更重要的也是中国传统的中药材,含有很多药用化学成分,能够治愈由多种病毒和细菌引起的疾病,并增强人体的抵抗力。此外,菘蓝还具有抗烟草花叶病毒和菌核病的特性,因此,这些有利性状对于改良栽培作物的品质有着非常重要的意义。在本研究中,已通过有性杂交成功的合成了白菜、甘蓝型油菜与菘蓝的部分有性杂种,也通过体细胞杂交成功的合成了白菜、萝卜、芥蓝与菘蓝的体细胞杂种和愈伤,并对它们进行了形态学、细胞学和基因组组成的分析,其主要结果如下。1白菜、甘蓝型油菜与菘蓝的有性杂交在白菜×菘蓝组合中,成功的获得了很多来源于成熟种子的杂种植株。它们表现为不同的形态学特征,但是大多数的植株都死亡,只有三棵植株生长到成熟。这3个杂种长势很弱,植株较两个亲本矮小,表现出一些菘蓝的形态学特征,且包含有菘蓝的化学成分。一株具有2n=10,是白菜的单倍体染色体数目,花粉败育。另两株杂种染色体分别为2n=20和2n=22,它们的花粉是败育的,但通过白菜的回交能产生回交后代。2n=20杂种的回交后代经几个世代的自交后在形态上表现为白菜类型,但也出现一些形态上的变异和脂肪酸含量的改变。2n=22的杂种经白菜回交后产生2n=21和2n=22的后代。GISH分析2n=22的杂种中含有两条染色体来自菘蓝的染色体,而2n=20的杂种中不含有菘蓝的染色体。但是,经AFLP分析发现两个杂种及其后代中都检测到菘蓝的特异带、两亲本的新增带和白菜的缺失带,它们的后代植株是基因组发生改变的新白菜类型。产生这些杂种和后代的机制为外源染色体的消除、附加和渗入。在甘蓝型油菜×菘蓝组合中,产生了45个成熟种子和85个未成熟胚,但产生的植株中只有5株(H1-H5)表现出菘蓝的一些特征,花粉育性低,且具有不同染色体数目。H1-H4具有相似的且变化的染色体数目(2n=25-30),H5杂种具有2n=19,为甘蓝型油菜单倍体的染色体数目。GISH分析发现H1和H5杂种中一条染色体和一个或两个染色体的末端片段被菘蓝的探针所标记。通过将H1与甘蓝型油菜回交后自交多代,或将H5自交多代后形成了在表型和基因组上都发生改变的新甘蓝型油菜类型。H1杂种通过与菘蓝连续回交两次后得到2n=20的白菜型油菜类型的后代,这表明在与菘蓝的远缘杂交中随着外源染色体的消除,甘蓝型油菜中的C基因组被消除而A基因组则被保留。本文还对这些部分杂种形成的原因和芸苔属多倍体中各基因组染色体的稳定性进行了讨论。2白菜、萝卜和芥蓝与菘蓝的体细胞杂交在白菜+菘蓝组合中,通过叶肉原生质体的对称融合获得了5株在田间成活并开花的杂种。在整体上它们的形态表现出中间型,但在花粉育性和一些表型上也出现一些差异,其中有两个杂种是部分可育的。经细胞学和GISH检测发现这些杂种都含有2n=48(AAIIII),花粉母细胞在减数分裂终变期形成24个二价体,后期Ⅰ主要表现24∶24均等分离,但在第二次减数分裂过程中出现了落后染色体和很多微核等异常的染色体行为。经AFLP分析表明这些杂种的DNA带型都是两个亲本之和并附加一些改变。一些杂种可用于白菜和菘蓝的遗传改良。在萝卜+菘蓝组合中,通过叶肉原生质体对称融合获得了一棵在田间成活并开花的杂种。杂种的形态表现为中间型,花粉完全败育,GISH分析表明具有预期的染色体数目2n=32(RRII),减数分裂终变期形成16个二价体,后期Ⅰ呈16∶16分离,而在减数分裂第二次分裂中出现明显的染色体紊乱现象。在芥蓝+菘蓝组合中,利用聚乙烯乙二醇(PEG)和二甲基亚砜(DMSO)共同诱导融合的方法,成功获得芥蓝与菘蓝原生质体融合的再生愈伤。随机挑取5个再生愈伤,利用9对随机引物对其进行SRAP分析,表明再生愈伤的DNA组成几乎是双亲之和,但是也有少量新增带和缺失带出现。类平均法(UPGMA)聚类分析表明,5个杂种愈伤明显聚在一起,其遗传组成十分相似,与菘蓝的遗传关系更近,与芥蓝的遗传关系更远。更多还原

【Abstract】 Wide hybridization plays an important role in crop genetic improvements.Related or distant species of cultivated crops contain a large reservoir of genes covering a variety of desirable traits like resistance to biotic or abiotic stresses.Gene transfer from these species to cultivated crops via wide hybridization including sexual and somatic hybridization could create novel plant forms and improve qualities of crops,also broaden genetic basis of cultivated crops and increase their genetic variability in breeding programes.Isatis indigotica Fort.(Chinese woad or tein-cheing,2n=14,II),belonging to the Isatideae tribe of the Brassicaceae family,is a dye crop with a greater indigo-production capacity.More importantly,as a traditional Chinese medicine,Ⅰ. indigotica contains compounds which may cure many diseases caused by bacteria and viruses and increase the protective immune response.Additionally,Ⅰ.indigotica also shows resistance to tobacco mosaic virus(TMV) and stem rot(Sclerotinia sclerotiorum), the most serious disease of rapeseed in China.So these available traits may be very beneficial for improving quality of cultivated crops.In the present study,intertribal sexual partial hybrids of Brassica rapa,B.napus with I.indigotica as pollen parent and intertribal somatic hybrids or hybrid calli between B.rapa,Raphanus sativus,B.oleracea L.var.alboglabra Bailey and I.indigotica were produced and characterized for their morphology,cytology and genomic components.The main results were as follows.1.Sexual hybridizations between B.rapa,B.napus and I.indigoticaIn B.rapa×1.indigotica combination,many hybrid plants were obtained from the mature seeds harvested and then exhibited different morphological traits.However, majority of them did not survive and only three plants grew to maturity.These 3 hybrids showed poor growth and much smaller stature than the two parents,but had some morphological traits and chemical composition of I.indigotica.One plant had 2n=10,the haploid chromosome number of B.rapa and was absolutely sterile.The other two plants had 20 and 22 somatic chromosomes and were male sterile but produced seeds following pollinations with B.rapa.All backcross progenies over several generations maintained a B.rapa-type phenotype and also displayed some variations in morphological characters and fatty acid compositions.They were all 2n=20 and showed good seed-set.The hybrid with 2n=22 produced some progeny plants with 2n=21 and 2n=22.Genomic in situ hybridization(GISH) detected two chromosomes of I.indigotica in the hybrid with 2n=22 but none in the one with 2n=20.AFLP bands specific for I.indigotica,novel for two parents or absent in B.rapa were detected in the two hybrids and their progenies.These progeny plants were novel B.rapa types with altered genomic constitution or alien additions.The hybrids and progenies were the outcome of the chromosome elimination, addition and introgression.In B.napus×I.indigotica combination,45 mature seeds and 85 immature embryos were produced,but only five hybrids(H1-H5) were distinguished morphologically from female B.napus parents by showing low fertility and some characters of I.indigotica,and also having different chromosome numbers.H1-H4 had similar but variable chromosome numbers in their somatic and meiotic cells(2n=25-30),H5 had 2n=19,the same number as the haploid of B.napus.GISH analysis detected one I.indigotica chromosome and one or two chromosome terminal fragments in H1,H5.New B.napus types with phenotypic and genomic alterations were produced by H1 after pollination by B.napus parent and selfing for several generations,and by H5 after selfing.The B.rapa-type progeny(2n=20) was derived from H1 after pollination by I.indigotica twice,showing that the A-genome chromosomes in B.napus were preferentially maintained and those from C-genome were inclined to be lost under the induction of alien chromosome elimination in wide cross with I.indigotica.The reasons for the formation of the partial hybrids with unexpected chromosomal complements and for the genome-specific chromosome stability in Brassica allopolyploids were discussed.2.Somatic hybridizations between B.rapa,R.sativus,B.alboglabra and I.indigotieaIn B.rapa+ I.indigotica combination,five mature hybrids were produced during symmetric fusions of mesophyll protoplasts.They were morphologically intermediate but showed some differences in phenotypic traits and fertility,two were partially fertile. Cytological and GISH investigations revealed that these hybrids had 2n=48 with AAIIII complement and their pollen mother cells(PMCs) showed normal pairing of 24 bivalents and mainly equal segregation 24:24,but meiotic abnormalities of lagging chromosomes and micronuclei appeared frequently during second divisions.AFLP analysis showed that all of these hybrids had mainly the DNA banding pattern from the addition of two parents plus some alterations.Some hybrids should be used for the genetic improvement of B. rapa and the dye and medicinal plant.In R.sativus+I.indigotica combination,one mature hybrid established through symmetric fusions of mesophyll protoplasts had intermediate morphology but was totally sterile.It had the expected chromosome number(2n=32,RRI I) and parental chromosomes were distinguished by GISH analysis,and these chromosomes were paired as 16 bivalents in PMCs at diakinesis and mainly segregated equally as 16:16 at anaphase I(A I),but the meiotic disturbance presented obviously in second division.In B.alboglabra+I.indigotica combination,intertribal hybrid calli were induced and produced by using polyethylene glycol(PEG,3350) and DMSO.The randomly selected 5 hybrid calli and their parents were subjected to SRAP analysis by using 9 pairs of primers randomly selected,and the results showed that the calli had mainly the DNA banding pattern from the addition of two parents,but also had a few novel bands for two parents.SRAP clustering analysis showed that their genomic compositions were similar but closer to I.indigotica than to B.alboglabra.更多还原