Comparative transcriptome analysis of cotton fiber development of Upland cotton (Gossypium hirsutum) and Chromosome Segment Substitution Lines from G. hirsutum × G. barbadense

Abstract

Background: How to develop new cotton varieties possessing high yield traits of Upland cotton and superior fiber quality traits of Sea Island cotton remains a key task for cotton breeders and researchers. While multiple attempts bring in little significant progresses, the development of Chromosome Segment Substitution Lines (CSSLs) from Gossypium barbadense in G. hirsutum background provided ideal materials for aforementioned breeding purposes in upland cotton improvement. Based on the excellent fiber performance and relatively clear chromosome substitution segments information identified by Simple Sequence Repeat (SSR) markers, two CSSLs, MBI9915 and MBI9749, together with the recurrent parent CCRI36 were chosen to conduct transcriptome sequencing during the development stages of fiber elongation and Secondary Cell Wall (SCW) synthesis (from 10DPA and 28DPA), aiming at revealing the mechanism of fiber development and the potential contribution of chromosome substitution segments from Sea Island cotton to fiber development of Upland cotton.

Results: In total, 15 RNA-seq libraries were constructed and sequenced separately, generating 705.433 million clean reads with mean GC content of 45.13% and average Q30 of 90.26%. Through multiple comparisons between libraries, 1801 differentially expressed genes (DEGs) were identified, of which the 902 up-regulated DEGs were mainly involved in cell wall organization and response to oxidative stress and auxin, while the 898 down-regulated ones participated in translation, regulation of transcription, DNA-templated and cytoplasmic translation based on GO annotation and KEGG enrichment analysis. Subsequently, STEM software was performed to explicate the temporal expression pattern of DEGs. Two peroxidases and four flavonoid pathway-related genes were identified in the "oxidation-reduction process", which could play a role in fiber development and quality formation. Finally, the reliability of RNA-seq data was validated by quantitative real-time PCR of randomly selected 20 genes.

Conclusions: The present report focuses on the similarities and differences of transcriptome profiles between the two CSSLs and the recurrent parent CCRI36 and provides novel insights into the molecular mechanism of fiber development, and into further exploration of the feasible contribution of G. barbadense substitution segments to fiber quality formation, which will lay solid foundation for simultaneously improving fiber yield and quality of upland cotton through CSSLs.

Keywords: Chromosome Segment Substitution Lines; Fiber development; G. hirsutum × G. barbadense; Gossypium hirsutum; Transcriptome analysis.

Fig. 1

Pearson Correlation Coefficient of the genes identified from the 15 samples. H1, H2 and Z36 represent MBI9915, MBI9749 and CCRI36, respectively. 10 = 10 DPA, 15 = 15DPA, 20 = 20DPA, 25 = 25DPA, and 28 = 28DPA

Fig. 2

Principal Component Analysis of the genes identified from the 15 samples. H1, H2 and Z36 represent MBI9915, MBI9749 and CCRI36, respectively. 10 = 10 DPA, 15 = 15DPA, 20 = 20DPA, 25 = 25DPA, and 28 = 28DPA

Fig. 3

A heat map of 1801 DEGs from 15 samples. H1, H2 and Z36 represent MBI9915, MBI9749 and CCRI36, respectively. 10 = 10 DPA, 15 = 15DPA, 20 = 20DPA, 25 = 25DPA, and 28 = 28DPA

Fig. 4

GO functional classification of 1801 DEGs

Fig. 5

GO enrichment and KEGG pathway analysis of the up-regulated DEGs

Fig. 6

GO enrichment and KEGG pathway analysis of the down-regulated DEGs

Fig. 7

Multiple comparisons between CCRI36 and two CSSLs at diverse stages of fiber development. H1, H2 and Z36 represent MBI9915, MBI9749 and CCRI36, respectively. 10 = 10 DPA, 15 = 15DPA, 20 = 20DPA, 25 = 25DPA, and 28 = 28DPA. Red numbers represent the up-regulated genes, while green ones represent the down-regulated genes

Fig. 8

Multiple comparisons of the DEGs between MBI9915 and MBI9749 at the same stage of fiber development. H1 and H2 represent MBI9915 and MBI9749, respectively. 10 = 10DPA, 15 = 15DPA, 20 = 20DPA, 25 = 25DPA and 28 = 28DPA. Red numbers represent the up-regulated genes, while green ones represent the down-regulated genes

Fig. 9

Different gene expression patterns in the three lines. Each square represents a trend of gene expression. The number in top left corner indicates the profile ID number; and the number in bottom left corner indicates the number of genes in that profile. The profiles were ordered based on the number of genes enriched thereof

Fig. 10

GO functional classification of the genes in profile 1 in the three lines

Fig. 11

A heat map of the 29 oxidation-reduction process-related genes in profile 1

Fig. 12

Validation of RNA-seq data by qRT-PCR. H1, H2 and Z36 represent MBI9915, MBI9749 and CCRI36 respectively. Columns indicate the results of qRT-PCR, and zigzag lines indicate the results of transcriptome sequencing

Fig. 13

Flow chart of construction of CSSLs