天然甘蓝型油菜C染色体组C1，C2缺体的创建及遗传分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0577

摘要/Abstract

摘要：

缺体（nullisomic，2（n-1））是相对于正常生物个体（euploid，2n）的染色体组缺失一对同源染色体的有机体，是一种不可多得的遗传材料，能够实现对单条染色体的遗传解析。本研究通过甘蓝型油菜（Brassica napus，2n=38，AⁿAⁿCⁿCⁿ）与先前获得的剥离白菜（restituted B. rapa，RBR，2n=20，AⁿAⁿ）进行杂交及连续回交，通过C基因组染色体特异分子标记及荧光原位杂交（flourescence in situ hybridization，FISH）技术首次筛选出了甘蓝型油菜C染色体组的两个缺体系：C1染色体（NC1）和C2染色体缺失的缺体系（NC2）。随后对两个缺体材料的花粉母细胞（pollen mathor cell，PMC）观察证实，在终变期的PMC呈现18个二价体，减I后期染色体以18:18均等分离为主，但在后期I及后期Ⅱ均出现不同频率的落后染色体。与亲本相比，两个缺体的生物量、花粉育性、结实率等显著降低，且表现一些特有性状，例如NC1全叶被覆毛刺，NC2开花时间比亲本提早近两个月，说明缺失染色体上携带有相关性状的抑制基因。这两个缺体材料有效降低了甘蓝型油菜基因组的复杂性，有助于目标性状的初步定位，或者基因定位结果的验证，为今后的甘蓝型油菜遗传研究提供便利。

关键词: 缺体, 甘蓝型油菜, 荧光原位杂交, 种质资源

Abstract:

The nullisomic（2（n-1））line losing a pair of chromosome from the normal organism chromosome set has been recognized as a rare and important genetic resources for revealing the missing chromosome. Herein, after screening the offsprings from the cross and backcross between Brassica napus and the restituted B. rapa（RBR, 2n =20, AⁿAⁿ）, we used the integration of specific C chromosome molecular markers and fluorescence in situ hybridization technology to screen two nullisomic lines of chromosome C1（NC1）and chromosome C2（NC2）in natural B. napus（2n =38, AⁿAⁿCⁿCⁿ）for the first time. Both two nullisomic lines had 18 bivalents at diakinesis and a dominant segregation of 18:18 at meiosis anaphase I（MA I）in the pollen mother cells. However, some lagging chromosomes were observed at MA I and MA II in both two lines. Compared to their parental B. napus, the two lines presented significantly smaller phenotype at biomass, pollen stainability, and seed setting, also showed some specific features. For example, the NC1 had the leaves with obvious burrs, and NC2 showed significantly early flowering time of two months earlier than its parental B. napus, indicating some genes inhibited such characters were locating on the missing chromosome. The two nullisomic lines effectively reduce the complexity of B. napus genome, which can be used to map the target traits, or to verify the gene function, and thus to provide convenience for future genetic analysis of B. napus.

Key words: nullisomic, Brassica napus, fluorescence in situ hybridization, germplasm resources

蔡梦鲜, 高作敏, 胡利娟, 冯群, 王洪程, 朱斌. 天然甘蓝型油菜C染色体组C1，C2缺体的创建及遗传分析[J]. 生物技术通报, 2023, 39(3): 81-88.

CAI Meng-xian, GAO Zuo-min, HU Li-juan, FENG Qun, WANG Hong-cheng, ZHU Bin. Development and Genetic Analysis of Two Nullisomic Lines（NC1 and NC2）in Natural Brassica napus[J]. Biotechnology Bulletin, 2023, 39(3): 81-88.

图/表 5

图1 天然甘蓝型油菜C染色体缺体创建流程

Fig. 1 Construction process of nullisomic of C chromosome in natural B. napus

图2 NC1和NC2缺体单株的C染色体特异分子标记鉴定结果 M：Marker标记大小分别为2 000、1 500、1 000、750、500、250、100 bp；Oro是甘蓝型油菜；NC1，NC2代表甘蓝型油菜C染色体缺体；C1c，C2g，…C9e为C基因组染色体特异性引物

Fig. 2 Identification of C chromosome nullisomic-specific markers in NC1 and NC2 M: Marker. The mark sizes were 2 000, 1 500, 1 000, 750, 500, 250 and 100 bp. Oro is B. napus. NC1 and NC2 refer to the nullisomic of chromosome C in B. napus. C1c, C2g, …C9e are chromosomal specific primers for C genome

图3 甘蓝型油菜“ Oro ”，NC1及NC2的体细胞及花粉母细胞荧光原位杂交分析 A：“Oro”有丝分裂细胞，有38条染色体（蓝色），包括18条C亚基因组染色体（红色）；B-C：NC1，NC2有丝分裂细胞，有36条染色体（蓝色），包括16条C亚基因组染色体（红色）；D：C2缺体终变期，显示8个红色杂交信号；E-F：NC1及NC2中处于减数分裂后I期的PMC检测到16个杂交信号均等分离。蓝色表示DAPI染色，红色表示BAC BoB014O06探针信号。标尺=10 μm

Fig. 3 FISH analysis of somatic cells and pollen mother cells of B. napus “Oro”，NC1，and NC2 A: “Oro” mitotic cells with 38 chromosomes（blue）, including 18 C subgenomic chromosomes（red）. B-C: NC1, NC2 mitotic cells with 36 chromosomes（blue）, including 16 C subgenomic chromosomes（red）. D: C2 showing 8 red hybridization signals at diakinesis. E-F: The PMC of NC2 and NC1 show equal separation of 16 hybridization signals at anaphase I. Blue indicates DAPI staining and red indicates BAC BoB014O06 probe signal. Bar: 10 μm

图4 亲本甘蓝型油菜“Oro”，NC1，NC2表型及花粉育性 A：亲本甘蓝型油菜“Oro”；B：NC1植株，放大框中显示NC1叶脉、叶片及叶边缘被覆白色毛刺；C：NC2植株，放大框中显示NC2已现蕾；E-F：“Oro”，NC1和NC2的花粉育性

Fig. 4 Phenotype and pollen fertility of parental B. napus “Oro”，NC1，and NC2 A: Parental B. napus “Oro”. B: NC1 plants. The enlargement shows that blade, vein, and leaf margin cover with burrs. C: NC2 plants. The enlargement shows that NC2 plant has early squaring stage. E-F: The pollen fertility of “Oro”, NC1, and NC2

图5 甘蓝型油菜NC1与 NC2减数分裂染色体行为观察 A-D：依次为NC1体细胞染色体数（2n=36），花粉母细胞减数DK、AP I及AP II时期的细胞；E-H：依次为NC2体细胞染色体数（2n=36），花粉母细胞减数DK、AP I及AP II时期的细胞；I：正常配对的缺体中I期细胞；J，K：NC1及NC2中出现染色体滞后现象的后I期细胞；L：NC2中AP II时期出现滞后染色体的PMC。箭头所指为落后染色体，标尺=10 μm

Fig. 5 Observation of chromosome behavior of at meiosis of NC1 and NC2 in B. napus A-D: Somatic chromosome number（2n=36）of NC1, PMCs of NC1 at DK, AP I, and AP II, respectively. E-H: Somatic chromosome number（2n=36）of NC2, PMCs of NC2 at DK, AP I, and AP II, respectively. I: the PMC of nullisomic show normal pairing at metaphase I. J, K: The PMCs of NC1 and NC2 with delaying chromosome at AP I. L: The PMC of NC2 with delaying chromosome at AP II. The arrows show the delaying chromosomes. Bar: 10 μm

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