Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (10): 1950-1958.doi: 10.3864/j.issn.0578-1752.2020.10.003

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

Improvement of the Resistance Against Sclerotinia sclerotiorum in Ogu CMS Restorer in Brassica napus Using Wild B. oleracea as Donor

WAN HuaFang,DING YiJuan,CHEN ZhiFu,MEI JiaQin(),QIAN Wei()   

  1. College of Agronomy and Biotechnology, Southwest University/Academy of Agricultural Sciences, Southwest University, Chongqing 400716
  • Received:2019-09-16 Accepted:2019-12-18 Online:2020-05-16 Published:2020-05-22
  • Contact: JiaQin MEI,Wei QIAN E-mail:jiaqinmey@163.com;qianwei666@hotmail.com

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Abstract:

【Objective】Sclerotinia stem rot (SSR) is a devastating disease in rapeseed (Brassica napus L.). The resistance breeding is greatly limited due to the lack of resistance resources in rapeseed. Ogu CMS is one of the most effective pollination control systems in Brassica. Up to date, however, there has no Ogu CMS restorer with high resistance against SSR reported in rapeseed. High resistant source was reported in a wild B. oleracea which carried two major resistant loci, C1-and C9-QTL. The objective of this study is to transfer the resistance loci from the resistant B. oleracea and develop resistant Ogu CMS rapeseed restorer.【Method】Resynthesized rapeseed line was developed via interspecies cross between a SSR-resistant wild B. oleracea (namely B. incana) and B. rapa by embryo rescue and chromosome doubling. The hybridization was carried out between the resynthesized line and a rapeseed Ogu CMS line, followed by successive self-pollination. Marker assisted selection (MAS) was carried out in each generation to select individual plants carrying resistant loci from B. incana. These plants were screened for resistance level to SSR by inoculation test on detached branches. The plants which exhibited significant lower susceptibility (S) than rapeseed cultivar Zhongshuang 9 (the tolerant control) were then chosen for seed quality analysis. Fertile plants with relatively lower erucic acid and glucosinolate content were self-pollinated for the next generation development. Finally, self-pollinated lines carrying resistant QTL, exhibiting high resistant to SSR, performing good seed quality and yield were considered as SSR-resistant rapeseed Ogu CMS restorer.【Result】Resynthesized rapeseed line AC1-4 was successfully developed from the resistant B. oleracea and B. rapa, which showed significantly higher resistance level to SSR than the tolerant control Zhongshuang 9. From 146 F1 plants derived from AC1-4 and a rapeseed Ogu CMS line 4Q292, two individual plants (4C99 and 4C115) were selected to develop F2 generation, which carried resistant QTLs (C1- and C9-QTL) and exhibited normal seed setting and low susceptibility values (0.66 and 0.40, respectively). Among 124 F2 plants, eight individual plants with good self-fertility were selected to develop 982 F2:3 plants, from which a total of 50 individual plants were identified to carry resistant QTL C1-QTLand C9-QTL. Especially, two fertile plants (5X82-2 and 5X82-7) exhibited ‘single low’ seed quality (6.5% erucic acid in 5X82-7, and 45.38 μmol·g-1 meal glucosinolate in 5X82-7) and low susceptibility to SSR (S = 0.48 and 0.33, respectively).【Conclusion】It successfully improved SSR-resistance in Ogu CMS rapeseed restorer using wild B. oleracea as donor, which will be helpful for developing Ogu CMS restorer with high resistance against SSR and good seed quality in rapeseed.

Key words: Brassica napus, sclerotinia resistance, Ogu CMS, restorer

Table 1

SSR markers used in marker-assisted selection for resistant QTL"

序号
No.		 标记
Marker		 抗病QTL*
Resistant QTL		 连锁群
Linkage group		 R2(%) 引物序列
Primer sequence (5′-3′)
1 Na10-H06 qLR09-3/qLR10-1 C1 13.6 F：AGAATGAGACCCAGAAACCG
R：GCCACACTCTCTCTTACTAGGGC
2 SWUC150 qLR09-3/qLR10-1 C1 13.6 F：AGATCCTTGCAATTTCGCAC
R：TCCTGCAAAGCTTCATCCTC
3 SWUC455 qLR09-4/qLR10-2 C3 10.8 F：GACAACACAACAGACGCA
R：GCATTTCCCATTACTTCCA
4 SWUC427 qSR10-2 C7 3.4 F：CCTCTGTTTCTTTGCTCTTTG
R：GATTCATTGTGTGTGTGATGT
5 SWUC260 qSR09-1/qSR10-3 C9 16.1 F：AACGGCGCTCTAGAGGAAAT
R：AGCTGAACAGCTTGATTGGG
6 SWUC663 qSR09-1/qSR10-3 C9 16.1 F：TGCAGTCGGAATAACCATCA
R：TTCCTATTCTCCAACCGCAG
7 SWUC934 qLR09-2 C9 24.1 F：TTCACACTCCCGATCTCTCC
R：AGATTCCCTCCCCAACAAGT

Fig. 1

Characteristics of AC1-4 (resynthesized Brassica napus) with high resistance against Sclerotinia sclerotiorum A: Seedling of Brassica incana; B: Seedling of 6Y733 (Brassica rapa); C: Seedling of AC1-4; D: Seedling of 4Q292; E: Seedling of F1 (4Q292 × AC1-4); F: Chromosome of haploid (Brassica incana × 6Y733); G: Chromosome of AC1-4 (chromosome doubling); H: Pollen fertility identification of haploid (Brassica incana × 6Y733); I: Pollen fertility identification of AC1-4 (chromosome doubling); J: Resistance identification of leaves of Zhongshuang 9, 6Y733 and AC1-4 (from left to right); K: Resistance identification of branches of Zhongshuang 9, 6Y733 and AC1-4 (from left to right)"

Fig. 2

Verification of the relatives and F1 hybrid individual plants with SSR primer SWU260"

Table 2

Results from molecular marker analysis of F2:3 families"

编号
Code		 家系大小（株）
Size of family
(plants)		 含/不含QTL植株数
Amount of plants inherited/non-inherited QTL		 同时含有C1和C9 QTL植株数/百分
Amount/percentage of plants inherited both QTL on C1 and C9
C1 C9
5X77 72 20/52 5/67 1/1.3
5X78 46 21/20 27/25 20/43.5
5X79 70 23/47 0/70 0/0.0
5X80 184 96/88 99/85 68/37.0
5X81 129 30/98 7/122 3/2.3
5X82 144 70/74 74/70 58/40.3
5X83 184 34/150 26/158 16/8.7
5X84 153 81/72 72/81 56/36.6
共计Total 982 - - 222/22.6

Fig. 3

The Sclerotinia resistance level of selected individuals in F2:3 families"

Table 3

Relative susceptibility and seed quality of some individuals in F2:3 generation"

序号
No.		 编号
Code		 相对感病度
Relative susceptibility		 芥酸
Erucic acid (%)		 硫苷
Glucosinolate (μmol·g-1 meal)
1 5X82-2 0.33±0.07 25.62±1.02 45.38±2.04
2 5X80-29 0.43±0.27 20.69±1.21 96.85±0.82
3 5X80-77 0.47±0.04 19.27±0.63 92.66±1.53
4 5X82-7 0.48±0.10 6.50±1.41 74.88±0.25
5 5X84-89 0.48±0.22 30.37±1.59 107.99±2.35
6 5X82-6 0.5±0.24 20.22±2.79 105.30±0.72
7 5X78-18 0.51±0.01 46.43±0.66 122.93±0.88
8 5X82-136 0.58±0.00 25.26±1.00 96.80±2.52
9 5X82-65 0.63±0.31 28.26±0.94 96.90±1.01
10 5X80-50 0.65±0.19 32.44±2.00 101.39±2.19
11 5X82-43 0.67±0.10 25.33±1.13 79.09±0.68
12 5X84-28 0.71±0.06 16.41±2.24 94.41±0.61
13 5X80-7 0.74±0.10 3.03±1.77 78.74±0.94
14 5X80-161 0.81±0.00 22.39±1.60 114.42±1.11
15 5X80-61 0.82±0.05 23.99±1.76 87.40±1.18
16 5X80-28 0.84±0.02 17.49±1.17 76.48±1.17
17 5X80-110 0.89±0.05 20.08±1.11 68.34±2.01
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