论文部分内容阅读
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., G01, G02, G03 and G04, 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 G11, G12, G13 and G14 and four populations of the second reproduced generation G21, G22, G23 and G24. 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 G01, and population G04 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 G02, G11 and G21 showed number of alleles per locus (A), genetic diversity index (H) and Shannon index without significantly differences from the control population G01; populations G12 and G22 had the number of alleles per locus (A) significantly decreased; and the above genetic diversity parameters of populations G03, G04, G13, G14, G23 and G24 decreased significantly or very significantly. The results of χ2 test showed that there were almost no differences in the allelic frequency distribution between populations G02, G11 and G21 and the control populaiton G01; and populations G03, G04, G12, G13, G14, G22, G23 and G24 differed in allele frequency distribution, and the lower the vitality level, the greater the differences. Compared with the control population G01, populations G02, G11 and G21 had no significant changes in number of rare alleles, while populations G03, G04, G12, G22, G13, G14, G23 and G24 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%.
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 (G0) are shown in Fig. 1.
Fig. 1SSR electropherograms obtained with primer Satt307 in populations G01, G02, G03 and G04
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 G02, G03 and G04 among the original materials (G0), and their reproduced populations G12, G13, G14 and G22, G23, G24, the genetic diversity parameters were lower than the control population G01, 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 G11 and G21 of the control population, various genetic diversity parameters also most had no differences from the control group G01, 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 G02 and the control population, while its first reproduced generation G12 and second reproduced generation G12 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 G03 and G04 were very significantly different from the control population G01. The three genetic diversity parameters A, H and I of the progeny populations of populations G03 and G04, i.e., populations G13, G23 and G14, G24, were significantly different, or very significantly different from the control population, but these parameters were slightly higher than populations G03 and G04, 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 G02, G03 and G04 of the original materials (G0) with significant or extremely significantly differences in allele frequency from the control population G01, decreased with the decrease of vitality. Specifically, population G04 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 G03 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 G11 and G21 reproduced from population G01 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 G12 and G22 reproduced from population G02 were significantly higher than population G02 in number of loci with a significant or very significant difference from the control population. Specifically, population G12 had three loci with a significant and one locus with an extremely significant difference from the control population, respectively, and population G22 had three loci with a significant and two loci with an extremely significant difference from the control population, respectively. The first reproduced generations G13 and G14 and the second reproduced generations of populations G03 and G04 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 G11 had the highest genetic identity with the control population G01, which was 0.999 9, followed by population G21, which shared 0.999 8 genetic identity with control population G01; and populations G04 and G03 had the lowest genetic identity with the control population G01, which was 0.996 2 and 0.997 0, respectively. It could be seen from Fig. 2 that although population G04 had the lowest genetic identity with the control population G01, its absolute value was still high, and it was also clustered at 0.996 5 with the control population G01, 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 G11 had the highest genetic identity with the control population G01, and the genetic distance was the closest, followed by population G21. This is because population G11 was reproduced from the control group G01, and population G21 was again reproduced from population G11, 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 G02 with a germination rate decreased to 95.0% had higher genetic identity with population G01, while populations G03 and G04 with a germination rate decreased to 81.0% and 79.0%, respectively, had lower genetic identity with the control population G01, 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 G12 and population G22, population G13 and population G23, and population G14 and population G24, because the latter was reproduced from the former. The genetic identify of these 6 populations with the control population G01 was higher than that of population G03 and population G04 with the control population G01, 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 (G0), populations G03 and G04 with a germination rate of 81.0% and 79.0%, respectively, had significantly fewer rare alleles than the control population G01, the number of rare alleles shared with the control population G01 decreased with the decrease of vitality level, and the number of lost/increased alleles also had the same trend. However, population G02 with a germination rate of 95.0% had no big differences in above three indicators from the control population G01, 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 G01, its first and second reproduced populations G11 and G21 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 G12, G13, G14 which were regenerated from populations G02, G03 and G04, respectively, and populations G22, G23 and G24 which were regenerated from populations G12, G13, and G14, respectively, showed number of rare alleles significantly lower than the control population G01, and the number of rare alleles shared with the control population G01 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 lowvitality 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 selfpollinated crop with a natural selfcrossing 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% (G03 and G04 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 worlds 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 worlds 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 minicore 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)
(Continued on page 72)
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 (G0) are shown in Fig. 1.
Fig. 1SSR electropherograms obtained with primer Satt307 in populations G01, G02, G03 and G04
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 G02, G03 and G04 among the original materials (G0), and their reproduced populations G12, G13, G14 and G22, G23, G24, the genetic diversity parameters were lower than the control population G01, 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 G11 and G21 of the control population, various genetic diversity parameters also most had no differences from the control group G01, 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 G02 and the control population, while its first reproduced generation G12 and second reproduced generation G12 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 G03 and G04 were very significantly different from the control population G01. The three genetic diversity parameters A, H and I of the progeny populations of populations G03 and G04, i.e., populations G13, G23 and G14, G24, were significantly different, or very significantly different from the control population, but these parameters were slightly higher than populations G03 and G04, 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 G02, G03 and G04 of the original materials (G0) with significant or extremely significantly differences in allele frequency from the control population G01, decreased with the decrease of vitality. Specifically, population G04 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 G03 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 G11 and G21 reproduced from population G01 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 G12 and G22 reproduced from population G02 were significantly higher than population G02 in number of loci with a significant or very significant difference from the control population. Specifically, population G12 had three loci with a significant and one locus with an extremely significant difference from the control population, respectively, and population G22 had three loci with a significant and two loci with an extremely significant difference from the control population, respectively. The first reproduced generations G13 and G14 and the second reproduced generations of populations G03 and G04 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 G11 had the highest genetic identity with the control population G01, which was 0.999 9, followed by population G21, which shared 0.999 8 genetic identity with control population G01; and populations G04 and G03 had the lowest genetic identity with the control population G01, which was 0.996 2 and 0.997 0, respectively. It could be seen from Fig. 2 that although population G04 had the lowest genetic identity with the control population G01, its absolute value was still high, and it was also clustered at 0.996 5 with the control population G01, 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 G11 had the highest genetic identity with the control population G01, and the genetic distance was the closest, followed by population G21. This is because population G11 was reproduced from the control group G01, and population G21 was again reproduced from population G11, 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 G02 with a germination rate decreased to 95.0% had higher genetic identity with population G01, while populations G03 and G04 with a germination rate decreased to 81.0% and 79.0%, respectively, had lower genetic identity with the control population G01, 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 G12 and population G22, population G13 and population G23, and population G14 and population G24, because the latter was reproduced from the former. The genetic identify of these 6 populations with the control population G01 was higher than that of population G03 and population G04 with the control population G01, 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 (G0), populations G03 and G04 with a germination rate of 81.0% and 79.0%, respectively, had significantly fewer rare alleles than the control population G01, the number of rare alleles shared with the control population G01 decreased with the decrease of vitality level, and the number of lost/increased alleles also had the same trend. However, population G02 with a germination rate of 95.0% had no big differences in above three indicators from the control population G01, 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 G01, its first and second reproduced populations G11 and G21 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 G12, G13, G14 which were regenerated from populations G02, G03 and G04, respectively, and populations G22, G23 and G24 which were regenerated from populations G12, G13, and G14, respectively, showed number of rare alleles significantly lower than the control population G01, and the number of rare alleles shared with the control population G01 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 lowvitality 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 selfpollinated crop with a natural selfcrossing 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% (G03 and G04 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 worlds 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 worlds 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 minicore 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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