Effects of Seed Vitality and Regeneration on Genetic Integrity in Soybean by SSR Markers

Dong WANG Xiaodong ZHANG Runfang LI Lingyun LU Xiaomu WANG Xiaohong GU Xia XIN Guangkun YIN Xinxiong LU Hanfeng DING

AbstractThe seeds of Zhonghuang 18 were selected as a test material, and subjected to artificial aging treatment (0, 112, 154 and 196 d), obtaining four 4 populations, i.e., G01, G02, G03 and G04, the germination rates of which were 98.0%, 95.0%, 81.0% and 79.0%, respectively. The four populations were reproduced twice in field, giving four populations of the first reproduced generation G11, G12, G13 and G14 and four populations of the second reproduced generation G21, G22, G23 and G24. The results showed that the number of alleles (Ae) per locus and genetic identity of all the treatment populations did not change significantly compared with the control population G01, and population G04 still shared 0.996 2 genetic identity with the control population, indicating that the genetic identity between the population with a germination rate of 79.0% and the control population was still high. The results of t test showed that populations G02, G11 and G21 showed number of alleles per locus (A), genetic diversity index (H) and Shannon index without significantly differences from the control population G01; populations G12 and G22 had the number of alleles per locus (A) significantly decreased; and the above genetic diversity parameters of populations G03, G04, G13, G14, G23 and G24 decreased significantly or very significantly. The results of χ2 test showed that there were almost no differences in the allelic frequency distribution between populations G02, G11 and G21 and the control populaiton G01; and populations G03, G04, G12, G13, G14, G22, G23 and G24 differed in allele frequency distribution, and the lower the vitality level, the greater the differences. Compared with the control population G01, populations G02, G11 and G21 had no significant changes in number of rare alleles, while populations G03, G04, G12, G22, G13, G14, G23 and G24 decreased significantly in number of rare alleles. The above results showed that compared with the control population, the progeny populations reproduced from the population with a germination rate of 98.0% had significant changes in genetic diversity and number of rare alleles, while the values of the progeny populations reproduced from populations having germination rates of 81.0% and 79.0%, respectively, decreased significantly, and the number of alleles per locus and number of rare alleles of the progeny populations reproduced from the population with a germination rate of 95.0% began to decrease. The decline in viability has a greater effect on the genetic structure of soybean germplasm populations than reproduction generation. It is recommended that the germination rate standard for regeneration of soybean germplasm with an initial germination rate of 98.0% should not be lower than 81.0%.

Key wordsSoybean; Vitality; Regeneration; SSR; Genetic integrity; Germplasm preservation

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Received: August 26, 2018Accepted: November 23, 2018

Supported by Key Project of the National TwelfthFive Year Research Program of China (2013BAD01B0106); Research Fund for Young Scholars in Shandong Academy of Agricultural Sciences (2016YQN19); China Agriculture Research SystemGreen Manure (CARS22); National Crop Germplasm Resources Platform of China (2012/2013032); Major Science and Technology Innovation Project of Shandong Province (2017CXGC0311); "Coarse Cereal Innovation Team" of The Modern Agricultural Industry Technology System of Shandong Province (SDAIT1501); Agricultural Science and Technology Innovation Engineering Team of Shandong Academy of Agricultural Sciences (CXGC2018E15).

Dong WANG (1982-), male, P. R. China, research assistant, PhD, devoted to research about crop germplasm resource preservation.

*Corresponding author. Email: dinghf@163.com.

Crop germplasm resources are the material basis for the development of crop breeding, biotechnology and crop science, and are valuable assets for human survival and development. All countries in the world attach great importance to the collection and preservation of germplasm resources. At present, more than 1 700 lowtemperature germplasm banks have been built around the world, and the number of preserved germplasms has exceeded 7.4 million［1］. The number of germplasm stored in China for a long term has reached 397 000［2］, and the number of longterm preserved germplasms ranked the second in the world, second only to the United States (508 000)［3］.

However, no matter how the preservation technology is improved, the vitality of the seeds in germplasm banks will inevitably decrease with the prolongation of storage time, and the number of stored samples will be reduced due to the monitoring of vitality and the supply of seeds for external units. To this end, when the germination rate of the preserved germplasms is reduced or the number of stored germplasms is reduced to a certain threshold, reproduction and regeneration is required, and the regenerated seeds form samples for the next round of germplasm preservation［4］. Many studies have reported that in the germplasm reproduction process, its genetic integrity is susceptible to changes in seed aging, reproduction generation, reproduction population size, reproduction site, reproduction system, pollination and harvesting methods［5-10］.

Early methods for detecting genetic integrity of germplasms include morphological markers, cell markers and isozyme markers, all of which have varying degrees of defects. With the rise of DNA molecular marker technology, RFLP, RAPD, SSR and AFLP have been applied to detection of genetic integrity. SSR marker technology has the advantages of abundant number of markers, uniform coverage of the whole genome, good experimental repeatability, high reliability and strong site specificity. And SSR marker is also a kind of codominant marker that can identify heterozygotes and homozygotes and is of great significance to individual identification. Russell et al.［11］, Powell et al.［12］ and Taramino et al.［13］ proved that SSR has higher polymorphism than other molecular markers. Therefore, SSR marker technology is an ideal method to study the variation of genetic integrity.

China is the origin of soybeans. There are more than 25 000 soybean germplasm resources in Chinas national longterm crop germplasm bank, including local varieties, bred varieties and imported varieties, and more than 8 500 wild soybean germplasm resources have been preserved［14］. These resources are valuable assets for soybean genetic research and crop breeding in China, and it is of great significance to preserve these resources. Previous studies on factors affecting the genetic integrity of soybean germplasms have mostly focused on the regeneration years［15］, but less on the decline of vitality and reproduction generation. In this study, the soybean variety Zhonghuang 18 was used as a material, and the SSR molecular marker technology was used to analyze and evaluate the impact of the decline of vitality and reproduction generation on the genetic integrity of soybean germplasm resources, aiming at providing an important theoretical and practical basis for the formulation of scientific strategy for the preservation and regeneration of soybean germplasms.

Materials and Methods

Experimental materials

The seeds of soybean variety Zhonghuang 18 were provided by Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. In 2001, the water content of the seeds was determined to be 8%-9%, after harvest in field. In February, 2002, the seeds were sealed in aluminum foil bags and subjected to artificial aging treatment (0, 112, 154 and 196 d) at (40 ± 2) °C, obtaining seed populations as the original materials (G0) at four different vitality levels (G01, G02, G03 and G04), and seeds free of artificial aging treatment (G01) served as the control (CK). In 2003, the above four seed populations were planted separately in field, obtaining four seed populations (G1) of the first reproduced generation (G11, G12, G13 and G14). In 2006, the seed populations of the first reproduced generation were seeded in field, obtaining four seed populations (G2) of the second reproduced generation (G21, G22, G23 and G24) (Table 1). The reproduction was carried out at the Dishang Experimental Station of Institute of Food and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, in Shijiazhuang City. The field reproduction adopted randomized block design, with population as the treatment unit, and each treatment had three replicates, each of which was sown with 200 seeds. The reproduction and regeneration were according to Technical Regulation on Characterization and Documentation for Crop Germplasm Resources［16］, and seeds were harvested according to the mixed method. All harvested seeds were airdried and stored at 4 ℃ for testing.

Determination of indoor germination rate

The germination test was carried out according to the germination conditions in the International Rules for Seed Testing［17］. The germination bed was made up of two layers of filter paper at a temperature of 25 ℃ with two replicates. The number of germinated seeds was checked day by day, the germination potential was counted on the third day, and the germination rate was counted on the fourth day.

DNA extraction and detection

From the population of each treatment, 200 seeds were randomly taken, planted in vermiculite and cultured in an incubator under a 14 L∶10 D photoperiod. The temperature was 25 ℃ in the light and 30 ℃ in the dark, and the relative humidity was 80%. When the seedlings grew to the threeleaf stage, 60 plants were randomly taken from different populations, respectively, and DNA was extracted from each plant by SDS method. SDS was diluted with 1×TE to the same concentration (20 ng/μl) and preserved at 4 ℃ for later use. The concentration of extracted DNA was determined, and its quality was detected with 0.8%Agarose gel.

SSR analysis

Related information of 60 pairs of SSR core primers selected by Xie et al.［18］ were searched on http://soybase.org/resources/ssr.php (Table 2). These 60 SSR loci were all located on the soybean genetic map, covering 20 linkage groups of soybean, with two to four loci distributed on each linkage group, averagely three.

The PCR system had a total volume of 20 μl, including 10×PCR Buffer (containing Mg2+) 2.0 μl, 2.5 mmol/L dNTP 1.8 μl, 2.5 U μL Taq polymerase 0.4 μl, 5.0 μmol/L Primerpairs (1+1) μl, 20 ng/μl DNA 5.0 μl and ddH2O 9.0 μl. The PCR was started at 94 ℃ for 4 min, followed by 35 cycles of 94 ℃ for 30 s, 47 ℃ for 30 s and 72 ℃ for 30 s, and extended at 72 ℃ for 10 min, and the product was taken out when the temperature decreased to 15 ℃, and stored at 4 ℃ for later use. The amplification product was then subjected to 6% denatured polyacrylamide gel electrophoresis (70 W, 45 min), followed by silver staining［19］. The Taq polymerase, dNTP and PCR buffer used in the PCR were all purchased from Tiangen Biotech (Beijing) Co., Ltd.

Data statistics and analysis

At the position of the same mobility, it was recorded as 1 with the appearance of a band, 0 for no band, and 9 in the case of a deletion, and the size of the band was estimated according to DNA marker I and pBR322 DNA/Msp I DNA Ladder marker. For each population, the number of alleles (NA), number of polymorphic loci (K), percentage of polymorphic loci (P), number of alleles per locus (A), number of effective alleles per locus (Ae), and genetic diversity parameters including genetic diversity index (H) and Shannon index (I) were computed using POPGENE Ver. 1.31［20］. The allele frequency of each locus obtained with SSR markers was calculated using Powermarker Ver. 3.25 for the 12 soybean germplasm populations. The genetic diversity parameters corresponding to all loci of the treatment and control populations were subjected to t test using SAS V9.0, and the allele frequencies of all loci of the treatment and control populations were subjected to χ2 test. Unweighted pair group mathematics average (UPGMA) clustering analysis was performed according to the Neis genetic consistency between populations using NTSYSpc 2.1 software［21］, and comparison of rare allele number and other statistical analysis were performed in Microsoft Excel.

Results and Analysis

Analysis of population genetic structure

Sixty pairs of SSR core primers were used to detect the molecular markers of 12 germplasm populations of soybean "Zhonghuang 18". A total of 138 alleles at 60 loci were detected, and the number of alleles per locus was 1-4, averagely 2.3. The SSR electropherograms obtained by amplifying the four populations of the original materials (G0) are shown in Fig. 1.



Fig. 1SSR electropherograms obtained with primer Satt307 in populations G01, G02, G03 and G04

It could be seen from Table 3 that the number of effective alleles per locus in each population was not significantly different from that of the control population. For populations G02, G03 and G04 among the original materials (G0), and their reproduced populations G12, G13, G14 and G22, G23, G24, the genetic diversity parameters were lower than the control population G01, and with the prolongation of the aging time, vitality level and the genetic diversity parameters both became lower. The results of the t test showed that for the reproduced generations G11 and G21 of the control population, various genetic diversity parameters also most had no differences from the control group G01, indicating that the genetic structure of the population with a regeneration germination rate of 98.0% was better maintained after two times of reproduction. There were no significant differences in the genetic diversity parameters A, H and I between group G02 and the control population, while its first reproduced generation G12 and second reproduced generation G12 showed significant differences from the control population in A, indicating that the progeny populations of the population with a regeneration germination rate of 95.0% had a significant change in the number of alleles per locus due to the decline in the regeneration germination rate of the parental generation. The three genetic diversity parameters A, H and I of populations G03 and G04 were very significantly different from the control population G01. The three genetic diversity parameters A, H and I of the progeny populations of populations G03 and G04, i.e., populations G13, G23 and G14, G24, were significantly different, or very significantly different from the control population, but these parameters were slightly higher than populations G03 and G04, respectively, indicating that for the populations with regeneration germination rates of 81.0% and 79.0%, respectively, due to the decline in vitality level, their own genetic diversity and the genetic diversity of their progeny populations were lower than that of the control population. The above results indicate that the decline in vitality has a higher effect on the population genetic structure of soybean germplasms than reproduction generation.

Analysis of differences in allele frequency

As shown in Table 4, the number of loci in populations G02, G03 and G04 of the original materials (G0) with significant or extremely significantly differences in allele frequency from the control population G01, decreased with the decrease of vitality. Specifically, population G04 with a germination rate of 79.0% had the most loci with a significant or an extremely significant difference from the control population, 10 with a significant and four with an extremely significant difference, respectively, and population G03 with a germination rate of 81.0% was next to it, having eight loci with a significant and one locus with an extremely significant difference from the control population, respectively, indicating that the decline in vitality significantly affected the allele frequency distribution in soybean germplasm material populations. There were no loci in generations G11 and G21 reproduced from population G01 with a significant difference from the control population, indicating that soybean germplasm materials with a germination rate of 98.0% showed hardly any difference from the control population in allele frequency of each locus after two times of reproduction. Populations G12 and G22 reproduced from population G02 were significantly higher than population G02 in number of loci with a significant or very significant difference from the control population. Specifically, population G12 had three loci with a significant and one locus with an extremely significant difference from the control population, respectively, and population G22 had three loci with a significant and two loci with an extremely significant difference from the control population, respectively. The first reproduced generations G13 and G14 and the second reproduced generations of populations G03 and G04 had more loci with a significant or an extremely significant difference from the control population, and the second reproduced generation had more loci with a significant or an extremely significant difference from the control population than corresponding first reproduced generation, indicating that soybean germplasm materials with germination rates of 95.0%, 81.0%, and 79.0% and their reproduced progeny populations were different from the control population in allele frequency on each loci, and the lower the vitality level, the greater the difference. The above results indicate that the decline in vitality has a higher effect on allele frequency distribution of soybean germplasm materials than reproduction generation.

Analysis of genetic identity

It could be seen from Table 5 that population G11 had the highest genetic identity with the control population G01, which was 0.999 9, followed by population G21, which shared 0.999 8 genetic identity with control population G01; and populations G04 and G03 had the lowest genetic identity with the control population G01, which was 0.996 2 and 0.997 0, respectively. It could be seen from Fig. 2 that although population G04 had the lowest genetic identity with the control population G01, its absolute value was still high, and it was also clustered at 0.996 5 with the control population G01, indicating that all the treatment populations had higher genetic identity with the control population, which is consistent with the analysis of difference in number of effective alleles per locus. Population G11 had the highest genetic identity with the control population G01, and the genetic distance was the closest, followed by population G21. This is because population G11 was reproduced from the control group G01, and population G21 was again reproduced from population G11, indicating that the genetic identity of the soybean germplasm material with a germination rate of 98.0% was well maintained after two times of reproduction. Population G02 with a germination rate decreased to 95.0% had higher genetic identity with population G01, while populations G03 and G04 with a germination rate decreased to 81.0% and 79.0%, respectively, had lower genetic identity with the control population G01, indicating that the decline in vitality has a greater impact on the genetic identity of soybean germplasms. There was a high genetic identity between population G12 and population G22, population G13 and population G23, and population G14 and population G24, because the latter was reproduced from the former. The genetic identify of these 6 populations with the control population G01 was higher than that of population G03 and population G04 with the control population G01, indicating that the decline in vitality has a higher effect on the genetic identity of soybean germplasm materials than reproduction generation.

Analysis on change of rare alleles (P<0.05)

A rare allele refers to an allele within a population whose gene frequency is less than 5%. The genetic diversity within a population is largely due to the binding or integration of rare alleles (P<0.05) into the genotype background. Soybean germplasm materials are prone to loss or increase of rare alleles after aging treatment and reproduction and regeneration, resulting in changes in the number of alleles in population. The changes in the number of rare alleles within various populations are shown in Table 6. It



could be seen from Table 6 that in the original materials (G0), populations G03 and G04 with a germination rate of 81.0% and 79.0%, respectively, had significantly fewer rare alleles than the control population G01, the number of rare alleles shared with the control population G01 decreased with the decrease of vitality level, and the number of lost/increased alleles also had the same trend. However, population G02 with a germination rate of 95.0% had no big differences in above three indicators from the control population G01, indicating that the decline in vitality can significantly affect the number of rare alleles in the soybean germplasm population, mainly to reduce its number. Compared with G01, its first and second reproduced populations G11 and G21 had smaller changes in the above three indicators, indicating that the soybean germplasm material with a germination rate of 98.0% had its rare alleles well maintained after two times of reproduction and regeneration. However, populations G12, G13, G14 which were regenerated from populations G02, G03 and G04, respectively, and populations G22, G23 and G24 which were regenerated from populations G12, G13, and G14, respectively, showed number of rare alleles significantly lower than the control population G01, and the number of rare alleles shared with the control population G01 showed the same trend, while the number of lost/increased rare alleles increased significantly, indicating that the progeny populations reproduced from the populations with germination rates of 95.0%, 81.0% and 79.0% had larger changes in the number of rare alleles than the control population. The above analysis shows that the decline in vitality has a higher effect on the change in number of rare alleles than reproduction generation.

Discussion

Effects of decline in vitality and reproduction generation on the genetic integrity of Zhonghuang 18

Genetic integrity refers to the complete maintenance of the genetic structure of a population, which means the genotype frequency distribution and the allele frequency distribution remain unchanged, the same as the original population［22］. Maintaining the genetic integrity of a germplasm is to minimize the hereditary change of the germplasm during storage, and maintaining the greatest genetic similarity between the progeny and the parent during the reproduction and regeneration process is the core of the germplasm preservation work. Parzies et al.［5］ applied isozyme markers to study local barley cultivars stored for different years and found that there were significant differences in the frequency of gliadin band between germplasm materials of different reproduced generations. Chebotar et al.［6］ studied the rye varieties of different reproduced generations by SSR molecular marker technology, and found that there were also significant differences in allele frequency, and the loss or increase of some alleles was detected. Roos［9］ studied a population formed from eight bean varieties with different seed coat colors and pod colors, and found that six of them were lost after 15 times of seed aging and regeneration cycles. In this study, SSR molecular marker technology was applied to detect soybean germplasm populations at different vitality levels that were reproduced twice, and it was found that the number of effective alleles per locus and the genetic identity of each treatment population had no big differences from those of the control population, which is related to the genetic structure of soybean itself. Moreover, the test material "Zhonghuang 18" is a cultivar belonging to homogeneous germplasm material, of homozygous genotype, and the individuals have basically the same genetic structure. During the reproduction and regeneration process, the probability of being contaminated by exotic pollen is also extremely small. Therefore, the genetic identity of each treatment population was better maintained than that of the control population.

Further analysis showed that the number of alleles, number of polymorphic loci, percentage of polymorphic loci, number of alleles per locus and the genetic diversity index and Shannon index all increased in the original material populations with germination rates of 95%, 81.0%, and 79.0%, respectively, compared with the control population, and the longer the aging time, the lower the vitality level, and the greater the decline of genetic diversity parameters, indicating that the genetic diversity in the lowvitality soybean populations was lower than the control population at a high vitality level. This is consistent with the results of Zhang et al.［23］. The study of rare allele changes reveals the nature of the problem. Rare alleles occupy a small share in a population, but greatly increase the genetic diversity within the population. Rare alleles can be lost easily due to factors such as seed aging, size of reproduction population, seed harvesting method, and reproduction generation. Changes in rare alleles can less change the genetic identity between populations, but greatly change the genetic diversity of populations. The study on the genetic structure and allele frequency distribution of each population, the genetic identity with the control population and changes in rare allele all indicate that the decline in vitality has a greater effect on the genetic integrity of soybean germplasms than reproduction generation. Therefore, it is important to determine a high germination rate standard when performing soybean germplasm reproduction and regeneration.

Germination rate standard for soybean germplasm reproduction and regeneration

The goal of germplasm reproduction and regeneration strategy research is to reduce the effects of genetic drift, genetic shift, heteromorphic pollen pollution and seed confounding on genetic integrity of germplasm resources. The research contents include updating germination rate standard, size of reproduction population and seed harvesting method［24-26］. Soybean is a typical selfpollinated crop with a natural selfcrossing rate of about 0.5%-1.0%, the size of reproduction population and seed harvesting method would not influence its genetic integrity, and the reproduction and regeneration strategy should focus on updating the germination rate standard. Appropriate germination rate index for reproduction and regeneration can reduce the effects of gene mutation in stored seeds and field selection on the genetic integrity of germplasm resources. According to the results of 30 ℃ aging test on soybean seeds, Li et al.［27］ recommended the germination rate of 80% as the vitality standard for soybean seed reproduction and regeneration, and 73% as the lower limit of the germination rate for regeneration. At present, the germination rate standard for reproduction and regeneration of germplasm gene banks at home and abroad is usually in the range of 65%-85%［4］. IPGRI recommends a germination rate of 85% as the standard, or a germination rate decreased to 85% of the initial germination rate［24,28］. In this study, there were no significant differences in the number of alleles, genetic diversity index, Shannon index, number of rare alleles and allele frequency distribution between the control population with a germination rate of 98% and its progeny generations, and the genetic consistency was relatively higher. However, compared with the control population, populations with a germination rate lower than 85% (G03 and G04 with germination rates of 81.0% and 79.0%, respectively) and its reproduced progeny generation showed the number of alleles, genetic diversity index, Shannon index and number of rare alleles significantly decreased and the allele frequency distribution increased, and the genetic consistency was relatively lower. This confirms the important guiding role of the germination rate of 85% recommended by IPGRI in germplasm regeneration.

Conclusions

The decline in vitality and reproduction generation did not significantly affect the number of alleles per locus and genetic identity in soybean germplasm populations, and the population with the germination rate decreased to 79.0% shared 0.996 2 genetic identity with the control population. The population with a germination rate of 98.0% had no significant changes in genetic structure, allele frequency distribution and number of rare alleles compared with its reproduced progeny populations, while the populations with a germination rate fallen below 85.0% (81.0% and 79.0%) and their progeny populations were significantly different from the control population in population genetic structure, allele frequency distribution and number of rare alleles. The decline in vitality has a greater effect on the genetic structure of the soybean germplasm population than reproduction generation. It is recommended that the germination rate standard for regeneration of soybean germplasm with an initial germination rate of 98.0% should not be lower than 81.0%.

References

［1］ FAO. The second report on the state of the worlds plants genetic resourcesnce for food and agriculture［C］. FAO, Rome, 2010.

［2］ WANG SM, LI LH, LI Y, et al. Status of plant genetic resources for food and agricultural in China (I)［J］. Journal of Plant Genetic Resources, 2011, 12 (1): 1-12. (in Chinese)

［3］ WANG SM, ZHANG ZW. The state of the worlds plant genetic resources for food and agriculture［J］. Journal of Plant Genetic Resources, 2011, 12 (3): 325-338. (in Chinese)

［4］ FRANKEL OH, BROWN AHD, BURDON JJ. The conservation of plant biodiversity［M］. Cambridge University Press, Cambridge, 1995.

［5］ PARZIES HK, SPOOR W, ENNOS RA. Genetic diversity of barley landrace accessions (Hordeum vulgare ssp. Vulgare) conserved for different lengths of time in ex situ gene banks［J］. Heredity, 2000, 84: 476-486.

［6］ CHEBOTAR S, RODER MS, KORZUN V. Molecular studies on genetic integrity of open pollinating species rye (Secale cereale L.) after longterm genebank maintenance［J］. Theor Appl Genet, 2003, 107 (8): 1469-1476.

［7］ STOYANOVA SD. Genetic shifts and variations of gliadins induced by seed aging［J］. Seed Sci & Technol, 1991, 19: 363-371.

［8］ RIO AH, BAMBERG JB, HUAMAN Z. Assessing changes in the genetic diversity of potato gene banks I. Effects of seed increase［J］. Theor Appl Genet, 1997, 95: 191-198.

［9］ ROOS EE. Report of the storage committee working population on effects of storage on genetic integrity 1980-1983［J］. Seed Sci & Technol, 1984, 12: 255-260.

［10］ TAO KL, PERRINO P, PIERLUIGI L, et al. Rapid and nondestructive method for detecting composition change in wheat germplasm accessions［J］. Crop Sci, 1992, 32: 1039-1042.

［11］ RUSSELL JR, FULLER JD, MACAULAY M, et al. Direct comparison of levels of genetic variation among barley accessions detected by RFLPs, AFLPs, SSRs and RAPDs［J］. Theor Appl Genet, 1997, 95: 714-722.

［12］ POWELL W, MORGANTE M, ANDRE C, et al. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis［J］. Mol Breed. 1996, 2: 225-238.

［13］ TARAMINO G, TINGEY S. Simple sequence repeats for germplasm analysis and mapping in maize［J］. Genome, 1996, 39: 277-287.

［14］ LIU YN, LI XH, WANG KJ. Analysis of the genetic variability for the minicore collection of Chinese wild soybean (Glycine soja) collection in the national gene bank based on SSR markers［J］. Journal of Plant Genetic Resources, 2009, 10 (2): 211-217. (in Chinese)

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