iFLAS: positive-unlabeled learning facilitates full-length transcriptome-based identification and functional exploration of alternatively spliced isoforms in maize.

The advent of full-length transcriptome sequencing technologies has accelerated the discovery of novel splicing isoforms. However, existing alternative splicing (AS) tools are either tailored for short-read RNA-Seq data or designed for human and animal studies. The disparities in AS patterns between plants and animals still pose a challenge to the reliable identification and functional exploration of novel isoforms in plants. Here, we developed integrated full-length alternative splicing analysis (iFLAS), a plant-optimized AS toolkit that introduced a semi-supervised machine learning method known as positive-unlabeled (PU) learning to accurately identify novel isoforms. iFLAS also enables the investigation of AS functions from various perspectives, such as differential AS, poly(A) tail length, and allele-specific AS (ASAS) analyses. By applying iFLAS to three full-length transcriptome sequencing datasets, we systematically identified and functionally characterized maize (Zea mays) AS patterns. We found intron retention not only introduces premature termination codons, resulting in lower expression levels of isoforms, but may also regulate the length of 3'UTR and poly(A) tail, thereby affecting the functional differentiation of isoforms. Moreover, we observed distinct ASAS patterns in two genes within heterosis offspring, highlighting their potential value in breeding. These results underscore the broad applicability of iFLAS in plant full-length transcriptome-based AS research.

Chloroplast Genes Are Involved in The Male-Sterility of K-Type CMS in Wheat.

The utilization of crop heterosis can greatly improve crop yield. The sterile line is vital for the heterosis utilization of wheat (Triticum aestivum L.). The chloroplast genomes of two sterile lines and one maintainer were sequenced using second-generation high-throughput technology and assembled. The nonsynonymous mutated genes among the three varieties were identified, the expressed difference was further analyzed by qPCR, and finally, the function of the differentially expressed genes was analyzed by the barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) method. A total of 16 genes containing 31 nonsynonymous mutations between K519A and 519B were identified. There were no base mutations in the protein-encoding genes between K519A and YS3038. The chloroplast genomes of 519B and K519A were closely related to the Triticum genus and Aegilops genus, respectively. The gene expression levels of the six selected genes with nonsynonymous mutation sites for K519A compared to 519B were mostly downregulated at the binucleate and trinucleate stages of pollen development. The seed setting rates of atpB-silenced or ndhH-silenced 519B plants by BSMV-VIGS method were significantly reduced. It can be concluded that atpB and the ndhH are likely to be involved in the reproductive transformation of 519B.

Mitochondrial genes are involved in the fertility transformation of the thermosensitive male-sterile line YS3038 in wheat.

Heterosis can improve the stress resistance, quality, and yield of crops, and the male sterility of wheat can be utilized to accelerate the breeding process of hybrid. To determine whether mitochondrial genes are involved in the fertility of K-type cytoplasmic male-sterile (CMS) line and the YS-type thermosensitive male-sterile (TMS) line in wheat, we sequenced and assembled the mitochondrial genomes of K519A, 519B, and YS3038 by next-generation sequencing (NGS). The non-synonymous mutations were analyzed, and the first-generation sequencing was conducted to verify the non-synonymous mutation sites. Furthermore, the expression patterns of genes with non-synonymous mutations were analyzed. Finally, the candidate genes were silenced by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) to test the functions of the candidate genes. The results revealed that the mitochondrial genomes of K519A, 519B, and YS3038 were 420,543, 433,560, and 452,567 bp in length, respectively. Besides, 33, 31, and 37 protein-coding genes were identified in K519A, 519B, and YS3038, respectively. There were 14 protein-coding genes and 83 open reading frame (ORF) sequences that differed between K519A and 519B and 10 protein-coding genes and 122 ORF sequences that differed between K519A and YS3038. At the binucleate stage, seven genes (nad6, ORF256, ORF216, ORF138, atp6, nad3, and cox1) were downregulated in K519A compared with 519B, and 10 genes (nad6, atp6, cox3, atp8, nad3, cox1, rps3, ORF216, ORF138, and ORF224) were downregulated in YS3038 compared with K519A. Besides, six genes (nad6, ORF138, cox3, cox1, rps3, and ORF224) were downregulated under fertile conditions relative to sterile conditions in YS3038. Gene silencing analysis showed that the silencing of cox1 significantly reduced the seed setting rate of YS3038, indicating that the cox1 gene may be involved in the fertility transformation of YS3038. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01252-x.