Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (8): 1579-1589.doi: 10.3864/j.issn.0578-1752.2021.08.001

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

Polymorphism Analysis Among Chromosomes of Dasypyrum villosum 6V#2 and 6V#4 and Wheat 6A and 6D Based on Wheat SNP Chip

XU ZhiYing1,2(),WANG BaiCui2,MA XiaoLan2,JIA ZiMiao2,YE XingGuo2,LIN ZhiShan2(),HU HanQiao1()   

  1. 1College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, Guangdong
    2Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2020-08-24 Accepted:2020-10-28 Online:2021-04-16 Published:2021-04-25
  • Contact: ZhiShan LIN,HanQiao HU E-mail:13414916566@163.com;linzhishan@caas.cn;huhanqiao@sina.com

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Abstract:

【Objective】 The polymorphism among the chromosomes of Dasypyrum villosum 6V#2 and 6V#4 and their wheat homeologous 6A and 6D in the DNA level was compared in this study based on big data to provide a theoretical basis for precision designing breeding of wheat-Dasypyrom villosum targeted chromosome translocation.【Method】A 6V#4(6D) alien disomic substitution line RW15 was crossed with a 6V#2(6A) alien disomic substitution line Nan 87-88, and their F2 plants were detected using 6V#4S/6V#2S/6AS/6DS/6VL specific molecular markers, and the new types of substitution lines were confirmed in F3 employing the above-mentioned markers again as well as genomic in situ hybridization (GISH) technique. Subsequently, polymorphism among the targeted chromosomes in the new types of the substitution lines and their parents Nan 87-88 and RW15 were detected by the probes of 6A and 6D-specific in the wheat 55K chip, and combined with the results of SNP analysis of two accessions of D. villosum with 6A and 6D probes in 660K chip to screen 6V specific SNPs. 【Result】 GISH analysis showed that 19EL124 and 19EL134 had 42 chromosomes including two complete foreign chromosomes in the somatic cells of root tips. Identification using molecular markers showed that 19EL124 had 6V#4S/6DS and missed 6V#2S/6AS specifically amplified bands, while 19EL134 displayed 6V#2S/6AS distinctive bands and lacked 6V#4S-specific bands; both 19EL124 and 19EL134 contained 6VL specific amplified bands, which confirmed that 19EL124 is a 6V#4(6A) disomic substitution line, and 19EL134 is a 6V#2(6D) disomic substitution line. The results of 55K chip showed that the detected efficiency of the key chromosome probes in the alien substitution lines was significantly lower than that of other chromosome probes, and the detected efficiency of different lines in the same type of substitution was also different. Among the 1 177 probes of 6A, the 6A substitution line Nan87-88 and 19EL124 could not be genotyped by 63.21% and 68.90% of the probes, respectively. And the polymorphism between 6V#2 and 6V#4 was detected by 22.51% of the probes. Among the 479 probes of 6D, 49.48% of the probes could not identify the 6D substitution line RW15, and 53.44% could not genotype the 6D substitution line 19EL134. The polymorphism between 6V#2 and 6V#4 was indicated by 16.70% of the 6D probes, there were 23 and 42 probes that could only detect 6V#2 and 6V#4 chromosome, respectively. Twenty-two and fifteen 6V specific SNPs were screened from 395 6A and 231 6D probes through integrating the same genotypes detected by probes which were shared by 55K and 660K chips in this study, and three of them showed polymorphism between 6V#2 and 6V#4 chromosomes. 【Conclusion】 The detection efficiency of the wheat chromosome probes was greatly reduced when the target wheat chromosome was replaced by the alien chromosome, and the proportion of NA genotyping was greatly increased, and most of the NA genotypes showed polymorphism between the two different alien chromosomes in the same type of substitution. The detection efficiency of probe for two alien chromosomes in the same type of alien substitution lines was different, wheat 6A probe could detect 6V#2 better, and wheat 6D probe could detect 6V#4 better. Thirty-seven SNP markers of 6V specific were obtained base on the association analysis of 6V genotyping between D. villosum and the alien substitution lines.

Key words: wheat, Dasypyrum villosum, alien disomic substitution, wheat SNP chip, polymorphism

Table 1

PCR amplification reaction system"

组分 Component 用量 Dosage (μL)
2×Taq master mix 7.5
dd H2O 5.9
正向引物 Forward primer (10 μmol·L-1) 0.3
反向引物 Reverse primer (10 μmol·L-1) 0.3
DNA (100 ng·μL-1) 1.0
总计Total 15.0

Table 2

Information on the molecular markers used in PCR amplification"

引物名称
Primer name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 检测类型
Types detected		 参考文献
References
6VS-06 GACAGGCAGCTATGAGGC AATCGTCGTTTGGAGTGG 6V#4S [20]
6VS-09 GTAAGAACAAGAGGCTAAACAG CCAGATGACGGTTATTACATAG 6V#4S [20]
6VS-10 GCCATAAGTGACGCTGAT GCATCCTGTGAAGTTGTTG 6V#4S [20]
6VS-12 TGTTGCCTCTCCTCATCA ATTGCTGTCCGCTCATAC 6V#2S
6V#4S		 [20]
6VS-15 AGGACCATACATTCACAGAG TTCCATGAGCAGATTAGCA 6V#2S
6V#4S		 [20]
6VS-18 AGCCAGTAAGATTCCGTATG TCTAACCTTCCTCACAACAC 6V#4S [20]
MBH1 GCCATTATAGTCAAGAGTGCACTAGCTGT AGCTCCTCTCGTTCTCCAATGCT 6V#2S
6V#4S
6AS;6DS		 [10]
P259-1 CGTGATTCAGGAAATGCGATAC TTGCGCCGCCATGTTAG 6V#4S [8]
P461-5a GCGTCATCCGCGCCCGTCAGGT GAGTGCTAATGATAGATGTG 6V#2S
6V#4S		 [8]
N-P5 GCGACCTGTTAGAATGCTATTACGATTAC ATGCTACTCTACCGATGCTTTGAACC 6AS
6DS		 [12]
6V-1 CCGTGCGACAGAACAGAAGTGA GCAATCAGCCACATACAGGTCATC 6VL [20]

Fig. 1

GISH patterns of somatic chromosomes in root tips of alien substitution lines Wheat chromosomes were stained with propidium iodide (PI) in red, and alien chromosomes in yellow-green, which were indicated by the arrows. a-d: RW15, Nan 87-88, 19EL124, 19EL134, 2n=42"

Fig. 2

Amplification patterns of alien substitution lines by molecular markers M: DL2000 marker; 1-7: 19EL124, 19EL134, RW15, Nan87-88, Wan7107, Pm97033, and 10SR3109"

Fig. 3

The SNPs between Nan87-88 and RW15 detected by 6A and 6D-specific probes A: 6A probe; B: 6D probes. a: Total probes and their distribution on the chromosomes; b: Base substitution-type SNP; c: Missing-type SNP(RW15-); d: Missing-type SNP (Nan87-88-)"

Table 3

The NA number of alien substitution lines detected by all probes"

探针数量
No. of probes		 RW15 南87-88
Nan87-88		 19EL124 19EL134
1A（1797） 20 10 51 51
1B（1808） 20 10 22 26
1D（1124） 10 6 8 6
2A（1597） 11 8 9 13
2B（1686） 27 14 27 35
2D（1034） 4 3 10 11
3A（1108） 4 7 11 12
3B（1804） 13 12 16 17
3D（945） 11 6 8 7
4A（1368） 8 11 8 5
4B（1334） 11 6 10 26
4D（477） 6 1 6 6
5A（1791） 25 9 28 6
5B（1637） 18 9 21 14
5D（870） 8 6 19 9
6A（1177） 29 744 811 30
6B（1628） 22 26 21 32
6D（479） 237 13 33 256
7A（1628） 9 13 30 15
7B（1601） 9 13 12 20
7D（1090） 7 4 16 10

Fig. 4

The NA proportion of each chromosome of four alien substitution lines detected by 28020 probes of 55K chip with physically located on wheat chromosomes"

Fig. 5

Differential genotyping between the two alien chromosomes 6V#2 and 6V#4 in the same type of substitution lines detected by 6A and 6D-specific probes, respectively A: Differential genotyping between two 6V(6A) substitution lines of 19EL124 and Nan 87-88 detected by 6A probes. a: Distribution of total differential probes; b: Distribution of differential probes with base substitution; c: Distribution of differential probes with missing-type (19EL124 -); d: Distribution of differential probes with missing-type (Nan 87-88 -). B: Differential genotyping between two 6V(6A) substitution lines of 19EL134 and RW15 detected by 6D probes. a: Distribution of total differential probes; b: Distribution of differential probes with base substitution; c: Distribution of differential probes with missing-type (19EL134 -); d: Distribution of differential probes with missing-type (RW15 -)"

Table 4

SNP between two accessions of Dasypyrum villosum CMM and No.1026 detected by 6A/6B/6D probes of 660K chip"

染色体
Chr.		 探针数量
No. of probes		 总SNP(占比)
Total SNP (percentage)		 碱基替换型SNP(占比)
SNP of base replacement
(percentage)		 CMM-NA SNP(占比)
CMM-NA SNP
(percentage)		 No.1026-NASNP(占比)
No.1026-NA SNP (percentage)
6A 4710 1710(36.31%) 888(18.85%) 414(8.79%) 408(8.66%)
6B 14352 5626(39.20%) 3037(21.16%) 1376(9.59%) 1213(8.45%)
6D 3051 1206(39.53%) 725(23.76%) 247(8.10%) 234(7.67%)
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