Crops ›› 2023, Vol. 39 ›› Issue (1): 30-37.doi: 10.16035/j.issn.1001-7283.2023.01.005

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Genetic Diversity Analysis of Sophora flavescens Ait. Germplasm Resources Based on cpSSR Markers

Song Yun1,2(), Zhang Xinrui1, He Jiaxin1, Li Zheng1, Sun Zhe1, Li Aoxuan1, Qiao Yonggang1,2()   

  1. 1College of Life Sciences, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China
    2Shanxi Key Laboratory for Modernization of TCVM, Jinzhong 030801, Shanxi, China
  • Received:2021-08-23 Revised:2021-10-22 Online:2023-02-15 Published:2023-02-22

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Abstract:

A total of 150 Sophora flavescens plants from 15 different habitats were used as materials, and cpSSR molecular marker technology was used to explore the genetic diversity of S.flavescens germplasm resources from different habitats. The results showed that, a total of 311 bands were amplified by 18 pairs of cpSSR primers, and 97.47 alleles were detected, an average of 17.28 bands were amplified per cpSSR primer pair. The average number of alleles (Na) detected was 5.415, effective alleles (Ne) was 4.395, Shannon's information index (I) was 1.535, the diversity index (h) was 0.748, the unbiased diversity index (uh) was 0.832, and the polymorphism information content (PIC) was 0.886, PIC > 0.5 indicated that 18 pairs of cpSSR primers had good polymorphism. The genetic diversity of S.flavescens from different habitats was abundant, with the I was 1.600, the Ne was 4.786, and other genetic diversity indexes of THP germplasms in Wuxiang County of Shanxi Province were all higher, which indicated that THP was the place with rich genetic diversity. Analysis of Molecular Variance (AMOVA) showed that there were great differences among individuals within germplasms, and the genetic variation rate within germplasms was greater than that of inter germplasms. The results of cluster analysis, principal coordinate analysis (PCoA) and genetic structure analysis by STRUCTURE software divided S.flavescens from different habitats into two groups, and the classification results had obvious geographical correlation. The first group of nine S.flavescens germplasms were mainly from Shanxi, Hebei, Shaanxi and Inner Mongolia, and the second group were mainly composed of germplasms from Shandong, Henan, Jiangsu and Anhui. The 18 pairs of cpSSR primers had good applicability for S.flavescens. The genetic diversity of S.flavescens germplasm resources provided a certain theoretical basis for the exploration, utilization and protection and the breeding of excellent varieties of S.flavescens.

Key words: Sophora flavescens Ait., Chloroplast molecular marker, Genetic diversity, Germplasm resources

Table 1

Source information of S.flavescens"

种质Germplasm 来源Source 经度Longitude (E) 纬度Latitude (N) 海拔Altitude (m) 样本量Sample
HD 河北省邯郸市 114°03′~114°40′ 36°20′~36°44′ 1899 10
THP 山西省武乡县土河坪村 112°26′~113°22′ 36°39′~37°08′ 1069 10
XHD 山西省武乡县西河底镇 112°26′~113°22′ 36°39′~37°08′ 1069 10
AG 河北省安国市 115°33′ 38°42′ 32 10
WGS 山西省长治市五谷山村 113°01′~113°40′ 35°50′~37°08′ 976 10
CF 内蒙古赤峰市 116°21′~120°58′ 41°17′~45°24′ 1000 10
TG 山西省太谷区 112°28′~113°01′ 37°03′~37°12′ 808 10
BJ 陕西省宝鸡市 106°18′~108°03′ 33°35′~35°06′ 618 10
YL 陕西省榆林市 109°77′ 38°03′ 1000 10
PY 山东省平邑县 117°37′ 35°30′ 146 10
LY 山东省临沂市 117°24′~119°11′ 34°22′~36°13′ 88 10
LNJZ 辽宁省锦州市 121°51′ 41°31′ 23 10
BZ 安徽省亳州市 115°53′~116°49′ 32°51′~35°05′ 35 10
SY 江苏省沭阳县 118°30′~119°10′ 33°53′~34°25′ 5 10
HNJZ 河南省焦作市 113°26′~113°40′ 35°10′~35°21′ 91 10

Table 2

Information of cpSSR primer"

引物名称
Primer
name		 正向序列
Forward sequence (5′-3′)		 反向序列
Reverse sequence (5′-3′)		 产物长度
Product
length (bp)		 退火温度
Annealing
temperature (℃)
KS1 AAACCGACACGGATTACTCG CCCAATGTACCCTGATACGG 271 54.8
KS2 CCCCCAATCCTTCTTTGATT ATTGGCTGTTCGTCAATTCC 227 52.1
KS3 ATGAGGTTGTGAATCCGAGG CGATTCGATAAACGGCTCAT 246 54.5
KS4 ACAGGATTTGAACCCGTGAC CCTTCCTTATAATTTCATATCCTTCC 223 55.2
KS5 ACCTTCCCGGAGACTGAACT AAAGCTTTTGTTTCGGCTCA 276 58.2
KS6 TCCAATAACCATCCTTCCCTT GAGTTTTCACACCGGAAAGC 213 52.9
KS7 ACTCCTTTGATGGGTGTTGC ACAAAGAAATTCCACGGTCG 228 55.5
KS8 GGAGCACGGAATATCGAAAA CCAGAACCACGATGATTGAA 194 52.3
KS9 AACAGGCTCCGTAAGATCCA CATCTCAGACCTTGCGATTG 268 55.7
KS10 GCCTACGGATCAATCGACAT TGACCACGAAAGTCAAAATGA 229 54.5
KS11 TTTTCACCTCATACGGCTCC TGGTTATATATGGATTGCTAAAATTG 225 54.7
KS12 TTGATGCCTTGATCGAATGA TGAGCCAGGATCGAACTCTC 229 51.1
KS13 GAATCGACAGATCCACCGAC GAATCGACAGATCCACCGAC 250 55.6
KS14 TGAACTAATTGATTTGATTATTTTCCA TTTGGAACTGCCATCCAACT 206 47.9
KS15 TCAAGGGGTTCTCACAAACA ATTCTCGGTTGACAGGGTTG 145 53.6
KS16 AGAAGATTAAGGAACCCCCG GCATGAAACAACTCGAAGCA 245 54.0
KS17 CCGTGGGACTTAAGGAATGA CTCAAAGCAAAGCCAAGGAG 246 54.2
KS18 GGGGGAAACGGATACTCAAT TTCTTCTGCAGTACCTCGCC 270 54.3

Table 3

Size information of cpSSR amplified band"

引物名称
Primer name		 扩增条带数
Number of amplified bands		 扩增条带大小
Amplified band size (bp)
KS1
 22
 244、247、248、249、250、251、253、262、263、264、265、266、267、268、269、270、271、272、273、274、276、278
KS2 11 224、225、226、227、228、229、230、231、236、237、238
KS3 17 240、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257
KS4
 25
 205、206、207、208、209、211、212、213、214、215、216、217、218、219、220、221、222、223、224、231、233、234、235、236、237
KS5
 26
 250、256、257、258、259、260、261、262、263、264、265、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281
KS6 13 207、208、209、210、211、212、213、214、215、216、217、218、219
KS7
 18
 218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、236
KS8
 24
 197、198、199、200、287、294、295、296、297、298、299、300、301、302、303、305、306、307、308、309、310、311、312、313
KS9 17 256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272
KS10
 25
 202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226
KS11 15 190、191、192、193、194、195、196、197、198、199、220、221、222、223、224
KS12 12 227、228、229、230、231、232、233、234、235、237、238、239
KS13 13 251、252、253、254、255、256、257、258、259、260、261、262、264
KS14
 18
 197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、215
KS15
 22
 128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、169、170、172
KS16 13 238、239、240、241、242、243、244、245、246、247、248、249、250
KS17 9 242、243、244、245、246、247、248、249、250
KS18 11 268、269、270、271、272、273、274、275、276、277、278
总数Total 311
平均Mean 17.28

Table 4

Genetic diversity of cpSSR loci in S.flavescens"

引物名称Primer name Na Ne I h uh PIC
KS1 6.400 5.308 1.739 0.801 0.890 0.915
KS2 4.200 3.405 1.281 0.685 0.761 0.803
KS3 5.867 4.639 1.625 0.773 0.860 0.890
KS4 6.200 5.155 1.690 0.787 0.874 0.917
KS5 6.000 5.093 1.625 0.749 0.833 0.931
KS6 6.267 5.290 1.738 0.807 0.898 0.897
KS7 6.267 5.112 1.712 0.793 0.881 0.888
KS8 5.133 4.040 1.483 0.737 0.819 0.921
KS9 5.067 4.041 1.462 0.731 0.812 0.907
KS10 6.267 5.320 1.730 0.804 0.893 0.940
KS11 4.133 3.203 1.259 0.675 0.750 0.837
KS12 4.667 3.546 1.356 0.698 0.777 0.825
KS13 5.467 4.432 1.559 0.761 0.846 0.858
KS14 5.467 4.281 1.538 0.748 0.831 0.898
KS15 3.667 2.887 1.120 0.620 0.691 0.928
KS16 5.333 4.248 1.532 0.753 0.840 0.862
KS17 5.267 4.181 1.526 0.755 0.840 0.860
KS18 5.800 4.936 1.663 0.793 0.881 0.875
平均Mean 5.415 4.395 1.535 0.748 0.832 0.886

Table 5

Genetic diversity of 15 different germplasms of S.flavescens"

种质Germplasm Na Ne I h uh
HD 5.500 4.542 1.567 0.761 0.874
THP 5.667 4.786 1.600 0.769 0.855
XHD 5.167 4.096 1.470 0.726 0.806
AG 5.000 3.872 1.426 0.714 0.794
WGS 5.167 4.076 1.483 0.737 0.819
CF 5.444 4.387 1.556 0.759 0.844
TG 5.500 4.540 1.566 0.761 0.847
BJ 5.833 4.629 1.591 0.753 0.839
YL 5.556 4.573 1.565 0.754 0.838
PY 5.333 4.385 1.542 0.759 0.843
LY 5.278 4.329 1.502 0.739 0.823
LNJZ 5.556 4.580 1.584 0.768 0.853
BZ 5.278 4.421 1.475 0.713 0.793
SY 5.500 4.405 1.552 0.753 0.837
HNJZ 5.444 4.310 1.555 0.760 0.845
平均Mean 5.415 4.395 1.535 0.748 0.832

Table 6

AMOVA of S.flavescens germplasms"

项目Item 自由度df 平方和SS 均方MS 估计方差Estimated variance 遗传变异率Genetic variation rate (%)
种质间Inter germplasms 14 785.573 56.112 2.611 8
种质内Within germplasms 135 4049.900 29.999 29.999 92
总计Total 149 4835.473 － 32.610 100

Fig.1

Genetic structure analysis of 15 S.flavescens germplasms (a): Estimation number of populations for K ranging (2-10) by ΔK values, (b): K=2, genetic structure of S.flavescens germplasms"

Fig.2

PCoA of 15 S.flavescens germplasms"

Fig.3

UPGMA clustering results of 15 S.flavescens germplasms based on Nei's genetic distance"

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