| Tartary buckwheat is widely planted all over the world,with its yield increasing year by year.Its nutritive composition is balanced and rich in rutin,minerals,amino acids and so on.It is a natural dietary source of flavonoids and is deeply loved by the people.At the same time,it is also an important material for the study of plant stress tolerance because of its excellent properties of salt resistance,barren resistance and resistance to heavy metals.However,since the epicarp is so close to the seed and hard that cannot crack which contributes to difficulty to effectively crack in production.At present,the high temperature curing and recooling method is adopted,which not only reduces the nutrient content,but also increases the production cost and affects the economic value.Tartary buckwheat’s further development and utilization is seriously restricted by the difficulty of cracking.MQ is a new variety of buckwheat by physical and chemical mutagenesis.Its epicarp is thin and loose without any grooves.A systematic analysis of the molecular mechanism of easy cracking of MQ will contribute to the genetic improvement of Tartary buckwheat and promote the development of modern agriculture in China.In this study,physiological and biochemical analysis,multi-group combined analysis,CRISPR/Cas9 and other methods were used to systematically reveal the characters and molecular mechanism of easy cracking of MQ.The main results are as follows:(1)Through the morphological observation of MQ,it was found that MQ had the characteristics of typical Tartary buckwheat,with small triangular fruits and rough brown-yellow epicarp.The flowers are small,tasteless and yellowish green.The stem is smooth,the leaf is triangular,and the raceme is sparsely distributed among the branches.Further,the chloroplast genome of MQ with a total length of 159 272 bp was obtained through high-throughput sequencing,and it was found to have typical chloroplast genome structure,and the GC content was 37.9%.There were 2 reverse repeat regions,1 large single copy region and 1 small single copy region.Combined with the database information,113 chloroplast genes were successfully annotated,including 79 coding genes,4 r RNA and 30 t RNA.Finally,based on the chloroplast genome sequence information,it was found that Tartary buckwheat and MQ formed a subgroup according to the phylogenetic trees of 9 different plants constructed from buckwheat,rhubarb and mouse-ear mustard.(2)Through mechanical property analysis,it was found that the breaking force and hardness of MQ fruit were significantly lower than that of XQ fruit,and the brittleness of the MQ epicarp was slightly higher than that of XQ fruit,and the strength and toughness were significantly lower than that of XQ fruit.The wave peak trend of FTIR spectra of MQ and XQ was basically same,and the peak shape and position were similar,but there were differences in the intensity of some absorption peaks,indicating that the main components of MQ and XQ epicarp were similar,and the relative content of some components was different.The results showed that the content of lignin,cellulose and hemicellulose was less and pectin was more.Through the appearance observation,it was found that the surface of MQ was rougher than that of XQ.Through anatomical structure,it was found that the thickness of the first epicarp was much lower than that of the second epicarp,and the tissue characteristics of the epicarp were not obvious.Through the microstructure of the epidermis,it was found that the part of the epicarp of MQ was cracked,the structure was damaged and the overall structure was loose,while the structure of XQ was compact,crack-free and without damage.(3)64 MADS-box genes were identified in Tartary buckwheat genome.According to the different structural domains,they were divided into two subgroups,in which the M-type subgroup had 27 genes and the Mick-type subgroup had 37 genes.It was found from the gene structure that all Ft MADS genes contained multiple exons,while a small number of M-type subgroup genes did not contain introns.Further,MEME analysis found that motif1 and motif2 were highly conserved in all Ft MADS genes and belonged to the MADS-box domain.Based on the physical location information of genes in Tartary buckwheat genome data,64 Ft MADS genes were mapped into the chromosomal location map of Tartary buckwheat MADS family.It was found that Ft MADS genes were not evenly distributed on 8 Tartary buckwheat chromosomes,with at least 3 genes on Chr8 and up to 15 genes on Chr7.Repeat event analysis showed that 2 MADS genes participated in 1tandem repeat event and 12 MADS genes participated in 9 fragment repeat events.These MADS genes are mainly distributed on Chr1,Chr3,Chr4,and Chr7,and they all belong to the Mick-type subfamily.The multi-genome collinearity analysis was used to construct the relationship between Ft MADS gene and known functional genes.A total of 118 collinearity gene pairs were found,of which the M-type subgroup of Tartary buckwheat and the M-type subgroup of beet and grape were the most,mainly distributed on Chr1.The collinearity gene pairs of Mick-type subgroup and grape were the most,mainly distributed on Chr4.Finally,the phylogenetic trees of multi-plant M-type subfamily and Mick-type subfamily containing 5 dicotyledons and 1 monocotyledon were constructed.It was found in different branches that the genes of the same plant were clustered together,and the genes of different plants were clustered together according to their monocotyledon and dicotyledon.Through the analysis of MEME,it was found that most of the genes with similar evolutionary relations had similar motifs.(4)The 155 G transcript library of Tartary buckwheats covering three important stages of fruit development was established and a total of 13 750 differential genes were acquired.According to the difference of gene expression patterns in fruit maturation,these differential genes were divided into four groups: the differential genes up-regulated along with the fruit development(205),the differential genes quickly down-regulated along with the fruit development(746),the differential genes expressed not with the fruit development(5 975)and the differential genes slowly down-regulated along with the fruit development(6 824).Among the 13 750 differential genes,1 119 genes belonged to 72 transcription factor families,among which 31 genes most of them were down-regulated with fruit development and mainly involved in transcription and regulation of substance metabolism through nuclear binding with DNA were in MADS family.The expression patterns of 31 candidate genes were verified by q RT-PCR,and it was found that most of the genes were expressed in reproductive organs(flowers and fruits),among which 15 genes were highly expressed in fruits.At the later stage of Tartary buckwheat fruit development,gene expression differences were significant,and the expression levels of Ft MADS4,Ft MADS5,Ft MADS11,Ft MADS13,Ft MADS30,Ft MADS35,Ft MADS36,Ft MADS37,Ft MADS51 and Ft MADS61 were higher in MQ.By combining the relationship between the Ft MADS gene constructed in(3)and the known functional genes,the known functional genes with similar evolutionary relationship and the same motif were screened.It was found that the Ft MADS4 was in the same branch as AT3G58780 and AT2G42830 genes that regulate the fruit dehiscence of Arabidopsis thaliana.(5)CRISPR/CAS9 system was used to simultaneously knock out AT2G42830 and AT3G58780 genes.The results showed that 17 of the 25 independent lines were successfully mutated at AT2G42830 target sites,and 4 lines were found to be successfully mutated at AT3G58780 target sites.Through agrobacterium infection,Ft MADS4 gene was supplemented into the deletion Arabidopsis thaliana,and GUS staining and PCR identification showed that the double mutant of Ft MADS4 gene was successfully supplemented in Arabidopsis thaliana.Compared with wild Arabidopsis thaliana,it was found that wild Arabidopsis thaliana fruits cracked naturally after ripening,but the fruits of the double mutant plants did not crack after ripening until they were completed with Ft MADS4 gene.Therefore,Ft MADS4 had the ability to positively regulate fruit cracking.Conclusionly,this study determined the classification status of MQ by using multi-level,multi-group and multi-method correlation analysis.It was found that poor mechanical properties were the direct cause of easy cracking characters,and low contents of lignin,cellulose and hemicellulose which provided mechanical properties,were the material cause of easy cracking characters.64 MADS-box family genes of Tartary buckwheat and 118 collinearity gene pairs were identified from the genome,and the relationship between Ft MADS genes and known functional genes was established.A 155 G transcription library covering three important stages of fruit development of tough and easy cracking buckwheat was constructed,and 13 750 differential genes were found.Through the genetic transformation system of Arabidopsis thidopsis,it was finally confirmed that Ft MADS4 was involved in transcription by binding with DNA in the nucleus,regulating the downstream target genes and influencing the accumulation of substances providing mechanical properties,so as to positively regulate the function of Tartary buckwheat fruit cracking. |
