Molecular Plant Breeding 2020, Vol.11, No.24, 1-8
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2 Conclusions
The anther of Pepper (Yin Chuan Cavel) Male Sterility Lind analysis by RNA-Seq, compared with the reference
genome, a total of 3 319 genes were detected, which showed that the difference in transcriptome between male
sterile line and fertile line was very significant. The results showed that the differentially expressed genes in
Yinchuan Yangjiao pepper male sterile dual-purpose line played a regulatory role in its metabolic activities, and
the accumulation of differential genes involved in metabolic regulation was rich (An et al., 2014; Trapnell et al.,
2010). There were 34 down regulated and 68 up regulated differential metabolites detected in the anthers of fertile
and sterile lines. The main metabolites were amino acid and derivatives, nucleotides and derivatives, glycerol ester,
flavonoids, phenolic acids and sugar metabolism related substances (Lu et al., 2020). Amino acids, nucleotides,
flavonoids and sugars are all important substances affecting pollen development, the significant difference
accumulation between fertile and sterile lines indicated that pollen abortion of male sterile lines was closely
related to the differential metabolism and accumulation of the above substances. The results of transcriptome and
metabolome analysis showed that the differential gene expression and metabolites in mature anthers of Pepper
(Yin Chuan Cavel) Male Sterility Lind were mainly concentrated in the protein synthesis pathway of amino acid
biosynthesis, pyrimidine metabolism, tryptophan metabolism, cysteine and methionine metabolism and flavonoid
biosynthesis pathway. These results indicate that the significant differential expression of transcriptional genes in
the above pathways plays an important role in regulating the accumulation of metabolites. The differential
expression of transcriptional genes regulated the metabolism of key metabolic pathways, resulting in significant
differential accumulation of glycosides and energy substances closely related to pollen development, which
affected pollen development of male sterile lines and led to pollen abortion; The regulation of flavonoid
metabolism is related to the scavenging of reactive oxygen species (ROS), which may lead to pollen abortion. It
can be inferred that the differential expression of differential genes can regulate the metabolism and accumulation
of lipid, fenac and carbohydrate metabolites related to pollen development, and then lead to pollen abortion (Li et
al., 2016; Li et al., 2015).
3 Materials and Methods
3.1 Materials
The male sterile line of Yinchuan Yangjiao pepper was bred by pepper breeding research group of Germplasm
Resources Research Institute of Ningxia Academy of agricultural and Forestry Sciences. It will be planted in May
2019. At the full flowering stage, the pollen abortion was identified by the acetic acid magenta staining
microscope, and the fertile and sterile lines were identified. The mature anthers were sampled. The anthers of each
sample were collected from ten materials and stored in the refrigerator at -80℃.
3.2 RNA extraction and transcriptome sequencing
After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads, while prokaryotic mRNA
was enriched by removing rRNA by Ribo-Zero TM Magnetic Kit (Epicentre). Then the enriched mRNA was
fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random
primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the
cDNA fragments were purified with Qia Quick PCR extraction kit, end repaired, poly (A) added, and ligated to
Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR
amplified, and sequenced using Illumina HiSeq TM 2500 by Gene Denovo Biotechnology Co. (Li et al., 2020;
Limin Sun, 2019) .Three replicates were set for each sample, and the extraction and sequencing were entrusted to
Gene Denovo Guangzhou Biology Limited.
3.3 Extraction and analysis of metabolites
The freeze-dried anther was crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30
Hz. 100 mg powder was weighted and extracted overnight at 4℃ with 0.6 ml 70% aqueous methanol. Following
centrifugation at 10 000 g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250
mg, 3 ml; ANPEL, Shanghai, China,
and filtrated (SCAA-104, 0.22 μm pore size;
