Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (20): 4103-4112.doi: 10.3864/j.issn.0578-1752.2020.20.001

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

Development and Utilization of KASP Marker for Se Concentration in Synthetic Wheat SHW-L1

WEI GuangHui1,2(),LI Zhi2,CHEN Qiang2,LI Yang2,CHEN ShiHao2,PEI Ying2,ZHOU Yong3,CHENG MengPing3,TANG Hao3,WANG JiRui1,3,WEI YuMing1,3,LIU DengCai1,3,CHEN Li4,ZHENG YouLiang1,3,PU ZhiEn1,2()   

  1. 1State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Chengdu 611130
    2College of Agronomy, Sichuan Agricultural University, Chengdu 611130
    3Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130
    4Yibin Product Quality Supervision and Inspection Institute, Yibin 644000, Sichuan
  • Received:2019-12-02 Accepted:2020-03-07 Online:2020-10-16 Published:2020-10-26
  • Contact: ZhiEn PU E-mail:18437957800@163.com;puzhien@sicau.edu.cn

Abstract:

【Objective】As one of the essential elements of the human body, selenium plays an important role in disease prevention and antioxidants. Wheat, as one of the main crops, is the main source for humans to obtain plant selenium sources, so increasing the selenium content of wheat grains is an efficient, inexpensive and easy way to supplement selenium. This study intends to develop KASP markers for the identified SNP loci for the breeding of high-selenium wheat.【Method】Using the completed 660K wheat SNP markers of synthetic wheat SHW-L1 recombinant inbred lines population ( recombinant inbred lines, RILs), scanning the RILs population, further narrowing the QTL interval related to the selenium content of the previously located grains, and developing the SNP loci in the stable gene as KASP markers.【Result】Two successfully developed KASP markers AX-1 and AX-2, markers can be used for genotyping and identification of selenium content in the grain of the material. Both markers can be used to classify test materials according to genotype into two categories, namely high-selenium and low-selenium materials, meets the 1:1 allele separation ratio. However, according to the phenotype, the screening rate (greater than 90%) for low-selenium materials is much higher than that of high-selenium materials (less than 30%), and it should be clear that 81% of the phenotype materials with high selenium content have high selenium genotypes, indicating the reliability of the label. Therefore, the KASP markers developed can be used to improve selection efficiency. At the same time, the KASP marker can also be used for genotyping in other derived offspring lines with artificial wheat SHW-L1 as the parent, and the typing results are consistent with the phenotypic results, indicating the validity of the marker.【Conclusion】AX-1 and AX-2 can be used as a practical molecular marker for breeding and germplasm resource creation of high selenium wheat varieties with SHW-L1 as the parent.

Key words: synthetic wheat, selenium content, KASP

Table 1

Variance analysis of RIL population selenium concentration"

变异来源
Source of variation
平方和
Type Ⅲ SS
自由度
df
均方
MS
F
F value
Sig.
V 0.02995031 114 0.00026272 13.410 **
T 0.00009015 1 0.00009015 0.4890
V×T 0.02100010 110 0.00019091
误差Error 0.00415800 110 0.00003780

Table 2

QTL results after screening LOD values"

籽粒硒含量
Grain selenium content
编号
Number
染色体
Chromosome
标记
Marker
位置
Position (cM)
R2 加性效应
Additive
LOD
Grain se-1 1 5A 22 43.62 0.0765 0.0003 2.5551
Grain se-2 1 5A 13 29.54 0.0654 0.0003 2.7476
Grain se-2 2 5A 16 32.66 0.0774 0.0003 3.4722
Grain se-2 3 5A 21 35.58 0.0877 0.0004 3.9723

Fig. 1

SNP comparison of genotyping and grain selenium content X-axis: All materials of the RILs population, arranged according to the selenium content of the grains in ascending order; Y-axis: Markers initially selected; Arrows: 2 SNP markers; Blue: Type A; Purple: Type B; White: Missing"

Table 3

SNP markers used in this study"

编号
Number
染色体
Chromosome
变异碱基
Base
序列
Sequence (5′-3′)
AX-1 5A A/G CGTACCATGCGATCACACTTGGAGATGCGTGTGAG[A/G]GAAAAAGAGTACTCCATCCATCCAAATTCCATCCC
AX-2 5A A/G TGTAAATTAATTTGTTGTGGGTAGGGCATCTTGAG[A/G]AATGTTTACCTTCTTGCTGCTGCCTGGTTCGGTTT
AX-3 5A A/C GGGTGATAGCAAGGAAGCTGAAGAGCATGATGGAC[A/C]AGGGACAGACGGAAAAGAGTTGCAGGATGGACAAG
AX-4 5A A/G GATGACTTTGCGGCAATATTAAGAGATGTGCATCC[A/G]TTAGCAAGCAAGCTAGCGGACAAAACCAGCTTAAC
AX-5 5A T/C ACCCCTTGTTTAGTACCACCTCACCAAGTGAGAGT[C/T]ATTTCAACCGGTTTATAAGATTCAGCGTTCCAACC

Table 4

KASP primers and sequences"

Fig. 2

Initial KASP-SNP typing of selenium content samples from different grains by molecular markers These spots within the axis represent different single plant, and data spots might be overlapped; Black dots: Controls (ddH2O); Orange dots: High selenium gene genotypes; Blue dots: Low selenium genotypes. The same as below"

Fig. 3

KASP-SNP typing of selenium content in grain of RILs 138 population by molecular markers"

Fig. 4

Distribution of KASP-labeled AX-1 and AX-2 alleles in 118 materials AA: Selenium-deficient genotype; GG: Selenium-rich genotype"

Fig. 5

Qualitative detection of selenium content in grain of different validation samples by molecular markers"

Table 5

Comparison of typing test results and phenotypic data"

品种或自交系
Cultivar or inbred lines
籽粒硒含量
Selenium content in grains (mg·kg-1)
SNP基因分型
SNP genotyping
蜀麦969 Shumai 969 0.020 高硒High Se
蜀麦580 Shumai 580 0.011 低硒Low Se
蜀麦980 Shumai 980 0.009 低硒Low Se
蜀麦830 Shumai 830 0.017 高硒High Se
蜀麦114 Shumai 114 0.013 高硒 High Se
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