| Medicago sativa L.‘Qingshui’is the only validated and registered rhizome-rooted alfalfa species in China,and is an excellent forage for establishing ecological and covering grasslands in arid areas.The rhizome of Qingshui has relatively high resistance to stress and trampling as well as high renewal capacity,with better ground cover formation.Qingshui has high nutrition,but its yield is low.In order to improve the low yield performance of Qingshui,we have obtained valuable rhizome-rooted hybrid strains(RSA-01,RSA-02,and RSA-03)by crossing of Qingshui and the high yielding‘WL168’.Due to the optimal utilization period of alfalfa being 5～6 years,this study selected 1st and 5th age alfalfa plants.In this study,three hybrid strains,i.e.,RSA-01,RSA-02,and RSA-03,were used as the research objects,while the parents(Qingshui and WL168)were used as the control.The differences in the stability of high yield traits between hybrid materials and their parents were studied from the aspects of biomass,photosynthetic physiological,and longevity characteristics.At the same time,the relationship among biomass,longevity,and photosynthetic physiological characteristics were explored.Screening out‘high and stable’biomass and long-lived hybrid offspring materials to provide new and excellent germplasm resources for the establishment of grazing and ecological grassland.Further,transcriptomic sequencing analysis was used to explore important genes related to longevity and high biomass,and explore the sustainability of high biomass traits in hybrid materials from the perspective of longevity.The research results have important and far-reaching significance for delaying the aging process and prolonging the lifespan of alfalfa.The main results are summarized as follow:(1)Through hybridization with WL168,the biomass traits(plant height,stem diameter,leaf length,leaf width,leaf area,and biomass)of Qingshui have been improved to varying degrees.At age 1,the stem diameter,stem leaf ratio,leaf length,leaf width,leaf area,and dry weight of RSA-02 were significantly higher than Qingshui by 5.80%～23.14%,while the plant height,stem diameter,leaf length,leaf area,branch number,and dry weight of RSA-03 were significantly higher than Qingshui by 7.89%～22.12%,respectively.At age 5,the stem diameter,stem leaf ratio,leaf length,leaf area,and dry weight of RSA-02 were significantly higher than that of Qingshui by 7.49%,19.01%,8.19%,9.44%,and 7.97%,respectively,while the plant height,stem diameter,leaf length,leaf area,branch number,and dry weight of RSA-03 were significantly higher than that of Qingshui by 9.25%,7.33%,7.17%,9.37%,and 19.71%,respectively.Through correlation analysis,it was found that dry weight had the greatest correlation with plant height,followed by stem diameter,both of which were positively correlated.indicating the biomass of Qingshui has been improved to varying degrees through hybridization with WL168.Based on the analysis of the interannual stability of biomass in hybrid materials,the dry weights of 5-year-old RSA-01,RSA-02,and RSA-03increased by 9.03%,14.35%,15.23%,and 9.99%compared to the values of 1-year-old RSA-03,respectively.The interannual variation rate of dry weight of RSA-03 was lower than that of RSA-01 and RSA-02,indicating that the biomass of RSA-03 was prominent and stable.Analysis of the variability of biomass characteristics of hybrid materials indicated that the number of branches,dry weight,and leaf width of RSA-01,the number of branches,and leaf width of RSA-02,and the plant height of RSA-03 varied significantly,indicating that hybrid materials have a large selection space,while the stem thickness and leaf thickness of the three showed relatively uniform.(2)By hybridization,hybrid materials with rich photosynthetic characteristics are produced.At age 1,compared to Qingshui,the P_n,G_s,WUE,and SS contents of RSA-01increased by 23.64%,7.44%,33.71%,and 6.50%,respectively,while the P_n and T_r values of RSA-02 increased by 59.01%and 38.80%,respectively.The Chl(a+b),P_n,G_s,and WUE values of RSA-03 increased by 12.19%,32.27%,46.38%,and 31.92%,respectively.At age5,compared to Qingshui,the Chl(a+b)and SS contents of RSA-01 increased by 4.52%and14.45%,the Chla and Tr values of RSA-02 increased by 9.23%and 14.05%,respectively.and the Chl(a/b)and Suc contents of RSA-03 increased by 19.89%and 23.28%,resulting in hybrid materials with rich photosynthetic characteristics.The contents of Chl(a/b),P_n,G_s,WUE,and Suc in RSA-03 of 5-year-old were higher than those in RSA-03 of 1-year-old by9.99%～44.24%.Interestingly,Chlb,Chl(a/b),P_n,T_r,G_s,C_i,Suc,SP,and SS were more influenced by age than genetics,while the opposite was true for Car and Sta.(3)In order to clarify the longevity characteristics of the test material,the telomerase activity and telomere length of its somatic cells were measured,and the results were as follows,(1)The measured values of telomerase activity of the test materials at age 1 were as follows:WL168>RSA-02>Qingshui>RSA-03>RSA-01;the measured values of telomerase activity of the test materials at the age of 5 are WL168>RSA-01>RSA-03>Qingshui>RSA-02,indicating that hybridization can produce hybrid materials with abundant telomerase activity.Except WL168,all varieties have significant differences in telomerase activity between plants aged 1 year and plants aged 5 years,and the rate of change in telomerase activity does not differ reliably with age.(2)Relative telomere length of alfalfa tended to increase with age.Compared to plants in aged 1 year,relative telomere lengths of Qingshui,WL168,RSA-01,RSA-02,and RSA-03 in plants aged 5 years were 11.41,11.24,9.21,10.23,and 11.41,respectively.Accordingly,alfalfa varieties can be classified according to relative telomere length as long-lived(Qingshui,WL168,and RSA-03),medium-lived(RSA-02)and short-lived(RSA-01).Through contribution analysis,it was found that the formation of telomere length is influenced not only by genetic differences within the material itself,but also by interannual changes,with age exerting a greater effect.(4)In order to reveal the stability mechanism of high yield traits in alfalfa hybrid materials and further study the relationship among biomass,lifespan,and photosynthetic physiological characteristics of alfalfa,the following conclusions can be obtained:Ⅰ.Except for Qingshui,the dry weight variation characteristics of WL168,RSA-01,RSA-02,and RSA-03 are similar to telomere length,indicating that the dry weight of alfalfa is closely related to telomere length.Ⅱ.The direct path coefficient of photosynthetic physiological indicators on their dry weight is as follows:G_s>Chl(a+b)>Chlb>WUE>Chla>SS>Suc>SP>Car>T_r>NSC>C_i.G_s and Chl(a+b)have a significant positive effect on their dry weight,while SS and SP both have a significant negative effect on their dry weight through G_s,indicating that G_sand Chl(a+b)are the direct factors for alfalfa dry weight,and SS and SP both indirectly affect their dry weight through G_s.Ⅲ.Telomerase activity was strongly correlated with Chla,Chl(a+b),and WUE by-0.632,-0.680,and 0.846,respectively.Among them,telomerase activity was negatively correlated with Chla and Chl(a+b),and positively correlated with WUE.Telomere length has a strong correlation with Chl(a+b),Chla/b,Car,and SS by 0.659,0.686,0.854,and-0.685,respectively.Among them,telomere length has a negative correlation with SS,and a positive correlation with Chl(a+b),Chla/b,and Car,indicating that photosynthesis interacts with telomerase activity and length to jointly regulate the stability of high-yield traits in alfalfa hybrid materials.(5)Transcriptome results showed that 6375 differentially expressed genes were found in the RSA-03 Vs.RSA-01 comparison group,of which 2249 Unigenes were up-regulated and4126 were down regulated.Through the joint analysis of photosynthetic physiological characteristics and transcriptome of the stability of high-yield traits of hybrid materials,the results showed that:Ⅰ.compared with RSA-01,RSA-03 regulates the biomass traits(stem diameter,leaf area,fresh and dry weight)and longevity of alfalfa hybrid materials by altering the differential expression of genes involved in carbohydrate metabolism pathway and photosynthesis pathway by affecting photosynthetic products(Sta,NSC,and SP)and photosynthetic parameters(T_r,G_s,C_i and WUE).ⅡCompared with RSA-01,RSA-03 is involved in carbohydrate metabolism(fructose and mannose metabolism pathways and pyruvate metabolism),energy metabolism pathways(oxidative phosphorylation pathway,photosynthetic antenna protein pathway,carbon fixation pathway in photosynthetic organisms,photosynthesis pathway),lipid metabolism pathway,glycan biosynthesis and metabolism genes related to the metabolism of terpenes and polyketones(carotenoid biosynthesis,limonene and pinene degradation)and other secondary metabolism(isoflavone biosynthesis,isoquinoline alkaloid biosynthesis,and pantothenate and Co A biosynthesis)regulate key genes for longevity and high biomass in alfalfa.Nine differentially expressed genes were verified by fluorescence quantitative PCR,including photosynthetic related genes(MS.gene 47994,MS.gene 32425,MS.gene 058723,novel.811,MS.gene 71518,MS.gene87427,MS.gene 37999,MS.gene 76234,and MS.gene019953.The results showed that the expression trends of RNA-Seq and q RT-PCR were consistent,indicating that the high throughput sequencing results were reliable. |
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