Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (16): 3304-3316.doi: 10.3864/j.issn.0578-1752.2025.16.013

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

Molecular Cytogenetic Characterization of Wheat-Thinopyrum intermedium 7JS Addition Line CH71 with Resistance to Both Powdery Mildew and Stripe Rust

ZHANG ShuWei1(), SHI WenYu1, LI BingJie1, CHANG SongHe1, QIAO LinYi1, LI Xin1, CHANG LiFang1, CHEN Fang1, GUO HuiJuan1, CHANG ZhiJian1, ZHANG XiaoJun1,2,*()   

  1. 1 College of Agriculture (Institute of Crop Sciences), Shanxi Agricultural University, Taiyuan 030031
    2 Key Laboratory of Sustainable Dryland Agriculture (Co-Construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Taiyuan 030031
  • Received:2025-02-25 Accepted:2025-04-22 Online:2025-08-11 Published:2025-08-11
  • Contact: ZHANG XiaoJun

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Abstract:

【Objective】Thinopyrum intermedium, a tertiary gene pool of wheat, harbors valuable genetic resources for wheat improvement. This study aimed to develop novel wheat germplasms by transferring elite chromosomes from Thinopyrum intermedium into wheat via distant hybridization, investigating their impacts on disease resistance and agronomic traits to establish a theoretical foundation for wheat breeding. 【Method】A wheat-Thinopyrum intermedium disomic addition line, CH71 (2n=44), was developed from the BC1F6 progeny of a cross between common wheat cultivar Yannong 999 and the partial amphidiploid TAI8047 (2n=58). Non-denaturing fluorescence in situ hybridization (ND-FISH) with oligonucleotide probes (Oligo-pSc119.2, Oligo-pTa535, Oligo-B11, Oligo-pDb12H) was employed for karyotype analysis. Synteny-based (Synt) Oligo-FISH painting and Th. intermedium-specific STS markers were utilized to identify the homologous group and validate the origin of the alien chromosome. Disease resistance to powdery mildew (race E09) and stripe rust (mixed races CYR32, CYR33, CYR34) was evaluated under artificial inoculation. Agronomic traits, including plant height, spike length, spikelet number, thousand-grain weight, grain length, and grain width, were systematically measured.【Result】The wheat parent Yannong 999 exhibited a standard karyotype of 21 wheat chromosome pairs. TAI8047 contained 58 chromosomes (21 wheat pairs + 8 alien pairs), while CH71 harbored 44 chromosomes (21 wheat pairs + 1 alien pair, JS-1). Synt-FISH and PCR amplification using 183 STS primers specific to Th. intermedium chromosome 7JS confirmed JS-1 as a 7JS-derived chromosome. Twelve STS markers consistently amplified diagnostic bands in Th. intermedium, TAI8047, and CH71. CH71 displayed moderate resistance to powdery mildew and immunity to stripe rust, with genetic analysis indicating both resistances were conferred by the 7JS chromosome. Compared to Yannong 999, CH71 exhibited significant increases in plant height (+24.0 cm), spike length (+3.44 cm), spikelet number (+1.6), and grain length (+0.68 mm), but reductions in thousand-grain weight (-6.78%) and grain width (-2.70%). 【Conclusion】The novel disomic addition line CH71 (wheat-Th. intermedium 7JS) demonstrates dual resistance to powdery mildew and stripe rust, serving as a valuable germplasm resource for disease-resistant breeding and the cloning of alien resistance genes. The 12 STS markers identified herein provide an efficient molecular tool for rapid tracking of the 7JS chromosome in wheat backgrounds.

Key words: wheat, Thinopyrum intermedium, alien addition line, fluorescence in situ hybridization, STS markers

Table 1

Oligonucleotide probe sequences"

探针名称
Probes		 修饰类型
Modification types		 探针序列
Probe sequences (5′-3′)		 参考文献
References
Oligo-B11 5′6-FAM TCCGCTCACCTTGATGACAACATCAGGTGGAATTCCGTTCGAGGG [22]
Oligo-pDb12H 5′TAMRA TCAGAATTTTTAGGATAGCAGAAGTATTCGAAATACCCAGATTGC TACAG [23]
Oligo-pSc119.2 5′6-FAM CCGTTTTGTGGACTATTACTCACCGCTTTGGGGTCCCATAGCTAT [24]
Oligo-pTa535 5′TAMRA AAAAACTTGACGCACGTCACGTACAAATTGGACAAACTCTTTCGGAGTATC
AGGGTTTC		 [24]

Table 2

Sequences of STS markers specific to the chromosomes of Thinopyrum intermedium"

序号
No.		 引物名称
Primer name		 染色体
Chromosome		 正向引物
Forward sequence (5′-3′)		 反向引物
Reverse sequence (5′-3′)		 退火温度
Annealing temperature (℃)
1 C19-08 7JS CTTAGTCTGCAGCCTCTGTCC AGAGCATCGTCAATAGCCCAG 60
2 C19-22 7JS AAACATAGCCTAAGACGGGC GGTCATTGGGTGCAGAATAG 60
3 C19-39 7JS GAATCTGCGGTGTGCTATTT TCGGTCGACAACATAGTGTG 60
4 C19-54 7JS ACGCAAGTTTCAGATAAACAAGACT TCTCTTGATCGGTTGGACACC 58
5 C19-61 7JS CGTGTATCGAACGTGAAATG CAGTTGGAAATCCAGGAATG 60
6 C19-68 7JS CTCTTGTCGTCGAGGGTCT GTAGAGGTACTTAGCGGCGA 58
7 C19-83 7JS TTTGCTCACTCTCTGGTTCC GAAAGTTGGAGAACGCTCAA 60
8 C19-103 7JS GGATGAAGCATCTCCACAAC ATCAGGAAGCTCAGCATCAG 58
9 C19-105 7JS TTCTCATGTGTGTGACCCGC TTGGTAGAACGGCCACAAGA 60
10 C19-109 7JS AGTAGTGCTGAACTTGCCACT GCAAACGTCTTCGGCGTATC 60
11 C19-136 7JS GCTCACCATGAGAAGGAAAA GGATGAATGTTAATGGCGAG 60
12 C19-138 7JS CCTAGAAGTTCACGGCCTC GGCACTTCTGTAGCTTGCAT 60

Fig. 1

Karyotype identification of TAI8047, Yannong999 and CH71 a, b: Karyotype of TAI8047 chromosomes with different probes. a: The probes used are Oligo-B11 (green) and Oligo-pDb12H (red), b: The probes used are Oligo-pSc119.2 (green) and Oligo-pTa535 (red); c: Karyotype of Yannong 999 chromosomes with wheat probes, d-f: Karyotype of CH71 chromosomes with different probes. d: The probes used are Oligo-B11 (green) and Oligo-pDb12H (red), e: The probes used are Oligo-pSc119.2 (green) and Oligo-pTa535 (red), f: The probe used is the 7th homologous group of wheat probe sleeve Synt7 (red); g: Detailed karyotypes of CH71; h: Karyotype comparison between JS-1 chromosome of TAI8047 and foreign chromosome of CH71. The four karyotypes in each group were DAPI staining, probe Oligo-B11 (green)/probe Oligo-pSc119.2 (green), probe Oligo-pDb12H (red)/probe Oligo-pTa535 (red), and the first three combined images. Scale: 10 μm"

Fig. 2

Amplification results of molecular markers and their genetic map on 7JS chromosome M: Marker; 1: Thinopyrum intermedium; 2: TAI8047; 3: CH71; 4: Yannong 999; 5: Chinese spring. The white arrows indicate the specific band positions. a-l indicate the amplification results of marker C19-08, C19-22, C19-39, C19-54, C19-61, C19-68, C19-83, C19-103, C19-105, C19-109, C19-136, and C19-138, the figure of m indicates the genetic map of markers on 7JS chromosome"

Fig. 3

Identification of disease resistance of CH71 and its parents a: Identification of resistance to powdery mildew during the seedling stage; b: Identification of resistance to powdery mildew during the adult stage; c: Identification of resistance to stripe rust during the adult stage. The numbers at the bottom indicate the resistance level. 0: Immunity, 0;: Near immunity, 1: High resistance, 2: Moderate resistance, 3: Moderate sensitivity, 4: High sensitivity"

Table 3

Investigation of disease resistance of CH71 and its parents at seedling and adult stages"

材料名称
Material name		 染色体数目
Number of
chromosome		 外源染色体
Alien
chromosome		 条锈菌小种
CYR32/CYR33/CYR34
Stripe rust species
CYR32/CYR33/CYR34		 白粉菌小种E09 Powdery mildew species E09
苗期
Seedling resistance		 成株期
Adult-plant resistance
太原768 Taiyuan 768 42 — 4 4 4
系76（64）Line 76(64) 42 — 4 4 4
TAI8047 58 J+JS+St 0 0; 0
CH71 44 7JS 0; 2 2
烟农999 Yannong 999 42 — 4 4 4
SY95-71 42 — — 4 4
TC29 42 — 4 — —

Table 4

Investigation of agronomical traits between CH71 and its parents"

材料名称
Material
name		 外源染色体
Alien chromosome		 株高
Plant height
(cm)		 穗长
Spike length
(cm)		 小穗数
Spikelet number		 千粒重
Thousand grain weight (g)		 粒长
Grain length
(mm)		 粒宽
Grain width
(mm)
太原768 Taiyuan 768 — 76.60±3.51a 8.88±0.43a 20.80±1.10a 49.43±1.67b 6.84±0.17a 3.75±0.11c
系76（64）Line76 (64) — 77.20±3.90a 9.06±0.36a 20.40±0.89a 49.03±1.21b 6.86±0.14a 3.72±0.08bc
TAI8047 J+JS+St 94.80±4.44b 17.20±0.57c 21.60±1.67a 29.89±3.09a 8.10±0.17c 2.95±0.09a
烟农999 Yannong 999 — 77.40±3.96a 8.30±0.35a 20.40±0.89a 52.39±5.09b 6.82±0.24a 3.70±0.12bc
CH71 7JS 101.40±7.78b 11.74±1.28b 22.00±1.33a 48.84±2.43b 7.50±0.18b 3.60±0.09b

Fig. 4

Comparison of agronomic traits between CH71 and its parent Yannong 999 a: Comparison of plant types at harvest time; b: Comparison of spike type; c: Comparison of grain morphology. 1: CH71, 2: Yannong 999"

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