Increased boll number contributes to the yield heterosis of H318 in different years.
H318, as an elite cotton cultivar, showed an obvious yield increase over the control variety, with an average 3,664.5 kg/ha seed cotton and 1,521 kg/ha lint yield production (9.2% higher than control) in two years (2007–2008) of regional testing.
To explore the effects of heterosis on yield, we conducted a four-year field investigation about the traits like the number of bolls, the number of fruit site, abscission rate and the number of fruit branches (Fig. 1). At the same time, considering the climate influences to plant growth and production, the climatic changes from May to October (the whole growth period of cotton) in Wuhan from 2010 to 2013 were also obtained from Statistics Bureau of Hubei Province (Figure S1).
In 2010, a long-term lasting rainfall and large volume of precipitation in July (Figure S1 C, D) extremely affected the fruit setting efficiency (July and August are the flourishing flowering stage), which causes high abscission rate of 4–5 (paternal line) and B0011 (maternal line) (Fig. 1C). However, relatively lower abscission rate and more fruit sites was found in the H318 (Fig. 1B-C), which might be the reason for its final higher boll numbers (Fig. 1A).
In later relatively ideal climate conditions in 2011, more water in growing stage (June in Figure S1 C, D); suitable sunshine hours, raining and warming temperature during flourishing flowering stage (July and August in Figure S1 A-D), little difference could be found in the abscission rates between the hybrid and both parents (Fig. 1B), but much more fruit sites were produced in hybrid H318, which directly causing the increased boll number of H318 (Fig. 1A).
In 2012 and 2013, long-term high temperature stress occurred in July and August (Figure S1 A) caused higher abscission rate than the other years, but H318 still showed much lower abscission rate than its parents and more bolls were produced at last (Fig. 1A, C). No obviously differences were found about fruit site in 2012 and 2013, and fruit branches have no difference in all years (Fig. 1B, D). Thus, we speculate that the higher yield production of H318 directly derives from the stable higher number of bolls, and which is the result of the increased fruit sites or decreased abscission rate in different years.
Hybrid H318 shows heterosis on biomass and growth speed at seedling stage
Besides the yield heterosis, H318 also showed growth vigor than its parents at seedling stage (Fig. 2A). The fresh and dry weight (the whole plant) of H318 and its parents were measured at two-leaf stage, and results showed that H318 had obviously higher fresh weight than its parents and dry weight than 4–5 (Fig. 2B). Moreover, the fresh weight of B0011 was higher than 4–5, but without obviously changes about dry weight. To evaluate the plant growth status in detail, the cotyledon area was calculated every 2 days after cotyledons spreading until 14 DAS (Fig. 2C). At 6 DAS, the hybrid H318 had just a little larger area of cotyledon than both parents. After the 8 DAS, the cotyledon area was significantly increasing and reached its maximum area until the 14 DAS. The cotyledon area of H318 remarkably kept largest than its parents after 8 DAS. These results suggested that H318 have obviously heterosis about biomass production and growth speed at seedling stage.
Global analysis of differential gene expression of H318 and its parents
To analyse the global gene expression patterns of these three genotypes, a total of 9 RNA sequencing libraries (containing 3 independent biological repeats for each genotype) were constructed for Illumina sequencing using the whole seedlings of 8 DAS. In total, more than 43,000,000 clean reads (occupy 95% of raw reads) were generated from each sample (Table S1). All clean reads then were mapped to cotton genome (G. hirsutum TM-1 (AD)1) to obtain the genome location and annotation information (Table S2). Nearly 89% reads were mapped to genome and more than 80% reads were uniquely mapped. Most of mapped reads (> 85%) were found located in exon; reads in intergenic region occupied 11%-12%; and the rest (2.5%) located in intron (Figure S2).
The reads mapped to genome then were used for transcript assembling and gene expression level calculation. The FPKM of all genome annotated genes (70,478) and novel genes (6,564) were listed in Table S3. The correlation analysis showed perfect consistency between biological repeats (Figure S3). The differential expressed genes (DEGs) then were screened out with condition of P values < 0.05. Finally, a total of 17,308 DEGs containing 16,407 annotated genes and 1,261 novel genes were found between three genotypes (Table S4), and much less DEGs (306) between H318 and B0011 were found relatively (Figure S4).
To remove the low expressed or differences DEGs, a more stringent criterion (FPKM > 50 and log2(fold changes) > 1 in at least one sample) was performed and left 945 DEGs (Fig. 3A). Among these, 931 DEGs were found between H318 and its paternal line (4–5), most DEGs (532) were up-regulated in H318; only 78 DEGs were found between H318 and its maternal line (B0011, Fig. 3B). The DEGs distribution in three genotypes showed that most DEGs (844/945) is different expressed between H318 and 4–5 or B0011 and 4–5; 77 DEGs were shared in three genotypes (Fig. 3C). These results indicated that the gene expression pattern of H318 is more similar to its maternal line and difference to its paternal line.
To validate the reliability of RNA-Seq, 26 DEGs was selected randomly for qRT-PCR confirmation. A correlation analysis was made using data from RNA-Seq and qRT-PCR (Fig. 3C). And the result shows that the R2 value reaches 0.9791, indicating the high quality for RNA-Seq.
GO And KEGG Analysis
To gain a deeper understanding of the potential functions of these DEGs (945) in the three genotypes, GO and KEGG analyses were carry out for gene functional classification (Figure 4, Table S5 and Table S6). For GO analysis, the most significant enriched biological processes were Photosynthesis related processes (red, 67 DEGs); Energy production process (blue, 43 DEGs); Oxidation-reduction process (green, 84 DEGs); Carbohydrate metabolic related processed (yellow, 54 DEGs) and Metabolic process (purple, 341 DEGs) (Figure 4, Table S5). At the same time, Oxidoreductase activity (green) and Photosystem related components (red) mostly enriched in Molecular function and Cellular component respectively. For KEGG analysis, the significant enriched pathways were various metabolic related pathways (purple) with 211 DEGs (Figure 4, Table S6). And Photosynthesis (red,) and Carbon metabolism related pathways (green) were also found enriched with 45 DEGs and 62 DEGs respectively.
Photosynthesis, carbohydrate metabolic and oxidation-reduction pathways are enriched in H318.
From above GO and KEGG analyses, photosynthesis, oxidation-reduction and carbohydrate metabolic were speculated significantly changed between H318 and its parental lines. Of the 67 DEGs involved in photosynthesis, all of them were up-regulated in H318 and B0011 relative to 4-5 (Table S7). However, most of these DEGs were slightly down-regulated (32) or no change (31) in H318 compared to maternal line (B0011, Table S7). In oxidation-reduction process (84 DEGs), 68 DEGs were highly up-regulated in H318 relative to 4-5, and 55 DEGs slightly up-regulated in H318 relative to B0011 (Table S7). For carbohydrate metabolic process (54 DEGs), 30 and 37 DEGs were up-regulated in H318 relative to 4-5 and B0011 respectively. Results indicated that the photosynthesis pathway were both enhanced in H318 and B0011 related to 4-5, oxidation-reduction process and carbohydrate metabolic process were enhanced in H318. These results indicated that hybrid H318 might have stronger capability on photosynthesis, carbohydrate metabolic and oxidation-reduction than its parents especially to the paternal line. We speculated that enhanced capability of these functions might contribute to higher fresh and dry weight and faster growth speed of H318 at seedling stage (Figure 2).
Higher photosynthesis rate and sugar accumulation were found in H318 at seedling stage.
To validate the authenticity of this inference, the photosynthesis rate and the content of sucrose and starch at two leaf stage were assessed in both H318 and its parental lines (Figure 5). Results showed that the photosynthesis rate was significantly enhanced in H318 relative to its parental lines especially to the paternal line 4-5 (Figure 5 A). Correspondingly, the content of sucrose and starch were also found higher in H318 (Figure 5 B). Moreover, the relative expression level of six genes related to starch biosynthesis and photosynthesis process was also detected and showed in Figure 5 C including GRANULE BOUND STARCH SYNTHASE 1 (GBSS1), RUBISCO ACTIVASE (RCA), PROTON GRADIENT REGULATION 5 (PGR5), PHOTOSYSTEM II SUBUNIT X (PSBX), PHOTOSYSTEM I SUBUNIT L (PSAL) and PHOSPHORIBULOKINASE (PRK). The expression trends of these genes were highly consistent to the photosynthesis rate and the content of sucrose and starch, which indicated an enhanced photosynthesis rate and sugar biosynthesis in H318 relative to its parental lines. All these results implied that stronger photosynthesis and metabolic processes contributes to the heterosis in H318 at seedling stage.