| Medicago ruthenica L.,one kind of excellent forage grass,is a genus ofMedicago,with strong stress resistance and wide ecological adaptability,but with comparatively poor productivity.As a comprehensive agronomic trait,plant type is closely related to breeding potential,yield and harvest mode of forage.M.ruthenica germplasm materials have a large variation in plant type,and the relationship between plant type structure characteristics and yield remains to be unclear.In addition,most of the stems of M.ruthenica are prostrate or oblique,which is not conducive to mechanized harvesting and cannot be popularized and applied in a large area.Therefore,the study of M.ruthenica plant type characteristics and its formation mechanism is an important guide for high-yield breeding and application of Medicago genus.M.ruthenica ’Zhilixing’(erect stem),M.ruthenica ’Mengnong No.1’(prostrate stem)and M.ruthenica ’Mengnong No.2’(oblique stem)were used as the main materials.By analyzing their morphological characteristics of stems and leaves,yield formation characteristics and photosynthetic physiological characteristics,the high-yield plant type characteristic system ofM.ruthenica was preliminarily constructed.We further analyzed the regulatory mechanism of erect-stem formation by analyzing the anatomical structure of stems and the components of secondary cell walls,as well as using high-throughput sequencing and molecular biology techniques.The main results are as follows:1.Among the three different Plant type M.ruthenica cultivars,the average hay yield of high-yielding Mengnong 1 at the three-year early flowering stage was 19.20%and 12.55%higher than that of Zhilixing and Mengnong 2,respectively(P<0.05).Meanwhile,its photosynthesis efficiency was the highest,and its adaptability to light intensity was also the strongest.To sum up,the plant type characteristics of high-yield Mengnong No.1 are tall plant height,large and thick leaves,developed palisade tissue and high cell compactness.2.Erect stem cultivar had guide hole diameter,wide lignin distribution and relatively developed fibers between vascular bundles;the lignocellulose content of Zhilixing was higher than that of Mengnong No.1 at different growth stages.At branching stage,the G-S lignin monomer content and cellulose content of Zhilixing were 35.56%(p<0.01)and 12.20%(P<0.05)higher than Mengnong No.1 respectively.3.Transcriptome sequencing analysis of erect stem cultivars and horizontal stem cultivars was performed.The differentially expressed genes related to secondary wall synthesis and lignin synthesis pathway between Zhilixing and Mengnong No.1 by qRT-PCR.Only the lignin polymerization gene MrLAC17 was highly expressed in Zhilixing,and the expression level was 5-8 times than that of Mengnong No.1.MrLAC17 gene was cloned from M.ruthenica,and its bioinformatics analysis and subcellular localization were carried out.The overexpression and functional verification of Arabidopsis lac4 lac17 double mutants confirmed that MrLAC17 is able to be involved in regulating lignin synthesis.4.A series of bioinformatic comparisons and transcriptional activation validation of the MrLAC17 gene and its promoter sequence were performed in erect stem and prostrate stem.This showed that transcriptional regulation did not cause differences in the expression of MrLAC17 between the two cultivars.The small RNA libraries of erect stem and prostrate stem were further constructed and validated by Stem-loop qRT-PCR analysis.It is basically determined that miR397a negatively regulated MrLAC17 expression at the posttranscriptional level,which means the miR397a-LAC17 module is involved in regulating M.ruthenica lignin synthesis,which contributes to the formation of its erect stem. |
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