Heterosis has been widely exploited as an approach to enhance crop traits during breeding. However, its underlying molecular genetic mechanisms remain unclear. Recent advances in RNA sequencing technology (RNA-seq) have provided an opportunity to conduct transcriptome profiling for heterosis studies. We used RNA-seq to analyze the flower transcriptomes of two F1 hybrid soybeans (HYBSOY-1 and HYBSOY-5) and their parents. More than 385 million high-quality reads were generated and aligned against the soybean reference genome. A total of 681 and 899 genes were identified as being differentially expressed between HYBSOY-1 and HYBSOY-5 and their parents, respectively. These differentially expressed genes (DEGs) were categorized into four major expression categories with 12 expression patterns. Furthermore, gene ontology (GO) term analysis showed that the DEGs were enriched in the categories metabolic process and catalytic activity, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis found that metabolic pathway and biosynthesis of secondary metabolites were enriched in the two F1 hybrids. Comparing the DEGs of the two F1 hybrids by GO term and KEGG pathway analyses identified 26 common DEGs that showed transgressive up-regulation, and which could be considered potential candidate genes for heterosis in soybean F1 hybrids. This identification of an extensive transcriptome dataset gives a comprehensive overview of the flower transcriptomes in two F1 hybrids, and provides useful information for soybean hybrid breeding. These findings lay the foundation for future studies on molecular mechanisms underlying soybean heterosis.

Male-sterile plants are used in hybrid breeding to improve yield in soybean (Glycine max (L.) Merr.). Developing the capability to alter fertility under different environmental conditions could broaden germplasm resources and simplify hybrid production. However, molecular mechanisms potentially underlying such a system in soybean were unclear. Here, using positional cloning, we identified a gene, MALE STERILITY 3 (MS3), which encodes a nuclear-localized protein containing a plant homeodomain (PHD)-finger domain. A spontaneous mutation in ms3 causing premature termination of MS3 translation and partial loss of the PHD-finger. Transgenetic analysis indicated that MS3 knockout resulted in nonfunctional pollen and no selfpollinated pods, and RNA-seq analysis revealed that MS3 affects the expression of genes associated with carbohydrate metabolism. Strikingly, the fertility of mutant ms3 can restore under longd conditions. The mutant could thus be used to create a new, more stable photoperiod-sensitive genic male sterility line for two-line hybrid seed production, with significant impact on hybrid breeding and production.

Nectar secretion of RN-type cytoplasmic male sterility three lines in soybean [Glycine max (L.

Poor seed‐set limits potential for soybean hybrid seed production. The objective of this study was to evaluate effects of different pollinators on the rate of seed set in three soybean cytoplasmic male sterile (CMS) lines, JLCMS9A, JLCMS82A, and JLCMS89A. The following five treatments were applied under net room isolation‐conditions: (i) insecticide application and release of alfalfa leafcutter bees (Megachile rotundata) during flowering; (ii) insecticide application in the absence of leafcutter bees; (iii) release of alfalfa leafcutter bees during flowering without insecticide application; (iv) the absence of insecticide or alfalfa leafcutter bees during flowering; (v) plants without a net cover and pesticide application. Results indicated that the rate of pod production and seed set differ significantly between CMS lines and between treatments. Alfalfa leafcutter bees were an effective pollinator as evidenced by increases in pod‐bearing rate and seed‐set rate respectively from 21.35 to 53.22% and from 19.91 to 58.77% in the three CMS lines. The three CMS lines also showed significant differences in seed set, with JLCMS82A exhibiting the highest rate in all five treatments, followed by JLCMS9A and then JLCMS89A. These results suggested that thrips (Sericothrips variabilis) might have some effect on soybean pollination, but did not elicit a major influence based on the low increases in seed production. The natural insect population served an important role in outcrossing and seed set in soybean.

The cytoplasmic male sterility/restorer-of-fertility (CMS/Rf) system plays a vital role in high-efficiency hybrid seed production in crops, including soybean (Glycine max (L.) Merr.). The markers linked to fertility restoration and the restorer-of-fertility (Rf) genes are essential because they can facilitate the breeding of new CMS lines and production of commercial hybrid soybean seeds. To date, several soybean Rf genes have been mapped to various genetic loci in diverse genetic populations. However, the mapping range of restorer genes remains narrow, with relatively limited practical applicability. Therefore, in the present study, F2 and F3 segregating populations derived from the CMS line JLCMS5A crossed with the restorer line JLR2 were developed and used for Rf3 gene fine mapping. Genetic investigation indicated that the restorer line JLR2 was controlled by a single dominant gene, Rf3. By integrating bulk-segregant analysis and next-generation sequencing, a 4 Mb region on chromosome 9 was identified, which was most likely the target region harboring the candidate gene responsible for fertility restoration. This region was further narrowed down to 86.44 Kb via fine mapping in F2 and F3 populations using SSR, InDel, and dCAPS markers. This region contained 10 putative genes (Glyma.09G171100–Glyma.09G172000). Finally, Glyma.09G171200, which encodes a mitochondria-targeted pentatricopeptide repeat protein, was proposed as the potential candidate for Rf3 using sequence alignment and expression analysis in restorer and CMS lines. Based on single-nucleotide polymorphisms in Glyma.09G172000, a CAPS marker co-segregated with Rf3 named CAPS1712 was developed. Our results will be fundamental in the assisted selection and creation of potent lines for the production and rapid selection of novel restorer lines.