| The perennial forage maize is a tall grass of the family Poaceae.Its plants are erect,like-maize,multi-tillersand the plant height is 2.5-3.5m.It is widely planted because of its advantages of high-biomass yield ability,strong adaptability,excellent green feed quality,and perennials.Plant anthocyanins have certain antioxidant properties,can befreely removed,and have the functions of improving animal immunity,enhancing the body’s antioxidant capacity,and improving the quality of dairy products.Anthocyanins have attracted much attention as feed additives and anthocyanin-rich plants as green feed.In this study,the tri-hybrid of Zea may,Tripsacum dactyloides and Zea perennis,MTP and MTPP were used as the female parent,and the Z.perennis(9475)was used as the male parent for backcrossing,forming 79 groups and 99 population,respectively.The strains whose leaf sheaths appear purple are the research objects.The chromosome composition,anthocyanin content,agronomic traits,and forage yield of purple strains in different populations are studied;meanwhile,the key genes of anthocyanin synthesis and main anthocyanin components in the purple leaf sheath of the purple strain were analyzed by RNA-seq and LC-MS/MS technology.The results are as follows:1.The 79 and 99 populations of purple strains are all aneuploidy containing the chromosomes of Z.mays,T.dactyloides and Z.perennis.The total number of chromosomes of 79 purple strains is 55～58,the chromosomes of Z.mays are 7～11,the chromosomes of Z.perennis are 28～32,the chromosomes of T.dactyloides are16～18,and the average chromosome composition is 2n=56.40=9.2M+30.40P+16.80T.The total number of chromosomes of 99 population purple strains is 63～67,the chromosomes of Z.mays are 9～12,the chromosomes of Z.perennis are 36～41,the chromosomes of T.dactyloides are 15～17,and the average chromosome composition is 2n=65.14=10.4M+38.86P+16.40T.Chromosome translocation analysis between the two populations found that the chromosome translocation types of 99 purple strains and their translocation rates were higher in 79 populations,indicating that the addition of a set of Z.perennis sub-genome to MTPP leads to the increase of ploidy,which can promote the exchange of chromosome fragments between species.2.Create the forage strains of weak purple,light purple,medium purple and deep purple leaf sheaths using the“backcross of purple leaf sheath MTP and MTPP with light purple leaf sheath 9475”.Based on the determination of total anthocyanin content in the whole plant,analysis of agronomic traits,yield and heterosis,the purple strains selected from the 79 population were 7930 and 7936,and the purple strains selected from the 99 population were 9916 and 9936.The total anthocyanin content of the whole plant was ranked as 7936,9916,7930,9936,the corresponding contents were25.25 mg/100g,14.09 mg/100g,8.95 mg/100g,7.61 mg/100g;the two-year forage yield ranking was 7936,9916,9936,7930,the corresponding yields are 144.59 t/hm~2,136.53 t/hm~2,134.75 t/hm~2,130.41 t/hm~2.7936 has the highest total anthocyanin content and strong forage potential.3.Correlation analysis showed that the total anthocyanin content of the entire population of 79 population was very significantly positively correlated with the number of maize chromosomes,but was significantly negatively correlated with the number of chromosomes of Z.perennis.Plant height,tillers and forage yield were extremely significantly correlated with the chromosome number of maize,and were extremely significantly or significantly negatively correlated with the chromosome number of Z.perennis and the chromosome of T.dactyloides.The total anthocyanin content of the of 99 population was extremely negatively correlated with the number of chromosomes of T.dactyloides,and the number of tillers,total stem thickness,and forage yield were extremely significantly positively correlated with the total number of chromosomes.It shows that anthocyanin content,forage yield and agronomic traits are affected by chromosome composition.Therefore,in the breeding of perennial forage maize varieties,assisted selection breeding can be carried out based on molecular genetics studies such as the chromosome number and composition of the material.4.Transcriptome sequencing analysis of 7936 leaf sheath showed that there were13783 differential genes in purple leaf sheath compared with green leaf sheath,of which 7974 genes were down-regulated(57.85%)and 5809 genes were up-regulated(42.15%).In the anthocyanin synthesis pathway,CHS,F3H,F3’H,ANS,UFGT genes were up-regulated,CHI,F3’,5’H genes were down-regulated,and DFR was not differentially expressed.Among the up-regulated genes,the differential expression of CHS and F3’H was larger.Transcriptome sequencing results also found 683differentially expressed transcription factors,of which 437 transcription factors were down-regulated(63.98%)and 247 transcription factors were up-regulated(36.02%).According to the functional annotation of the differential transcription factors in the NR,NT and KO databases,three differentially expressed transcription factors that regulate the synthesis of anthocyanins in plant vegetative organs:PL1,B1 and Lc.5.The total anthocyanin content of the leaf sheath at the beginning of 7936tasselling was 190 mg/100g,which was similar to the content of anthocyanin in the leaves of Napiergrass.Using LC-MS/MS technology,the anthocyanins in 7936 leaf sheaths were detected as 10 cyanidin,2 peonidin,5 pelargonidin,2 delphinidin,1rosin anthocyanin and 4 proanthocyanidins.6.Represented by the 79 population,q RT-PCR analysis of five anthocyanin synthesis structural genes in the leaf sheaths of different purple strains showed that the relative expression of CHS in 7930(medium purple leaf sheath)and 7936(deep purple leaf sheath)higher than light purple and weak purple leaf sheath materials,indicating that CHS is an essential gene for anthocyanin synthesis in purple forage maize.The relative expression levels of DFR,ANS and UFGT roughly corresponded to the level of anthocyanin in leaf sheath.The relative expression levels of DFR and ANS of 7930(medium purple leaf sheath)and 7936(deep purple leaf sheath)were not significantly different,but the relative expression levels of UFGT of the two showed significant differences,the expression of UFGT gene may play an important role in the accumulation of anthocyanins in the leaf sheath of purple forage grass. |
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