Transcriptome profiling of Elymus sibiricus, an important forage grass in Qinghai-Tibet plateau, reveals novel insights into candidate genes that potentially connected to seed shattering

Abstract

Background: Elymus sibiricus is an important forage grass in semi-arid regions, but it is difficult to grow for commercial seed production due to high seed shattering. To better understand the underlying mechanism and explore the putative genes related to seed shattering, we conducted a combination of morphological, histological, physiochemical and transcriptome analysis on two E. sibiricus genotypes (XH09 and ZhN03) that have contrasting seed shattering.

Results: The results show that seed shattering is generally caused by a degradation of the abscission layer. Early degradation of abscission layers was associated with the increased seed shattering in high seed shattering genotype XH09. Two cell wall degrading enzymes, cellulase (CE) and polygalacturonase (PG), had different activity in the abscission zone, indicating their roles in differentiation of abscission layer. cDNA libraries from abscission zone tissue of XH09 and ZhN03 at 7 days, 21 days and 28 days after heading were constructed and sequenced. A total of 86,634 unigenes were annotated and 7110 differentially expressed transcripts (DETs) were predicted from "XH09-7 vs ZhN03-7", "XH09-21 vs ZhN03-21" and "XH09-28 vs ZhN03-28", corresponding to 2058 up-regulated and 5052 down-regulated unigenes. The expression profiles of 10 candidate transcripts involved in cell wall-degrading enzymes, lignin biosynthesis and phytohormone activity were validated using quantitative real-time PCR (qRT-PCR), 8 of which were up-regulated in low seed shattering genotype ZhN03, suggesting these genes may be associated with reduction of seed shattering.

Conclusions: The expression data generated in this study provides an important resource for future molecular biological research in E. sibiricus.

Keywords: Abscission layers; Elymus sibiricus; Mechanism; Next-generation sequencing; Seed shattering; Transcriptome analysis.

Fig. 1

Different seed shattering habits of two E. sibiricus genotypes. (a1) High seed shattering type XH09. (a2) Low seed shattering type ZhN03. (b) Seed shattering degree of inflorescence in XH09 and ZhN03. Photos taken at 28 days after heading. (c) Time-course changes in the seed shattering degree of XH09 and ZhN03 at 7, 14, 21 and 28 days after heading. BTS was measured upon detachment of seed from the pedicels by pulling. Bars indicate the mean values ± standard deviation. Double asterisks (**) represent significant difference of BTS between XH09 and ZhN03 at p < 0.01 level

Fig. 2

Histological analysis of abscission zone. (a) and (d), (b) and (e), (c) and (f) show longitudinal sections across the abscission zone of XH09 and ZhN03 at 7 DAH, 21 DAH and 28 DAH, respectively. Sections were stained with safranine-fast green, and lignin in red. (g) and (k), (h) and (l), (i) and (m) show scanning electron microscopy photos of pedicel junction after detachment of seeds in XH09 and ZhN03 at 7 DAH, 21 DAH and 28DAH, respectively. (j) and (n) show close-up scanning electron microscopy photos corresponding to red boxes in (i) and (m). A peeled-off and smooth surface is observed in the high seed shattering genotype XH09 (j), whereas broken and rough surface is observed in the low seed shattering genotype ZhN03 (n)

Fig. 3

Specific activity of two cell wall-degrading enzymes: cellulase (a) and polygalacturonase (b) in abscission zone. Bars indicate the mean values ± standard deviation. Double asterisks (**) represent significant difference of enzyme activity between XH09 and ZhN03 at p < 0.01 level

Fig. 4

Heat map diagram of the expression levels of 27 differentially expressed transcripts (DETs) involved hydrolase activity. The DETs were found between high seed shattering genotype XH09 and low seed shattering genotype ZhN03 at 21 days after heading

Fig. 5

qRT-PCR validations of RNA-seq data. Expression profiles of the selected genes as determined by RNA-seq and qRT-PCR. Data were collected from high seed shattering genotype XH09 and low seed shattering genotype ZhN03 at 7, 21 and 28 days after heading. The left-hand y-axis indicates FPKM value. The right-hand y-axis indicates relative expression level. Bars indicate the mean values ± standard deviation