【作者】 文静；

【导师】 傅廷栋；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 在相同的遗传背景下,甘蓝型黄籽油菜比黑籽类型具有以下优点:种皮薄、含油量高、油质清澈透明、纤维素含量少,饼粕中蛋白质含量高,无论食用还是饲用都具有优势。然而自然界中并不存在天然的甘蓝型黄籽油菜。甘蓝型油菜（Brassicanapus,AACC）黄籽育种开展几十年以来,育种工作进展不大,世界范围内可供利用的黄籽种质十分有限。造成这种状况的原因主要有两个:一是黄籽性状的遗传行为比较复杂,二是在甘蓝（B.oleracea,CC）的C基因组中未发现有控制黄籽性状的基因存在。近年来,西南大学报道在羽衣甘蓝（B.oleracea var.acephala,CC）中发现并选育出了稳定的黄籽品系。本研究就是通过黄籽羽衣甘蓝与黄籽白菜型油菜（B.rapa,AA）,黄籽芥菜型油菜（B.juncea,AABB）与黄籽羽衣甘蓝,黄籽埃塞俄比亚芥（B.carinata,BBCC）与黄籽白菜型油菜之间的杂交,期望将这些物种A/C基因组中控制黄籽性状的基因转入或聚合到甘蓝型油菜中去,合成黄籽甘蓝型油菜,以此扩大甘蓝型油菜的遗传多样性,并为甘蓝型油菜黄籽育种提供新的黄籽种质。在此过程中借助于普通细胞学压片技术、GISH技术、AFLP和SSR分子标记技术,我们对甘蓝型油菜合成过程中的杂种、杂种自交或小孢子培养后代进行了形态学、细胞学、分子生物学方面的研究,以探明人工异源多倍体形成之初基因组的构成变化,追踪芥菜型油菜与羽衣甘蓝杂交合成甘蓝型油菜过程中B基因组染色体丢失的规律和机制。本研究主要结果如下:1.在3个黄籽白菜型油菜与5个黄/褐籽羽衣甘蓝间配置正、反杂交组合,经细胞学、形态学以及SSR分子标记鉴定,共在12个组合中获得加倍成功的杂种苗（甘蓝型油菜人工合成种）,以白菜型油菜为母本杂交成功的组合有5个,以羽衣甘蓝为母本杂交成功的组合有3个,另有4个组合来自于2对亲本正反交。对5个白菜型油菜×羽衣甘蓝组合和5个羽衣甘蓝×白菜型油菜组合分别进行了子房培养和胚培养的效果研究。结果表明:在白菜型油菜×羽衣甘蓝杂交组合,取授粉后4-7天的子房进行培养,杂种获得率最高,同时我们的结果暗示1/2MS培养基对于杂种胚的形成具有促进作用,在组合1151×T₂中,接种在1/2MS培养基中的子房杂种率高达45%。在羽衣甘蓝×白菜型油菜杂交组合,杂交后16-18天取胚,接种在MS+0.2 mg l^-16-BA的培养基上效果最好,最高杂种获得率可达48.1%（组合T₃×JB₂）。2.观察了其中10个组合人工合成种的减数分裂行为。其中4个组合的合成种减数分裂过程基本正常,花粉育性在92.7%以上。组合1151×T₂的A₁（F₁植株经染色体加倍获得A₁植株）和4个随机挑选的A₂单株（A₁自交得到A₂）在终变期染色体配对异常,单价体和多价体普遍存在,在减数分裂后期Ⅰ时,18.95-44.30%的PMC中发生了一对同源染色体的不分离（chromosome nondisjunction）,呈现18:20的染色体不均等分离,从而导致了非整倍配子的产生。其它5个组合的A₁植株在减数分裂的不同时期出现不同程度的染色体行为异常,比如姐妹染色单体提前分开、染色体落后、个别染色体的消除等。异常的减数分裂行为是导致花粉育性降低、结实率不高的主要原因。3.对组合1151×T₂的合成种进行小孢子培养,将得到的66个MD系（microspore-derived lines,来自于A₂植株）进行染色体计数,从中分离得到了7个非整倍体,非整倍体获得率高达10.6%。非整倍体中,其中4个是缺体（2n=36）,2个是缺体单倍体（2n=18）,一个是四体单倍体（2n=20）。对它们进行了形态学性状的观察,并采用AFLP技术推测了缺体及缺体单倍体丢失染色体的基因组来源。结果表明,5个缺体/缺体单倍体可能丢失了一对/条来自羽衣甘蓝的染色体,1个缺体单倍体可能丢失了一条来自白菜型油菜的染色体。对缺体MD-8和MD-9进行了脂肪酸含量测定,推测MD-9丢失了一对来自羽衣甘蓝C基因组的染色体,该染色体上携带有控制株高、花瓣形态和数目、分枝习性以及与芥酸和硫甙生物合成有关的基因。4.用SSR和AFLP分子标记考察了1151×T₂的A₁和A₂代合成种的基因组变化规律,并用AFLP分子标记考察了其它组合合成种（异源多倍体）全基因组水平的序列丢失情况。结果表明:各组合A₁植株的基因组均发生了较大的变化,其中包括亲本片段消失、新片段的出现,在1151×T₂中还观察到A₁代丢失的亲本片段在A₂代重新出现。丢失片断的比例总是大大多于新增片断的比例。每个组合的合成种都无一例外地表现出丢失更多的来自白菜型油菜亲本的片断,说明甘蓝型油菜人工合成种的序列消除具有倾向性,即:总是消除更多的来自于AA基因组中的片断,并且这种倾向性不受细胞质的影响,无论正反交均是如此,导致甘蓝型油菜人工合成种无论是在形态上还是基因组组成上,都偏向于甘蓝亲本,聚类时与甘蓝聚在一起。SSR分子标记的结果还表明:序列消除也发生于简单重复序列,而且合成种也是丢失了更多的来自于AA基因组的简单重复序列。5.进行了3个黄籽芥菜型油菜与1个黄籽羽衣甘蓝的种间杂交,获得了9个完全加倍成功的异源六倍体AABBCC,将其分别与甘蓝型油菜人工合成种（1个来自于1151×T₂的合成种的DH系）和羽衣甘蓝杂交,分别获得93个五倍体（AABCC）和27个四倍体（ABCC）植株。四倍体和五倍体的GISH结果表明:B基因组内以及B与A/C基因组染色体间存在一定的同源性,B基因组染色体之间最多可以形成2个二价体,在四倍体和五倍体中分别最多有2条和4条B基因组染色体参与了与A/C基因组染色体的异源配对。在减数分裂后期Ⅰ时,B基因组染色体通过染色体落后和着丝粒错分离在部分细胞中被丢失,但五倍体中这种异常发生的比例较四倍体高,这说明B基因组染色体在四倍体的减数分裂过程中,发生丢失的机率比五倍体低。通过五倍体自交能更快地获得新型甘蓝型油菜。6.调查了四倍体、五倍体自交及四倍体与甘蓝型油菜人工合成种杂交的结实情况。结果表明,五倍体单株自交结实性比四倍体的好,而四倍体在与甘蓝型油菜人工合成种杂交后,结角率和结实率大大提高,收获的杂交种中体细胞染色体数目接近于38条的更多。因此,将ABCC与AACC杂交,能加快B基因组染色体的丢失,尽快地获得新的甘蓝型油菜。7.对六倍体AABBCC及四倍体ABCC进行小孢子培养,分别获得了8个和17个加倍成功的三基因组非整倍体植株。用普通细胞学压片及GISH技术对这些植株进行了观察和分析,结果表明:从六倍体AABBCC获得了6个染色体数目超过54的非整倍体,它们具有完整的两个B基因组（8对染色体）,花粉育性较高（78.23-86.85%）,自交几代后有望稳定,形成新的亚种。从四倍体ABCC中也分离得到了一些非整倍体,具有1-6对的B基因组染色体,个别植株减数分裂基本正常,花粉可染性达82.43%,可以作为芸薹属遗传研究和作物遗传改良的工具材料。8.通过黄籽白菜型油菜与黄籽羽衣甘蓝、黄籽芥菜型油菜与黄籽羽衣甘蓝的种间杂交,获得了黄籽甘蓝型油菜。另外,通过黄籽埃塞俄比亚芥与黄籽白菜型油菜的种问杂交,也获得了部分甘蓝型油菜黄籽材料。在白菜型油菜与羽衣甘蓝组合中,有6个组合的合成种后代或DH系中出现黄籽或暗黄籽株系,在2个互为正反交的组合中,正交组合（JB₂×T₃）所有A₁植株均结黄籽,而反交组合（T₃×JB₂）一直自交到A₃代都未见有黄籽株系出现,表明黄籽性状的表现在种间杂交中受到细胞质的影响。另外,还从这些组合中获得一些长角果、早花、大粒、双低的新种质。更多还原

【Abstract】 Yellow-seeded Brassica napus possesses a thiner seed coat,which is associated with higher oil,higher protein and lower fibre contents when compared to black-seeded ones in the same background.However,no naturally yellow-seeded forms of B.napus exist. Though many investigations to breed yellow-seeded B.napus have been carried out in the last several years,only few stable yellow-seeded forms have been developed.Several reasons have been attributed for the complicated inheritance of this trait and the failures to obtain truly yellow-seeded B.oleracea genotypes.Recent years,it was reported that yellow-seeded B.oleracea var.acephala mutants were found and breed true in Southwest University,China.The present investigation is to combine the genes for yellow seed color in A/C genome of other species into B.napus via interspecific hybridization of yellow-seeded species viz.B.oleracea var.acephala×B.rapa,B.rapa×B.oleracea var. acephala,B.juncea×B.oIeracea var.acephala,and B.carinata×B.rapa.By taking advantage of cytogenetic,genomic in situ hybridization（GISH）,amplified fragment length polymorphisms（AFLP） and simple sequence repeat（SSR） technology,the morphological,cytological and molecular characterizations of the hybrids and their progenies were conducted.This paper provides new evidence for the genomic changes in synthetic B.napus,and also provides clues for the rules of B-chromosomes elimination in progenies of B.juncea×B.oleracea.The main results were as follows:1.Reciprocal crosses were conducted between three yellow-seeded varieties of B. rapa and five yellow- or brown-seeded accessions of B.oleracea var.acephala.Hybrids were obtained from five crosses of B.rapa×B.oleracea,three crosses;of B.oleracea×B. rapa and two reciprocal crosses.To eatablish an efficient reproducible culture system for different Brassica interspecific crosses,ovary culture and embryo culture were conducted in crosses B.rapa×B.oleracea and oleracea×B.rapa,respectively.A higher rate of hybrid production was recorded when ovaries were cultured at 4-7 days after pollination （DAP）.Of different culture media,medium E（MS with half strength macronutrients） showed good response for ovaries from all the crosses,the highest rate of hybrid production reaching 45%in B.rapa（1151）×B.oleracea（T₂）.In embryo culture,the hybrid rate was significantly enhanced at 16-18 DAP,up to 48.1%in B.oleracea（T₃）×B.rapa（JB₂）.2.For ten crosses,meiotic behavior of resynthesized B.napus was observed.PMCs of newly allotetraploids from four crosses presented normal meiotic:progression.The pollen stainability of these plants was above 92.7%.Meiosis in the amphidiploids of cross 1151×T₂ was characterized by high frequencies of univalents and multivalents per PMC at diakinesis/metaphase I and notably unbalanced chromosome segregations at anaphase I （AI）.In all the plants observed in 1151×T₂,18.95-44.3%of PMCs exhibited a segregation of 18:20（n-1:n+l） at AI,which was caused by nondisjunction of one bivalent or the distribution of two homologous univalents to the same pole.Meiosis proceeded normally after AI then,which led to the formation of viable n-1 and n+1 gametes and high pollen fertility of these plants.Amphidiploids from other crosses presented abnormal chromosome behavior at different meiotic phases,such as precocious division of centromere,chromosome laggards and elimination which led to the poor pollen stainability and seed set.3.Micro@ore culture was carried out using amphidiploids of cross 1151×T₂ in an attempt to isolate Brassica nullisomics for the first time.Four nullisomics（2n=36）,two nullihaploids（2n=18） and one tetrasomic haploid（2n=20） were identified cytologically and characterized for their morphology and by using AFLP molecular markers.Five nullihaploids/nullisomics were supposed to be absent of one or one pair of C-genome chromosome（s）,and one nullihaploid was supposed to be absent of one A- genome chromosome.Moreover,we presumed that MD-9 lost one pair of C-genome chromosomes carrying the genes for plant height,the size and number of petals,branch habit,erucic acid and glucosinolates biosynthesis.4.To study the dynamics of genome change after allopolyploidization,we surveyed the amphidiploids（A₁ and A₂ plants） of 1151×T₂ with AFLP and SSR molecular markers. Also amphidiploids（A₁ plants） of other crosses were surveyed by AFLP molecular markers.Extensive genomic changes were detected in all the amphidiploids,involving loss of parental restriction or SSR fragments,gaining of novel fragments and reappearance of parental fragments in A₂ plants that lost in A₁ plant.The frequency of losing parental fragments was much higher than that of gaining novel fragments.For all the reciprocally synthetic B.napus,the data showed that the frequency of sequence losing in B.rapa was much higher than in the B.oleracea.The results suggested that the biased sequence elimination was not caused by cytoplasmic factors.5.Nine hexaploids were produced from cross B.juncea×B.rapa and a subsequent chromosome doubling.Ninety-three pentaploids（AABCC） and twenty-seven tetraploids （ABCC） were obtained after hybridize the hexaploids with resynthesized B.napus and B. oleracea,respectively.GISH analysis of pentaploids and tetraploids revealed that a maximum of two bivalents formed by autosyndesis within B genome at diakinesis,and a maximum of two and four B-chromosomes paired with A/C chromosomes in pentaploids and tetraploids,respectively.B-chromosomes could be eliminated through B-chromosome laggards and precocious division of chromatids at anaphase I.However, the frequency of the B-chromosome abnormity in pentaploids was much higher than in tetraploids,indicating that new-type B.napus can be obtained more easily from selfed progenies of pentaploids.6.Pentaploids（AABCC） and tetraploids（ABCC） were selfed to eliminate B-chromosomes and generate newly B.napus.Pentaploids had better seed set than tetraploids.However,siliques per flower and seeds per silique of tetraploids were greatly enhanced when pollinated with the pollen of resynthesized B.napus.More derivatives of the ABCC×AACC cross exhibited a chromosome number adjacent to 38.Thus, hybridization between tetraploids and resynthesized B.napus can also be conducted to accelerate the elimination of B-chromosome and to generate newly B.napus.7.Eight and seventeen trigenomic aneuploids with doubled chromosomes were obtained through microspore culture of hexaploids（AABBCC） and tetraploids（ABCC）, respectively.Out of the eight aneuploids derived from hexaploids,six aneuploids with 2n＞54 were revealed by GISH to contain eight pairs of B-chromosomes in it.They exhibited normal meiosis procession in most cases.Higher pollen fertility（78.23-86.85%） of these plants indicates that they are promising materials to develop new subspecies in Brassica.GISH analysis found 1-6 pairs of B-chromosomes in aneuploids from tetraploids.Some of them had 82.43%stainable pollen,and seed set was achieved from self-pollinated.These aneuploids are important materials in rapeseed improvement and genetic analysis.8.For the first time,yellow-seeded B.napus were obtained through interspecific hybridization between yellow-seeded B.rapa and yellow-seeded B.oleracea var. acephala,and between yellow-seeded B.juncea and yellow-seeded B.oleracea var. acephala.Meanwhile,hybridizations of yellowed B.carinata and yellowed B.rapa also succeeded in developing yellow-seeded B.napus.In crosses between B.rapa and B. oleracea var.acephaIa,yellow and yellow-brown seeded B.napus plants segregated in the advanced generations or doubled haploid（DH） populations of six crosses.The fact that A₂ plants of JB_×T₃ produced yellow seeds,whereas in its reciprocal crosses no yellow seeds but brown or black seeds were obtained,indicated cytoplasmic effect on seed color.Additionally,the prevalence of various interactions between the two parental genomes gave rise to novel variations which may be of practical interest.For example, new genotypes of long-pod,earliness,big-seed and double low materials were connected with the newly resynthesized B.napus in this study.更多还原