花生栽培种和野生种Arachis sp.30119六倍体杂种的创制与鉴定

doi:10.3864/j.issn.0578-1752.2024.10.003

中国农业科学 ›› 2024, Vol. 57 ›› Issue (10): 1870-1881.doi: 10.3864/j.issn.0578-1752.2024.10.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

花生栽培种和野生种Arachis sp.30119六倍体杂种的创制与鉴定

刘华¹(), 曾凡佩²(), 王倩³, 陈国权³, 苗利娟¹, 秦利¹, 韩锁义¹, 董文召¹, 杜培¹(), 张新友¹()

¹ 河南省农业科学院河南省作物分子育种研究院/神农种业实验室/农业农村部黄淮海油料作物重点实验室/河南省油料作物遗传改良重点实验室，郑州 450002
² 郑州大学农学院，郑州 450001
³ 郑州大学生命科学学院，郑州 450001

收稿日期:2023-11-27 接受日期:2024-02-23 出版日期:2024-05-16 发布日期:2024-05-23
通信作者:
张新友，Tel：0371-65729560；E-mail：haasz@126.com。
杜培，E-mail：dupei2005@163.com
联系方式: 刘华，Tel：0373-7366706；E-mail：liuhua703@126.com。曾凡佩，E-mail：fanpei006677@163.com。刘华和曾凡佩为同等贡献作者。
基金资助:
河南省农业科学院优秀青年基金(2022YQ16); 国家自然科学基金(32272153); 河南省科技研发计划联合基金(232301420025); 国家现代农业产业技术体系(CARS-13); 河南省现代农业产业技术体系(S2012-05); 河南省农业科学院基础科研项目(2023JC03)

Development and Identification of an Interspecific Hexaploid Hybrid Between an A. hypogaea Cultivar and a Wild Species Arachis sp. 30119 in Peanut

LIU Hua¹(), ZENG FanPei²(), WANG Qian³, CHEN GuoQuan³, MIAO LiJuan¹, QIN Li¹, HAN SuoYi¹, DONG WenZhao¹, DU Pei¹(), ZHANG XinYou¹()

¹ Institute of Crop Molecular Breeding, Henan Academy of Agricultural Sciences/The Shennong Laboratory/Key Laboratory of Oil Crops in Huanghuaihai Plains, Ministry of Agriculture and Rural Affairs/Henan Provincial Key Laboratory for Oil Crops Improvement, Zhengzhou 450002
² School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001
³ School of Life Sciences, Zhengzhou University, Zhengzhou 450001

Received:2023-11-27 Accepted:2024-02-23 Published:2024-05-16 Online:2024-05-23

摘要/Abstract

摘要：

【目的】花生野生种包含许多优异的抗病虫基因，是栽培种改良的重要基因资源库，将野生种染色质导入栽培种是花生远缘杂交研究的重要目的。野生种A. sp. 30119对多种花生病害具有抗性，研究其与栽培种的杂种后代，为明确A. sp. 30119的身份和未来能够利用其优异抗性提供依据。【方法】利用异源四倍体花生栽培品种白突131与二倍体野生种A. sp. 30119杂交，通过胚拯救获得种间杂种F₁（W1212），但F₁不结实。为了揭示W1212不结实的原因并获得可育的后代，先利用根尖细胞有丝分裂中期和花粉母细胞染色体制片，观察其染色体数目和减数分裂染色体联会情况，再利用试管苗染色体加倍方法对W1212进行加倍处理，最终收获4个单粒荚果，将其中一个荚果的种子组织培养成完整植株并命名为Am1212。利用顺序GISH/FISH和SSR标记分析Am1212的染色体组成，并观察调查其表型特征。最后，利用rDNA FISH随机分析8个Am1212的F₃代植株的有丝分裂中期染色体数目，评价其染色体遗传稳定性。【结果】白突131与A. sp. 30119的杂种一代W1212花粉母细胞减数分裂中期Ⅰ的染色体平均构型为1Ⅲ+6Ⅱ+15Ⅰ，其染色体不能正常配对，表现为高度不育，染色体加倍处理后W1212下针枝条的花粉育性显著提高。白突131、A. sp. 30119和Am1212的顺序GISH/FISH分析结果表明，A. sp. 30119可能为A基因组二倍体野生种。Am1212有60条染色体，包含白突131与A. sp. 30119的全部染色体，证实其为六倍体杂种后代。但Am1212自交F₃代有37.5%的植株发生了染色体数目变异。通过分子标记筛选和表型调查，获得17个特异追踪A. sp. 30119的显性和共显性标记，明确了Am1212的遗传特性。【结论】创制了一个新的六倍体花生Am1212，该六倍体整合了野生种染色质，同时，Am1212也拥有了小荚果等不利性状，且染色体遗传稳定性较差，因此，未来育种利用过程中，需要结合精准的染色体鉴定技术，打破不良连锁，获得具有补偿效应且包含有利性状的染色体变异材料。

关键词: 花生, 野生种, 六倍体, 寡核苷酸荧光原位杂交, 分子标记

Abstract:

【Objective】 Wild Arachis species containing many elite disease and insect resistant genes are an important gene pool for the improvement of cultivated peanut A. hypogaea L., so the introduction of chromatin from wild species into cultivated peanut remains an important task for distant hybridization of peanut. Unfortunately, only a few wild species have been successfully involved in the development of interspecific hybrids, despite the existence of a huge amount of wild germplasm resource. The wild species A. sp. 30119 contains multiple disease resistance. However, no interspecific hybrid between it and the cultivated peanut has been reported.【Method】 After crossing allotetraploid peanut cultivar Baitu 131 with diploid wild species A. sp. 30119 and followed by the embryo rescue, the interspecific hybrid F₁ (W1212) was obtained. However, interspecific hybrid F₁ did not produce seed when selfed. In order to reveal the reason for infertility and continue the generation, the chromosome number of root tip cells was counted and chromosome pairing in pollen mother cells during meiosis of W1212 observed. The chromosome of W1212 was doubled through colchicine treatment during the in vitro culture W1212 plantlet. Finally, we harvested four one-seeded pods from the established W1212 plants; one of the pods undergone in vitro cultured and a number of plants were established from the culture and named as Am1212. The chromosome composition of Am1212 was analyzed by sequential GISH/FISH and SSR marker. Additionally, phenotypic characteristics of Am1212 were investigated. The metaphase chromosome numbers of 8 randomly selected F₃ plants were analyzed by rDNA FISH to evaluate the genetic stability of Am1212.【Result】 The average chromosome configuration during meiosis of pollen mother cells of W1212 was 1Ⅲ+6Ⅱ+15Ⅰ, and the abnormalities in chromosome pairings lead to the high sterility of F₁ plants. The pollen viability of peg-setting branches of W1212 was significantly improved after chromosome doubling treatment. Sequential GISH / FISH of Baitu 131, A. sp. 30119 and Am1212 indicated that A. sp. 30119 may be a diploid with A genome. Am1212 has 60 chromosomes consisting of all chromosomes of both Baitu 131 and A. sp. 30119, which confirmed its nature of a hexaploid hybrid, but 37.5% F₃ plants derived from Am1212 exhibited chromosome number variation. After conducting the screening of SSR molecular markers and phenotypic investigation, we obtained 17 dominant or co-dominant markers for specifically tracking wild species chromosomes and clarified the genetic characteristics of Am1212.【Conclusion】 In this study, we successfully created a new hexaploid peanut Am1212 which incorporated A. sp. 30119 chromatin. However, Am1212 exhibited instability in chromosome numbers and inherited unacceptable agronomic traits from wild species, such as small pods. Therefore, it is necessary to develop more accurate and efficient chromosome manipulation techniques to minimize the unfavorable gene linkage and generate alien chromosomal lines that possess compensatory effects and favorable traits for future breeding purposes.

Key words: peanut, wild species, hexaploid, oligonucleotide fluorescence in situ hybridization, molecular markers

刘华, 曾凡佩, 王倩, 陈国权, 苗利娟, 秦利, 韩锁义, 董文召, 杜培, 张新友. 花生栽培种和野生种Arachis sp.30119六倍体杂种的创制与鉴定[J]. 中国农业科学, 2024, 57(10): 1870-1881.

LIU Hua, ZENG FanPei, WANG Qian, CHEN GuoQuan, MIAO LiJuan, QIN Li, HAN SuoYi, DONG WenZhao, DU Pei, ZHANG XinYou. Development and Identification of an Interspecific Hexaploid Hybrid Between an A. hypogaea Cultivar and a Wild Species Arachis sp. 30119 in Peanut[J]. Scientia Agricultura Sinica, 2024, 57(10): 1870-1881.

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编号 Number	单价体 Monovalent	二价体 Bivalent	三价体 Trivalent	棒状二价体 <BOLD>C</BOLD>lavate bivalent	环状二价体 <BOLD>C</BOLD>yclic bivalent
F₁-1	17	5	1	2	3
F₁-2	10	7	2	5	2
F₁-3	15	6	1	3	3
F₁-4	17	5	1	3	2
F₁-5	14	8	0	3	5
F₁-6	15	6	1	5	1
F₁-7	14	5	2	1	4
F₁-8	18	6	0	5	1
F₁-9	19	4	1	1	3
F₁-10	11	8	1	5	3
平均Average	15	6	1	3.42	2.58

标记 Marker	初始名称 Origin name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	特点 Characteristics
E467	ER974516	GCATGCAAAGGAGAAAGCTC	GAGTGGATAATGAGGTGAACAAAG	父本显性Male dominance
G21	AhTE0486	TGATTGGATCTCAATTCCCA	TTTGATCGCTCTCTCTTTCTTTC	父本显性Male dominance
E578	GM174	CGTCTTTGTCAGATGTTTCCAA	TTACCCAAAATCCCATAAGGTG	父本显性Male dominance
G41	AhTE0202	GAGAGCGGTAATTTTTATGATTTG	TACTTTTGCGACTTTGTCTCCA	父本显性Male dominance
F149	XX67	TTGCCATCACGCTTCTTG	TGTTGCCTTCTTCCTCCC	父本显性Male dominance
G43	AhTE0422	TGGCGTAATCTTTTAAGAACCAA	AGAATTAATGTCATCAAACGAATG	父本显性Male dominance
G19	AhTE0571	TTACATAAGGCATTAACGAGTGTT	AACAGCTTTGCAGGAGATGG	父本显性Male dominance
G78	AhTE0437	TGGCTTTTGGGTGTGTATGA	GCCACGAGAGAATCCAAAAA	父本显性Male dominance
E57	GM1628	AAACGTGCTCTAGAAACATACAAAA	CAACACATGCAATGCAACAA	共显性Co-dominance
E152	GM2831	TGAAGAAGAAGCTGAGGCAAA	AGAACCTTCCATCTCTGTTGTTG	共显性Co-dominance
E441	ER974469	CAACACAAGCCCACAACAAA	TCCATCATCACCCTCATCAA	共显性Co-dominance
E723	GM474	GAGGTCGCCATAGTCATCGT	AAAATGTTGGGTGTTGGCAT	共显性Co-dominance
A31	PM84	TGAATGCTAGGCAACCAAAA	TGTAGAGACGACACCCATGC	共显性Co-dominance
F6	1X18-F1	GGTGAGGCAATGAAAGGT	AGCAACAGCCGATGAAGG	共显性Co-dominance
F69	2K49-T3	GTTCCAAACTTGTTTTCC	GGTCTTCACGTTAATCTTC	共显性Co-dominance
F238	TC39C01	AGGCGCGGTGATAGAAAAC	GTTTTGCTGTTCGTCCCTAAAC	共显性Co-dominance
G31	AhTE0522	TTGCTGCTGTTATCTCCTTAAAAA	GCTAAAGCAAAGAGAATTGATTGG	共显性Co-dominance

性状Traits	白突131 Baitu 131	Am1212	t-test
叶柄长Petiole length (mm)	38.60±8.58	24.70±1.99	3.53**
主茎叶长Leaf length on main stem (mm)	33.60±3.93	28.70±3.34	n.s.
主茎叶宽Leaf width on main stem (mm)	16.10±0.96	19.50±3.67	n.s.
主茎高Height of main stem (cm)	44.20±4.79	95.40±13.81	7.01**
侧枝长Length of first primary branch (cm)	49.60±5.85	87.00±18.55	3.85**
总分枝数Number of branches	58.60±8.93	26.00±6.36	5.82**
结果枝数Number of bearing branches	18.80±3.12	8.80±1.92	5.61**
饱果Mature pod (pcs)	49.20±11.37	8.60±2.90	6.93**
幼果Immature pod (pcs)	2.50±0.50	6.80±2.95	6.96**
株型Plant type	直立Erect	半蔓生Semi-spread	/

花生栽培种和野生种Arachis sp.30119六倍体杂种的创制与鉴定

Development and Identification of an Interspecific Hexaploid Hybrid Between an A. hypogaea Cultivar and a Wild Species Arachis sp. 30119 in Peanut

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