白菜与埃塞俄比亚芥远缘杂交种质创制及黑腐病抗性转育

doi:10.16420/j.issn.0513-353x.2021-0151

摘要/Abstract

摘要：

以白菜（Brassica rapa ssp. chinensis,AA基因组）为母本,以起源于非洲对黑腐病免疫的埃塞俄比亚芥（Brassica carinata,BBCC基因组）为父本进行远缘杂交,通过胚挽救方法获得ABC基因组杂种植株。杂种植株营养体粗壮高大,杂种优势明显。分子标记和流式细胞检测确定是真杂种。F₁代植株初期无花粉,组培继代多代后育性逐渐恢复。与埃塞俄比亚芥回交的后代株系育性恢复速度高于与白菜回交的后代。流式细胞分析发现杂种染色质含量介于白菜和埃塞俄比亚芥之间,并且存在染色体和非整倍体加倍现象。花粉母细胞染色体压片显示减数分裂中存在染色体不减数、染色体滞后、染色体非对称分离、染色体粘贴和染色体桥等现象。从人工合成ABC植株与白菜回交的群体中筛选出高抗或免疫黑腐病的株系,抗性可以遗传。

关键词: 白菜, 埃塞俄比亚芥, 远缘杂交, 黑腐病, 种质创新

Abstract:

Present study synthesized the ABC genomic plant by interspecific hybridization and embryo rescue using pakchoi（Brassica rapa ssp. chinensis,AA genome）as maternal and Ethiopian mustard（Brassica carinata,BBCC genome）,which originated from Africa and was immune to black rot. The hybrid plant showed strong heterosis at the vegetative growth stage. The genomic composition was confirmed by molecular markers and flow cytometry analysis. The F₁ plants were sterile at beginning but gradually restored the fertility after several generation of tissue culture and propagation. The fertility restoration is faster in the backcross progeny with Ethiopian mustard than that with pakchoi. Flow cytometry analysis indicated that the chromosomal contents of the F₁ and backcross progeny distributed in the interval of pakchoi and Ethiopian mustard. The automatic chromosomal doubling was observed in the leaf tissue cells. The abnormal chromosome behaviors include non reduction,chromosome lagging,asymmetric segregation,chromosome stickiness and chromosome bridges were revealed in the pollen mother cells. The black rot immune or high resistant lines were screened from the backcross progeny of the synthesized ABC with pakchoi plants. The resistance was inheritable.

Key words: Brassica rapa ssp. chinensis, Brassica carinata, inter-specific hybridization, black rot, germplasm innovation

中图分类号:

S634.3

张双双, 苏维, 刘阳, 王海平, 宋江萍, 阳文龙, 贾会霞, 张晓辉, 李锡香. 白菜与埃塞俄比亚芥远缘杂交种质创制及黑腐病抗性转育[J]. 园艺学报, 2021, 48(7): 1304-1316.

ZHANG Shuangshuang, SU Wei, LIU Yang, WANG Haiping, SONG Jiangping, YANG Wenlong, JIA Huixia, Zhang Xiaohui, LI Xixiang. Germplasm Innovation and Black Rot Resistance Transferring in Brassica rapa ssp. chinensis Through Interspecific Hybridization with Brassica carinata[J]. Acta Horticulturae Sinica, 2021, 48(7): 1304-1316.

图/表 11

表1 分子标记引物及序列

Table 1 The primer sequences of the molecular markers

分子标记编号	上游引物（5′-3′）	下游引物（5′-3′）
Molecular marker ID	Forward primer	Reverse primer
1A	GAGAAAACTCGGTGGGATCA	TCCCATACGCTCTCCTTCAC
1B	GCAACACTTGCTGTTTTGGA	GCTGAGGTAGGAAGGGAAGG
2A2	GATCGACCTTCCATCACGTC	CCTTGTGCAAGAAGGTGTTG
2B	GCTTTGTGGGTTTGAGCTTG	GCCATTTTGAACCATGAACC
3A	ATAACGCTGAACTGGCGAAG	TTGATTGTCACACCGGAGAA
3B	CCGGACCATAAATTATCGCA	GCTCCTCCTCCTCCATCTTC
3B2	CCTCCGCTAAGGTAAATCTCG	GCCGGAAGAAGAGATCAGTTT
5B2	TTCTTGTCAGTCCTGTCCCC	TCCCGAGGTACTTCACTTGC
6D2	GCTTCATTGGATCCCACATC	GGGTTCGTGATTGATGGTAAA
7A2	TGCCCTCCAAAATCCAATTA	CAGAAGCTCGGGAAGACATC
7C	GGGAAAATTAAACCAAGCCA	GAACACATGGTGGACACAGC
7D	GGAGAAGAAAACAGCGATGC	GGAATAGCTCTTGACGCTCG
8B2	TGCTGCTAAGTCTAGTCCACAA	CCTCAAGATCCACAATGCCT
9D	TGACAGCATATGAAGCCTGC	GATCCTGCCACAAGAATTTGA

图1 白菜与埃塞俄比亚芥杂交F1代植株表型

Fig.1 The phenotype of the F1 hybrid of Brassica rapa ssp. chinensis and Brassica carinata

图2 分子标记鉴定白菜（Bra）与埃塞俄比亚芥（Bca）杂交F1代（H）植株 1A ~ 9D：分子标记编号。

Fig. 2 Analysis of the F1 hybrid（H）of Brassica rapa ssp. chinensis（Bra）and B. carinata（Bca）with molecular markers 1A-9D：Marker ID.

图3 流式细胞分析白菜与埃塞俄比亚芥杂交F1染色质含量

Fig.3 Flow cytometric analysis of chromatin content in Brassica rapa ssp. chinensis and Brassica carinata F1 hybrid plant

图4 白菜与埃塞俄比亚芥远缘杂交后代花、角果、种子的形态对比

Fig.4 Morphological comparison of flowers,siliques,seeds of progenies from interspecific hybridization between Brassica rapa ssp. chinensis and B. carinata

图5 白菜与埃塞俄比亚芥远缘杂交后代单株种子产量

Fig.5 Seed yield of progenies from interspecific hybridization between Brassica rapa ssp. chinensis and B. carinata

图6 白菜与埃塞俄比亚芥杂交后代幼嫩叶片细胞的染色质含量分布 BC2(A)：与白菜回交2代;BC1(U)：与埃塞俄比亚芥回交1代;BC1(U)-BC2(A)：埃塞俄比亚芥回交1代,再与白菜回交2代;BC1(U)S2- BC1(A)：与埃塞俄比亚芥回交1代,自交2代,再与白菜回交1代。

Fig.6 Chromatin content in cells from young leaf of the hybrid of Brassica rapa ssp. chinensis and Brassica carinata BC2(A)：Second generation progenies of the inter-specific hybrid backcrossed with B. rapa ssp. chinensis;BC1(U)：Progenies of the inter-specific hybrid backcrossed with B. carinata;BC1(U)-BC2(A)：Inter-specific hybrid backcrossed with B. carinata for one generation and backcrossed with B. rapa ssp. chinensis for two generation;BC1(U)S2-BC1(A)：Inter-specific hybrid backcrossed with B. carinata for one generation,followed by two generation of selfing,and then backcrossed with B. rapa ssp. chinensis for one generation.

图7 BC2（A）植株减数分裂时花粉母细胞的染色体异常情况 A：染色体不减数;B：染色体滞后;C：染色体3条聚集;D：染色体非对称分离;E、F：染色体粘贴和染色体桥。

Fig. 7 The abnormal chromosome behavior of pollen mother cells during meiosis in BC2（A）plants A：Chromosome non reduction;B：Chromosome lagging;C：Three chromosomes organized in a group;D：Asymmetric segregation;E,F：Chromosome stickiness and chromosome bridges.

图8 白菜与埃塞俄比亚芥杂交后代的黑腐病抗性统计 BC1(A)S2是与白菜回交后自交2代,BC1(U)S2是与埃塞俄比亚芥回交后自交2代。

Fig.8 Black rot resistant assays of progenies from interspecific hybridization between Brassica rapa ssp. chinensis and B. carinata BC1(A)S2：Second generation selfing of plants backcrossed with B. rapa ssp. chinensis;BC1(U)S2：Second generation selfing of plants backcrossed with B. carinata.

图9 与白菜回交BC3代植株的表型（A）和接种抗性鉴定（B）

Fig.9 The phenotype（A）and black rot resistant identification（B）of the BC3 plants with Brassica rapa ssp. chinensis as recurrent parent

图10 转育的抗病株系BC3(A)-S2与白菜亲本接种黑腐病菌鉴定

Fig.10 The disease resistant BC3(A)-S2 line and the original pakchoi inoculated with Xanthomonas campestris pv. Campestris

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