甘蓝型油菜新型黄籽种质资源创新与鉴定

【作者】 李爱民；

【导师】 王幼平； 梁国华；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2010， 博士

【摘要】 甘蓝型油菜是我国油菜主要栽培品种,但其作为异源双二倍体种起源发生的历史较短,遗传背景也较狭窄,加之我国甘蓝型油菜引自国外,遗传基础更为单一,开展甘蓝型油菜种质资源创新工作显得十分必要。甘蓝型油菜没有天然的黄籽种质资源,目前培育出的黄籽甘蓝型油菜均来自芸苔属内种间杂交,还未见通过属间杂交获得黄籽甘蓝型油菜的报道,且现有的甘蓝型油菜黄籽性状有一个共同特点,它们的种皮色泽既不象白菜型油菜那样呈鲜黄色,也不象芥菜型油菜那样呈橙黄色,而是姜黄或土黄色,并且种皮上有黑色的斑点、斑块和褐色的环状带纹。同时种皮色泽的遗传不稳定,长期自交不能纯合,仍有少数单株出现黑籽,育种上应用较为困难。因此,开展甘蓝型油菜黄籽种质资源创新工作,对拓宽甘蓝型油菜黄籽性状遗传基础,丰富黄籽遗传资源,具有非常重要的意义。远缘杂交可以把不同种间、属间甚至亲缘关系更远的物种进行杂交,突破种属界限,扩大遗传变异,从而创造新的变异类型或新物种,是种质资源创新的重要手段;同时所获得的杂种不存在生物安全性问题,可直接应用于育种和生产。本研究以白芥和甘蓝型油菜属间杂种为基础材料,通过不断回交获得包括黄籽性状在内的大量甘蓝型油菜新种质,并对新型黄籽新种质进行较为深入的研究和鉴定工作,为在育种中的应用奠定基础。本研究取得的主要研究结果如下:1、通过对白芥和甘蓝型油菜原生质体融合后代不断回交,获得多个具有白芥优良性状和变异类型的后代,如结角密度高、黄籽和抗病等优良性状,同时部分后代也表现出长角果、多籽粒等变异类型。这些后代的获得,为丰富油菜种质资源提供了可能,并为培育高产、优质和抗病油菜新品种提供丰富的育种材料。同时,本试验首次通过属间杂交,获得新的黄籽甘蓝型油菜新种质,为研究黄籽性状的分子机理以及获得色泽稳定、纯合的黄籽性状提供了可能。2、通过对获得的黄籽新种质种子解剖学比较观察发现,所获的回交后代黄籽株系的种子解剖结构基本上与甘蓝型油菜相似,但也表现出黄籽亲本(白芥)的部分特征。如种皮色泽主要分布在栅栏层,甘蓝型油菜和部分后代株系中有色泽分布,而白芥和部分黄籽后代株系中没有色泽分布。栅栏层在甘蓝型油菜中最厚,在白芥中最薄,而后代株系介于两者之间。回交后代株系和甘蓝型油菜种皮表面纹饰为网-穴状,白芥为沟槽状或水疱状。回交后代株系胚子叶细胞面积和蛋白体面积指数介于两亲本之间,油体有大、小两种,其大小在亲本和后代间存在差异。说明白芥外源染色体或染色体片段的导入,改变了回交后代株系种子结构性状。3、用白芥基因组作探针,对回交后代减数分裂进行GISH分析,结果发现,甘蓝型油菜的染色体在回交各个世代中均能正常配对和分离,而白芥染色体多以单价体形式存在,部分白芥染色体会与甘蓝型油菜染色体形成三价体,在杂种后代减数分裂过程中可能会发生白芥与甘蓝型油菜的染色体重组,为易位系的产生奠定了基础。BC3F1黄籽材料根尖细胞和减数分裂细胞进行GISH结果显示,获得的黄籽材料染色体数目与正常甘蓝型油菜相同,且未获得杂交信号,排除获得的黄籽材料是异附加系或置换系的可能性。微卫星核心序列引物33.6扩增结果证明,在黄籽后代的基因组中存在与白芥相同的特异序列。TT2-2引物及根据TT2-2扩增特异产物测序结果设计的引物Sa1在白芥和黄籽后代中扩增的特异产物一致性,进一步确定了黄籽后代中具有白芥的DNA序列,结合细胞学鉴定结果,可以确认获得的黄籽回交后代株系是带有白芥DNA序列的易位系。同时获得黄籽特异性分子标记Sa1,为分子标记辅助育种奠定了基础。4、通过对6个黄籽回交后代株系的农艺性状、产量性状、品质性状及抗病性分析,结果表明,黄籽株系D244-18黄籽显性度、一致性高,成熟期早,籽粒大,单株产量较高,含油量较高,品质接近国家双低标准,抗菌核病能力中等,具有较高的育种利用价值。黄籽株系D244-6成熟期较早,一次有效分枝数多,结角密度较高,全株角果数多,单株产量高,含油量高,品质指标接近国家标准,抗菌核病能力强,但黄籽显性度、一致性较差。单从高产、优质、抗病品种选育角度看,也是一个不可多得的优良株系,但黄籽性状、双低品质性状需进一步改良。更多还原

【Abstract】 The genetic basis of Brassica napus L. (2n=38,AACC), one of the most important oilseed crops worldwide, is quite narrow. As an allopolyploid with a short phylogeny, the specie was originated in Europe and then introduced into China, thus it is important to improve B.napus germplasm.Previous studies showed that yellow seed resource does not exist in natural B.napus, amounts of yellow-seed B.napus obtained were derived from interspecific gene flow between B.napus and B.rapa, B.juncea, B. carinata , whereas yellow-seed related gene introgression from relative genus have never been found. Furthermore, B.napus with yellow seed character obtained by conventional methods behaves common feature—khaki or ginger seed capsule with black spots and brown circular fringe, which is genetically instable and hard to pure by selfling, unlike the delicate yellow of B.rapa and orange yellow of B.juncea. Thus, it is important to carry out germplasm enhancement and expand genetic basis of B.napus, which is significant to broaden yellow seed resources.Distant hybridization, as a valuable measure to germplasm reformation, may overcome genus boundary, enlarge genetic variation, and ultimately resulting the creation of new variants and species. On the other hand, the progenies of hybrids are exempt of bio-safety and available for B.napus breeding and production. In this study, somatic hybrids obtained by protoplast fusion of B.napus and S.alba were applied for meaningful creation of new B.napus germplasm. Through successive backcrossing with B.napus, several progeny lines with advantageous agronomic characters were received, such as yellow-seeded germ, which were taken for further identification and of great value to B.napus breeding. The major findings of this research are as follows:1、Progenies with improved characters from S.alba, such as high pod density, yellow seed, disease resistance, longer silique, multi-seed, were obtained through successive backcrossing, which may greatly contribute to enrichment of B.napus germplasm and act as materials for B.napus breeding of high output, quality and resistance varieties. Our research firstly obtained novel rapeseed with yellow seed color via intergeneric hybridization, which is interested for mechanism discussion of B.napus seed color and yellow seed breeding.2、Comparative anatomy analysis of novel yellow seed resulted that seeds of yellow seed progenies not only resemble to B.napus in anatomic structure, but behave some characters of S.alba. The seed coat pigments were mainly distributed in the palisade layer, the highest quantity of seed coat pigmentation was observed in B.napus and some backcross lines. On the other hand, the seeds of S. alba and several backcross progenies exhibited nearly no pigmentation. The thickness of palisade layer was highest in B.napus, thinnest in S.alba and intermediate in the hybrid progenies. Ornamentation characters of progeny seed surface is reticulation-carve shaped, the same as B.napus, whereas S.alba seed coat manifests the appearance of groove or blister. Cellular area and protein body area of scutellum from backcrossing progeny lines are intermediate of the parents. Oil body exists as two forms—big or small oil body, and its discrepancy is obvious between progenies and parents. These results indicate that introgression of S.alba chromosome or gene fragment is the cause of seed structural mutations in progeny lines.3、GISH analysis with S.alba genome as probe showed that chromosome of B.napus behaves normal pairing and segregation in every generation. While S.alba chromosome mostly exists as univalents, and occasionally form trivalents with B.napus chromosomes, making it possible for chromosome recombination between B.napus and S.alba. GISH of both meiotic and mitotic chromosomes from BC3F1 showed normal chromosome number and no hybrid signal was detected, demonstrating these materials are not addition lines or substitution lines. In addition, we used minisatellite core sequence 33.6 as primer also obtained a specific band of S.alba. Primers TT2-2 designed according to flavonoid biosynthetic genes of Arabidopsis thaliana and Sal resulted from specific product of TT2-2 resulted same sequences between S.alba and yellow seed progenies, illuminating our yellow seed germplasm possess DNA sequences of S.alba. The developed molecular marker Sal of yellow seed may contribute to B.napus breeding.4、Analysis of economical characters, yield traits, quantitative characters and disease resistance of six yellow seed progenies showed that, the dominant degree and uniformity of yellow seed line D244-18 is higher, as well as earlier age of maturity, bigger seeds, higher yield per plant, higher oil content, mediate Sclerotinia selerotiorum resistance. This line is proximal to the double-low quality standards and of great utility value. Line D244-6 possess earlier age of maturity, more effective branches, higher pod density, et al, also next to the nation quality standard, high Sclerotinia selerotiorum resistance,but the dominant degree, uniformity of yellow seed is not up to scratch. Further improvement of D244-6 is needed for making it valuable as breeding material.更多还原