Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (10): 1870-1881.doi: 10.3864/j.issn.0578-1752.2024.10.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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. 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 Online:2024-05-16 Published:2024-05-23
  • Contact: DU Pei, ZHANG XinYou

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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

Fig. 1

Observation of metaphase chromosomes, meiosis, and pollen viability of the interspecific hybrid W1212 A: Metaphase chromosomes of W1212; B: Meiotic chromosome pairing of W1212; C: Pollen viability of W1212; D: Pollen viability of peg-setting branches of W1212 after chromosome doubling"

Table 1

Meiotic chromosome pairing analysis of the interspecific hybrid F1"

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

Fig. 2

Sequence GISH/FISH and karyotype analysis of Baitu 131, A. sp. 30119 and Am1212 A-C: Sequential GISH/FISH using Multiplex # A (A, green), Multiplex # B (A, red), Multiplex # 1 (B, green), Multiplex # 2 (B, red), 45S rDNA (C, red) and 5S rDNA (C, green) as probes in Baitu131; D-E: Sequential GISH/FISH using Multiplex # A (D, green), Multiplex # B (D, red), Multiplex # 1 (E, green), Multiplex # 2 (E, red), 45S rDNA (F, red) and 5S rDNA (F, green) as probes in Am1212; G-H: DAPI staining (G) and sequential FISH using Multiplex # 1 (H, green), Multiplex # 2 (H, red), 45S rDNA (I, red) and 5S rDNA (I, green) as probes in A. sp. 30119; J: Karyotypes of Baitu 131, A.sp. 30119 and Am1212. G represented the GISH; OF represented the oligonucleotide FISH; RF represented rDNA FISH"

Fig. 3

PCR amplification pattern of Baitu131, A. sp. 30119 and Am1212 using SSR marker G82, G97"

Table 2

Dominant or co-dominant molecular markers of A. sp. 30119 in Am1212"

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

Fig. 4

Morphologies of plant (A), leaves (B) and legumes (C) of Baitu131, Am1212 and A. sp. 30119"

Table 3

Comparison of amphidiploid Am1212 with Baitu131 and A. sp. 30119 in some agronomic traits"

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 /

Fig. 5

Metaphase chromosome FISH and chromosome number of hexaploid Am1212 F3 A-H: FISH using 45S rDNA and 5S rDNA probes in Am1212 F3-1, Am1212 F3-2, Am1212 F3-3, Am1212 F3-4, Am1212 F3-5, Am1212 F3-6, Am1212 F3-7 and Am1212 F3-8. Blue showed DAPI staining, red showed 45S rDNA signal, and green showed 5S rDNA signal"

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