*4.4. Pollen Viability*

For pollen viability and pollen developmental progression, flowers collected from Col-0 and *AtBGLU20* antisense transgenic plants were fixed in Carnoy's fixative (6:3:1 alcohol:chloroform:acetic acid) for 2 h. Then, the anthers were detected and stained with a solution of Malachite green, acid fuchsin, and Orange G for approximately 12 h, as previously described [61].

## *4.5. Identification of BrBGLUs and Phylogenetic Tree Construction*

The protein sequence of 48 BGLU members were downloaded from TAIR (http://www. arabidopsis.org/tools/bulk/sequences/index.jsp) [6]. All putative protein sequences of *B. rapa* (version 3.0) were downloaded from BRAD (http://brassicadb.org/brad/index.php) [35] and used as queries to search against the Hidden Markov Model (HMM) profile (Version 3.1b2) with the Pfam HMM library (Pfam 32.0) [62]. A total of 64 protein sequences with PF00232 (E value below 1E−<sup>5</sup> ) were obtained, and these sequences were considered as *BrBGLUs* candidates and used for further analysis. Multiple sequence alignment of full-length BGLU proteins and phylogenetic tree construction were conducted using ClustalX2 [31]. The phylogenetic tree was generated by the MEGA6 program, using the neighbor-joining method with the 'pairwise deletion' option and 'Poisson correction' model, with a bootstrap test of 1000 replications [63].

## *4.6. Chromosomal Location, Nomenclature, and Gene Duplication of BrBGLUs*

The position of each *BrBGLU* on *B. rapa* chromosomes was identified from BRAD (http://brassicadb.org/brad/index.php). For nomenclature, the '*Br*' for *B. rapa* was added, followed by BGLU, and numbered according to its position from top to bottom on *B. rapa* chromosomes 1–10. MCScanX software was used to search potentially duplicated BrBGLUs [64]. All of the putative protein sequences of *B. rapa* (version 3.0) were compared with themselves by BLASTP, with a tabular format and an e-value of < 10−<sup>5</sup> . Then, tandem, segmental, and dispersed duplications were identified using MCScanX, using default criteria.

## *4.7. Co-Expression and Gene Ontology Enrichment Analysis*

*AtBGLU20* was used as bait gene for genome-wide co-expression analysis to identify genes of similar function from Expression Angler [65]. *BrBGLU10* was represented by two EST probes *Brapa\_ESTC004210* and *Brapa\_ESTC007739*, which were used as bait for co-expression analysis. A cutoff threshold of 0.90 for the Pearson correlation coefficient was used. The expression pattern analysis was performed using the Arabidopsis eFP browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb. cgi) [37]. Clustering analysis for categorization was performed with the TIGR Multi-Experiment Viewer (http://www.tm4.org/mev.html). GO enrichment analysis was performed using agriGO (http://bioinfo.cau.edu.cn/agriGO/index.php) [66].

## *4.8. Microarray Analysis*

To analyze the gene expression patterns of *BrBGLUs* in *B. rapa* during pollen development, the previously published microarray data relating to male sterility analysis were downloaded from NCBI's Gene Expression Omnibus (GSE47665) [29]. The microarray data were re-annotated using BLASTX by comparing with the newly improved *B. rapa* reference genome sequence (version 3.0) [35].

## **5. Conclusions**

In conclusion, 64 *BrBGLUs* have been identified in *B. rapa* genome, which were classified into 10 subgroups with *Arabidopsis* counterparts, and the GH1-i subgroup included putative pollen development-related *BrBGLU10*. Base on its known function in *Arabidopsis*, BrBGLUs may participate in various defense responses against biotic and abiotic stresses, flavonoid metabolism, and pollen development. This study has provided valuable information for a better understanding of BGLUs, and for their biotechnological application to crops.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/7/ 1663/s1.

**Author Contributions:** Designed the research scheme: X.D. and Y.H. Performed the experiments: X.D. and Y.J. Analyzed the data: X.D., Y.J., and Y.H. Wrote the manuscript: X.D. and Y.H. All authors read and approved the final manuscript.

**Funding:** This research was funded by the National Science Foundation of China, 31601771 and the Applied Basic Research Project of Yunnan, 2017FB056.

**Acknowledgments:** We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
