Backbone parents are important breeding materials for genetic improvement of soybean (Glycine max). Creating excellent backbone parents is conducive to obtaining groundbreaking soybean varieties. Therefore, in-depth research on the genetic characteristics and genotype characteristics of backbone parents is conducive to revealing the inheritance and mechanism of excellent genes and their allelic variations in backbone parents, and has an important role in creating and guiding soybean genetic breeding. Since the beginning of soybean cultivation in very early maturity areas in China's alpine regions, a series of soybean varieties with a large promotion area have been created through years of artificial improvement. Among them, Heihe54 is one of the parents that has made a significant contribution to the very early maturity areas in alpine regions, and the large varieties derived from it also account for a relatively large proportion. Heihe18 was approved in 1998 to have a promotion area of over 50 million mu, and Heihe43 has been promoted since 2007, with a cumulative promotion area of 120 million mu. Currently, it is still the largest variety planted in production, and these varieties are all derived from the contributions of the backbone parent Heihe54.

The development of whole-genome sequencing technology has provided the possibility for people to further reveal the genetic material basis of gene transmission in offspring of backbone parents. In recent years, many breeders have used molecular markers and association analysis to explore excellent allelic variations in chlorophyll in natural populations of wheat (Li et al., 2012). Liu et al. (2012) used this method to study the genetic contribution of soybean varieties to derived varieties; Tang et al. (2012) used STS-PCR technology to study the replacement trend of conventional rice varieties and the contributions of key parents; Xiong et al. (2008) used SSR (Simple sequence repeats) technology to study the genetic contribution of Chinese soybean cultivar germplasm. The development of molecular technology provides technical support for revealing the genetic essence of backbone parents at the genome-wide level. This study uses the specific-locus amplified fragment sequencing (SLAF-Seq) to conduct full gene molecular marker analysis of very early maturity backbone parents and their derived offspring in high and cold regions of China, clarify the transmission of genomic segments from the backbone parents in the very early maturity regions in the offspring derived varieties, and reveal the genetic characteristics of the backbone parents in the offspring derived varieties through gene information analysis at the whole genome level of soybean varieties in high and cold regions of China, and to reveal the genetic essence of key parents and provide technical support for promoting the northward migration of soybean varieties and selecting parents for breeding in very early maturity regions.

1 Results and Analysis

1.1 Genetic contribution of Heihe54 to its derived varieties

1.1.1 Cluster analysis and genetic improvement of Heihe54 derived variety

Heihe54 is the backbone parent of the 4~5 accumulated temperature zone in Heilongjiang Province. Nine provincial extension varieties have been derived from Heihe54, with a cumulative planting area of over 30 million mu. Heihe18, derived from Heihe54, is a key parent that has made outstanding contributions to the cultivation of early maturing varieties in the northern cold regions of Heilongjiang Province, with a cumulative promotion area of 46.063 million mu; Cluster analysis of Heihe54 and Heihe18 and their derivatives showed that the genetic distance between Heihe54 and Heihe18 was relatively close, with a genetic distance of 0.416 between the varieties. The genetic distance between Heihe54 and Beifeng11 is relatively far, and the genetic distance between varieties is 0.57; The genetic distance between Heihe18 and its derivatives is relatively close, with a genetic distance of 0.145~0.49; The genetic distance between Heihe18 and Heihe9 is the closest, with a genetic distance of 0.145 for the variety; The genetic distances between Keshan1, Dongsheng8, Dongsheng7, Beidou10, and Heihe43 were also relatively close, with 0.219, 0.217, 0.227, 0.245, and 0.27, respectively; The genetic distance between Heihe18 and Beifeng11 is the farthest, with a genetic distance of 0.492 between varieties. It can be seen that the suitable genotype in the northern high and cold regions is similar to Heihe18, and the offspring improved varieties have changed in stress resistance, adaptability, and yield traits (Figure 1; Table 1).

1.1.2 Distribution of genetic information on different chromosomes of Heihe54 derived varieties

The genetic information transmission rate of the same allele variation in Heihe54 among different chromosomes of the derived variety is lower than that of Heihe18, and there are differences between different varieties and different chromosomes. The same allele variation sites on different chromosomes between Heihe54 and the new variety range from 0.42 to 0.962, while Heihe18 ranges from 0.478 to 0.993. The same allele variation on chromosome 4 is higher than 78.6%; On chromosome 10, the same allele variation of Heihe54 and Heihe18 genetic information in the derived varieties varies greatly, indicating that the same allele variation of Heihe54 is less in the derived varieties, ranging from 0.492 to 0.828, while the same allele variation transmitted by Heihe18 genetic information in the derived varieties is higher, ranging from 0.766 to 0.971. This indicates that under the pressure of ecological selection, the variety demand and market demand of farming systems and production methods are affecting the changes in genetic information transmission on chromosome 10 (Table 2; Table 3).

1.1.4 Contribution of Heihe18's genetic material to Heihe43 and Keshan1

Heihe18 is a large variety with a larger promotion area and more derivative varieties after Heihe54. At present, the main varieties promoted in the northern alpine regions are all related to Heihe18. Heihe43 and Keshan1 are another variety with a large promotion area after Heihe18. The cumulative promotion area of Heihe43 has reached more than 90 million mu. Compared with Heihe18, the base substitution numbers of these two new varieties are 0.27 and 0.219, indicating a relatively close genetic distance. The proportion of identical alleles transmitted by Heihe18 genetic information between two varieties is significantly different on chromosomes 1, 3, 7, 8, 10, 11, 12, 13, 14, 15, 16, and 17. Some new alleles have emerged, and the proportion of identical alleles on chromosomes 4 and 19 compared to Heihe18 is as high as 95% or more (Figure 3). It inherits the early maturity, small pointed leaves, stem strength, plant height, resistance to sudden death, and heavy flower resistance of Heihe18. And Heihe43 and Keshan1 have made new breakthroughs in wide adaptability, high yield, and disease resistance (Figure 4).

2 Discussion

China's cold and very early maturity regions are new areas for soybean cultivation. With the enhancement of variety innovation capabilities, soybean cultivation has developed rapidly in the cold and very early maturity regions, and a unique variety ecological type has been formed. Based on the analysis of the whole genome SNP scan, the very early maturity backbone parents in high and cold regions have the same genome segment, and the genetic proportion of the backbone parents on chromosomes 4 and 9 is relatively high. From its variety characteristics, it can be seen that it preserves the characteristics of the backbone parents in early maturing, sharp leaves, strong stems, sudden death resistance, heavy stubble resistance, and wide adaptability, making its excellent characteristics suitable for the current changes in the growth environment and able to develop in the very early maturity regions. With the introduction of exogenous genes, varieties have made breakthroughs in terms of wide adaptability, high yield, and disease resistance.From the perspective of genetic improvement distance, the genetic distance of the backbone parent varieties in the cold and very early maturity regions is relatively close, and the genetic genes are narrow. In terms of genetic improvement, it is necessary to further strengthen the infiltration of exogenous specific genes, which is an important measure to achieve a higher level of new varieties.

Heihe18 is a widely cultivated variety derived from Heihe54. Compared to Heihe43, Heihe18 has more than 54.5% of the same allele variation on each chromosome, which reflects the contribution of Heihe18's genetic information at the genetic level. However, new variation sites are more common on chromosomes 1, 3, 14, 10, and 5, and Heihe43 retains more of the same allele variation sites on chromosomes 4, 6, and 19, which is more than 96%. This indicates that the artificial selection pressure in high and cold regions of China has led to the stable inheritance of traits such as early maturity, cold resistance, and adaptability to future generations, while characteristics such as cold tolerance, disease resistance, and so on that adapt to new production environments are continuously improved with the introduction of new genes.

3 Materials and Methods

3.1 Experimental materials

The tested varieties were the backbone varieties Heihe54 and their derivatives in the very early maturity spring soybean region: Heihe18, Heihe43, Dongsheng7, Beidou10, Longda1, Dongsheng8, Beifeng11, Keshan1, and Heihe9, a total of 10 varieties.

3.2 Test design

Planted in the field from 2012 to 2014, each variety was planted in three rows, with three repetitions. The agronomic traits were investigated: seedling stage, leaf type, flower color, hairy color, node number, branch number, plant height, seed coat color, hilum color, stem termination, and growth period; Yield traits: 100 grain weight, grain weight per plant, plot yield, number of pods per plant (Table 4); Inoculation identification: resistance to root rot (Han et al., 2006), gray spot (Ding et al., 2009), and viral disease (Zhang et al., 2015).

3.3 Measurement items and methods

Investigate the agronomic traits of the tested varieties: seedling stage, leaf type, flower color, hairy color, node number, branch number, plant height, seed coat color, hilum color, stem termination, and growth period; Yield traits: 100 grain weight, grain weight per plant, plot yield, number of pods per plant.

3.4 Disease identification

Standard identification methods for national and provincial variety certification were used: determination of root rot, gray spot, and viral diseases.

3.5 SLAF-Seq technology

The soybean germplasm resources under test were sequenced using SLAF-Seq technology, and the DNA of each sample was extracted. The DNA was digested and interrupted using selected enzyme digestion combinations. The enzyme digestion products were repaired at the 5'-terminal, and the 5'-terminal was phosphorylated. An A was added at the 3'-terminal to complement the 5'-terminal T of the Solexa connector. The Solexa sequencing connector was continued to be connected, and the crossover products were mistakenly located on the flowcell for bridge amplification. The agarose gel electrophoresis was used to select the fragment size, PCR amplification was used to enlarge the library, and the constructed library was sequenced with 111uminaHisegTM2500 (Zhang et al., 2020). Each germplasm resource obtained an average of 2 230 890 raw Reads. Compared with the reference genome soap, it obtained 312 398 SLAFs with a sequencing depth of 4.12. SNPs were detected based on SLAFs, and a total of 432 222 SNP loci were obtained, with a SLAF polymorphism of 56.5%. Genetic and principal component analysis was performed based on the SNPs of the variety (Saiton and Nei, 1987; Tamara et al., 2011). Genetic distance was calculated using Mega5.0 software, Neighbor Joining (N5), Maximum Composite Cikelihood model (Price et al., 2006; Tamura et al., 2011), and the same allelic variation was calculated using perl programming language.

Authors’ contributions

WL and LLJ were the experimental designers and executors of this study; WWW and BWW participated in data compilation and writing the first draft of the paper; ZGX and YHR participated in some experiments; LLJ is the project leader, guiding experimental design, data statistics, thesis writing, and revision. All authors read and approved the final manuscript.

Acknowledgements

This study was jointly supported by the National Soybean Industry Technical System Support (CARS-O4-PSO5), New Soybean Varieties with Resistance to Reversion Genes Cultivation Project (2016ZX08004002), the Agricultural Science and Technology Innovation Leaping Project (HNK2019CX01), the Harbin Science and Technology Bureau Project (2017RAQYJ034), the Heilongjiang Postdoctoral Fund (LBH-Z16184), and the Heilongjiang Academy of Agricultural Sciences Academic-level Project (2017BZ14).

Han Y.P., Li W.B., Terry R., Anderson V.P., Wen J.Z., and Gao J.G., 2006, Study on QTL Location of phytophthora root rot, Dadou Kexue (Soybean Science), 25(1): 23-27.

Jing J.J., Wen J.Z., Hua G.H., Jiang C.L., Chen Q.S., Liu C.Y., Gu X., and Yang X.H., 2009, Monitoring of physiologicalrace of soybean frogeyespot and analysis of variety resistance in Heilongjiang province, Dadou Kexue (Soybean Science), 28(1): 178-180.

Li W.Y., Zhang B., Zhang J.N, Chang X.P., Li R.Z., and Jing R.L., 2012, Exploring elite Alleles for Chlorophyll content of flag leaf in natural population of wheat by association analysis, Zuowu Xuebao (Acta Agronomica Simica), 38(6): 962-970.

https://doi.org/10.3724/SP.J.1006.2012.00962

Liu X.L., Si Q.L., Li Q.Q., Wang C.Y., Wang Y.J., Zhang H., and Ji W.Q., 2012, SSR analysis of genetic diversity and temporal trends of the Core Wheat (TriticumaestivumL.) Parent Funo and its Derivative Varieties (Lines), Nongye Shengwu Jishu Xuebao (Journal of Agricultural Biotechnology), 20(9): 983-995.

Price A.L., Patterson N.J., Plenge R.M., Weinblatt M.E., Shadick N.A., and Reich D., 2006, Principal components analysis connects for stratification in genome-wide association studies, Nat. Genet., 38(8): 904-909.

https://doi.org/10.1038/ng1847

Saiton N., and Nei M., 1987, The neighbor-joining method: A new method for reconstructing phylogenetic trees, Molecular Biology and Evolution, 4(4): 406-425.

Tamara K., Peterson D., Peterson N., Stecher G., Nei M., and Kumar S., 2011, Mega5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Molecular Biology and Evolution, 28(10): 2731-2739.

https://doi.org/10.1093/molbev/msr121

Tang S.X., Wang X.D., and Liu X., 2012, Study on the renewed tendency and key Backbone-Parents of inbred rice varieties (O. sativa L.) in China, Zhongguo Nongye Kexue (Scientia Agricultura Sinica), 45(8): 1455-1464.

Xiong D.J., Zhao T.J., and Gai J.Y., 2008, Geographical sources of germplasm and their nuclear and cytoplasmic contribution to soybean cultivars released during 1923 to 2005 in China, Zuowu Xuebao (Acta Agronomica Simica), 34(2): 175-183.

https://doi.org/10.3724/SP.J.1006.2008.00175

Zhang S., Li B., Chen Y., Shaibu A.S., Zheng H., and Sun J., 2020, Molecular-Assisted distinctness and uniformity testing using SLAF-Sequencing approach in soybean, Genes, 11(2): 175.

https://doi.org/10.3390/genes11020175

Zhang W., Liu Z.K., Su Q.F., Li H., Jia J., Meng L.M., Sui J., Wang L.X., Zhao Z.W., and Jin Q.M., 2015, Identification and evaluation of new soybean varieties (Lines) resistance to SMV in Jilin Province, Jilin Nongye Kexue (Journal of Jilin Agricultural Sciences), 40(4): 51-53, 59.