## 4 Discussion  
### Large-Scale Genic SSR Marker Development in the Soybean  
Geneticists have identified major genes and QTLs controlling pod maturity [32; 33; 34; 35; 36; 37; 38] to examine the genetic characteristics of soybeans. The SSR marker, a type of effective genetic marker with various applications, is one of the most widely used molecular markers for plant genotyping due to its high polymorphism [39]. Although many soybean SSR markers have been used for genotyping [40; 41; 42] and QTL mapping [32; 33; 34; 35; 36; 37], some of the existing methods for SSR genotyping exhibit low sensitivity, accuracy, and efficiency, yet have high costs [15]. To overcome these disadvantages of SSR markers, over 1000 genic SSR markers have been developed for soybean genetic research [43; 44]. Genic SSR markers are used as an effective tool to identify genetic resources, since SSRs are located in transcribed genes that are annotated with putative functions based on biological information and homology search [45], and as a selection tool for specific traits in breeding populations of diverse crops [46; 47; 48; 49]. As shown in Supplementary Table S1, we developed 211 genic SSR markers. Among the 211 SSR repeat motifs, tri-nucleotide repeats (TNRs) were found to be the most abundant, accounting for 75.4% (159), followed by di-nucleotide repeats (DNRs) at 16.6%, and tetra-nucleotide repeats (TtNRs) at 8.0%. Meanwhile, repeats longer than five nucleotides were not found. These results were similar to previously published data, with TNRs reported as the most frequent (54-78%), followed by DNRs (17.1-40.4%) and TtNRs (3-6%) among cereal species [50]. Expression Profiling Shows That Six Genes Were Linked to the Growth and Development of the Soybean Pod  
To develop genic SSR markers associated with the pod maturity of soybeans, we preferentially selected six genes found to be regulated in the soybean pod. LATE FLOWERING (LATE) encodes a C\({}_{2}\)H\({}_{2}\)-type zinc-finger transcriptional regulator that acts as a floral repressor and regulates the expression of genes that control flowering in the leaf vasculature of Arabidopsis [51]. Glyma.03g160900.1, annotated as a LATE, showed the highest expression levels in the leaves of LM ecotypes; however, it was decreased in the pods of all three ecotypes. Late embryogenesis abundant protein (LEA, PFAM:PF03168) genes are upregulated in maturing seeds and vegetative tissues by salinity, dehydration, cold or freezing stress, and ABA treatment [52; 53; 54]. Glyma.03g201600.1 and Glyma.19g198600.1 were annotated as LEAs. The expression of Glyma.03g201600.1 was increased in the pods of the MM and LM ecotypes, whereas that of Glyma.19g198600.1, encoding syntaxin-24 (SYP24), decreased only in the pods of the LM ecotype. These results demonstrate that although different isoforms belong to the same gene family in soybeans, the genes are differentially regulated with stage advancement [55] and by tissue [56]. The cupin_1 domain represents the conserved barrel domain of the cupin superfamily, which contains 11S and 7S plant seed storage proteins. Plant seed storage proteins are the major nitrogen sources for the developing plant [57]. A glycinin subunit Gy7, which has two cupin_1 domains, has been reported as upregulated in soybean pods during seed maturation [58]. Our results, however, showed that Glyma.19G164800.1, the corresponding gene of Gy7, was upregulated in the pod of only the LM variety. Non-specific lipid transfer protein (nsLTP) genes, including Glyma.19g158400.1, have been isolated and characterized extensively in soybeans [59]. These genes reportedly play important roles in plant growth and development, including seed development [60]. Glyma.19g158400.1 was upregulated in the pods of all three ecotypes, although the highest expression was observed in the pods of the LM ecotype, indicating that the gene is downregulated with stage advancement. Moreover, JA is a key player in the control of senescence [61].