Morphological Characteristics And Transcriptome Analysis Of Alfalfa Stem Development At Different Stages

With high yield and good quality,alfalfa is an important legume forage for livestock.Compared with leaves,stems have a greater impact on the forage quality of alfalfa.It was found that the structure of alfalfa stems at the same developmental stage was different,but the differences in anatomical structure,developmental physiology and metabolic regulation among different types of stems were rarely reported.Based on the stem characteristics,6alfalfa varieties including Zhaodong,Aohan,Zhungeer,WL363,Ladak and WL168 were selected as materials for this study.By comparing the nutritional quality,two alfalfa varieties,WL168 and Zhungeer,were selected for comparative analysis of mechanical strength,morphological characteristics,cell wall composition,anatomical characteristics and transcriptome of stems at four developmental stages(17,24,32 and 85 days after mowing).The main results are as follows:1.By comparing the contents of NDF,ADF,IVTD and hemicellulose in the stems of six alfalfa varieties at the early flowering stage,it was found that WL168 and Zhungeer alfalfa were the two varieties with the greatest difference.The contents of NDF and ADF in stems of WL168 alfalfa were significantly lower than those of Zhungeer alfalfa(P≤0.05),while the contents of IVTD and hemicellulose were significantly higher than those of Zhungeer alfalfa(P≤0.05).Therefore,follow-up studies were conducted with these two varieties.2.At the four developmental stages,the mechanical strength of the stems of Zhungeer alfalfa was significantly higher than that of WL168 alfalfa(P≤0.05).The morphological indexes of the stems of the two alfalfa varieties were different:Compared with WL168,the stems of Zhungeer was shorter but thicker.The cell wall components of the two alfalfa stems were also different.The contents of cellulose,hemicellulose,lignin and protopectin of Zhungeer alfalfa were higher than those of WL168 alfalfa,but the soluble pectin content was lower.These results suggest that the low in vitro true digestibility(IVTD)of stems in Zhungerer may be related to higher lignin or cellulose content,and the difference in mechanical strength of stems may be also related to stem morphology and cell wall composition.3.The anatomical characteristics of stems showed that the xylem areas of the two alfalfa stems were thickened and expanded with the stems development.At the same development stage,the staining area of stem xylem of Zhungeer was larger than that of WL168.The stem of WL168 appeared hollow structure from the third stage,while the stems of Zhungeer was always solid structure.4.Comparative transcriptome analysis showed that a total of 1,993 genes were differentially expressed at four developmental stages of the two alfalfa stems,with the most differentially expressed genes at the stage 4.There were four significant enrichment pathways in the first stage,which were starch and sucrose metabolism,phenylpropanoid biosynthesis,isoflavone biosynthesis and flavonoid biosynthesis.The expression patterns of PAL,4CL,HCT,COMT and POD genes involved in lignin biosynthesis were consistent with lignin content and distribution.5.The comparative analysis at different developmental stages of the same alfalfa variety showed that the number of differential genes was highest between stage 1 and stage 4.There were 1636 differential genes in H1 vs H4,and 3723 differential genes in S1 vs S4.Moreover,721 differential genes were found between stage 1 and stage 4 in both varieties.The differential genes were significantly enriched in starch and sucrose metabolic pathways.With the development of stems,the expression levels(FMPK)of differential genes glg C,INV,HK,AMY,bgl X and TPS involved in starch and sucrose metabolism pathways,INV,HK,bglX and TPS involved in sucrose synthesis and hydrolysis,and glgC involved in starch synthesis showed a downward trend,while the expression level of AMY involved in starch decomposition gradually increased.The expression change trend of related genes was consistent with the law of alfalfa stem development.6.Differentially expressed transcription factors showed that 7 transcription factors involved in cell wall and lignin biosynthesis including NAC,MYB,bHLH families.