| Blueberry is a new fruit tree with high nutrition,high health care and high medical value.It has broad development prospects and is called " the third generation of fruit king in the world".However,its adaptability to saline-alkali soil is insufficient,generally requiring the soil p H value to be below 5.0.And because blueberries are small berry fruit trees and are not storable,some large cities have built bases around them to supply and develop nearby.However,because the soil is not suitable and the p H value is more than 6.0,many areas adopt methods such as applying sulfur powder to reduce the p H value of the soil,but this not only increases the cultivation cost,but also damages the soil ecological environment and affects the nutritional status of blueberries.Therefore,it is particularly important to cultivate blueberries suitable for higher p H soil.In this study,blueberry loose callus culture and mutation(high p H+salt)and screening of saline-alkali resistant cells were used to obtain saline-alkali resistant mutant callus cells.Mutant strains were obtained through differentiation,rooting and molecular identification of adventitious buds.Transcriptome sequencing was used to analyze its differential genes and metabolic pathways involved in differential genes,and the genetic mechanism of saline-alkali resistance was studied.In addition,in this study,the mutants 1 and 2 screened from the greenhouse of 4000 mu of blueberry base in Jizhou District,and its control group,Lanfeng,Duke,Berkeley,Brigitta,and Lexie,were used to observe growth indexes,fruiting indexes,and intrinsic quality of the fruit.The leaves and seeds of mutant and control blueberries were dissected and observed.On this basis,SRAP polymorphism of genomic DNA in blueberry leaves was analyzed to verify whether mutants 1 and 2 really mutated.The test results are as follows:1.The cell mutagenesis steps are: soft callus induction-loose callus-cell mutagenesis under high saline-alkali stress-cell bud differentiation-root differentiation-mutant.2.With the increase of the concentration of 2,4-D in each explant,the induction rate of soft callus showed a trend of gradually increasing at first and then decreasing.The induction rate of soft callus of leaf explants was 22%,and that of stem explants was 100%,and the growth rate of callus was higher than that of leaves.The best scheme for soft callus induction is to cut 0.5cm long blueberry stem segments from 3-5cm high blueberry tissue culture seedlings,inoculate them into 1/2MS+2mg/L2,4-D+20g/L sucrose medium,and culture for2～3 weeks.3.Loose callus cannot be obtained directly from explants.It must be transformed from soft callus to loose callus.KT or 6-BA alone cannot complete the conversion,and 2,4-D must be added.The survival rate of callus treated with BA+2,4-D was only 24% at the highest level,and the callus was transparent at low or high concentration BA.With the increase of subculture,most of the callus hydrolyzed and died.Under KT+2,4-D treatment,the highest survival rate of callus was 94%,and after three subcultures,the callus was gradually transformed into loose callus with a conversion rate of 94%.The ideal medium for induction of loose callus is MS+1 mg/LKT+0.5 mg/L2,4-d.4.Determination of mutagenic conditions of saline-alkali tolerant cells: when p H value was 6 ～ 6.5,callus can grow normally;When p H value was 7 ～ 7.5,the death rate of callus was 27 ～ 41%,and most of them died after subculture for 3 times.At p H 8,the death rate of callus was 68%,and after 20 days of culture,most of them died.At p H 8.5,95% of the calli died.After subculture for 2 times,a very small number of calli survived.When p H value was9.0,all callus browning died.To sum up,the mutant callus must be able to grow at p H 8.5.Therefore,p H 8.5 was selected as the inducing pressure of alkali-resistant callus.5.Differentiation of Buds from Loose Callus: When BA was used as mitogen to induce callus regeneration,the regeneration effect of callus was not ideal.Regenerated shoots cannot be obtained when BA was used alone or at a lower concentration(0.5 mg/L).Although regenerated shoots can be induced on a small number of calli when high concentration BA(1～2mg/L)was used in combination with IAA,the number of regenerated shoots was small,growth was slow,most of the regenerated shoots were deformed,and vitrification phenomenon occurred when high concentration BA(3mg/L)was present,and normally growing regenerated seedlings cannot be basically obtained.When BA was replaced by ZT,the regeneration rate can reach 100% when the concentration of ZT was 1.0 mg/L.In most cases,the regenerated buds grew normally and can be obtained to continue to grow into seedlings.Therefore,Anderson+1.0mg/L ZT+0.5mg/L IAA medium was selected as the regeneration medium for blueberry callus.6.Root differentiation: Regardless of the presence or absence of IAA,callus adventitious buds can take root,and the rooting rate reached 100%.Therefore,MS medium without hormone IAA was selected for rooting induction of alkali-resistant callus adventitious buds.In addition,on MS medium,LFB saline-alkali tolerant blueberry callus can take root with rooting rate of 100%.7.The results of transcriptome sequencing of salt-tolerant blueberry LFB callus showed:(1)A total of 11.46 Gb Clean Data was obtained by transcriptome sequencing.The Clean Data of each sample reached 5.71 Gb,and the Q30 base percentage was 91.68% and above.A total of 73,078 Unigenes were obtained after assembly,of which 27,403 Unigene were annotated,and 18,789 Unigenes over 1 kb were in length.Among them,10,554 SSR markers were obtained from SSR analysis.(2)The number of differentially expressed genes screened was 233,of which 53 were up-regulated and 180 were down-regulated;and the SNP between samples was 68,547.(3)Assembly data After annotation by each database,a total of 126 differential transcripts were obtained,including 40 differential transcripts to the COG database,68 differential transcripts of the GO function database,and 44 differential transcripts of the KEGG database.69 differential transcripts of KOG database,103 differential transcripts of Pfam database,98 differential transcripts of Swiss＿Prot database,and differential transcripts of egg NOG database.113,123 differential transcripts of the nr database.(4)The annotations in the secondary functions of GO showed that there were distinct BP(biological processes)in terms of the enrichment trend of differentially expressed genes: cell killing,reproductive regeneration,growth and development,immune processes,cellular components or Biogenetic,multicellular biological processes,etc.;there were obviously different CC(cell components): macromolecular complex,extracellular region,organelle part,cell part,etc.;there are obviously different MF(molecular function): nutrition library Activity,antioxidant activity,electron carrier activity,structural molecular activity,and the like.⑸ COG function annotation and pattern clustering were carried out on differential genes.Annotation results mainly focus on carbohydrate transport and metabolism,amino acid transport and metabolism,cell wall/membrane/envelope biogenesis,inorganic ion transport and metabolism,biosynthesis of secondary metabolites,transport and catabolism,energy production and conversion and other metabolic pathways.⑹ The annotation results of differentially expressed gene KEGG were classified into 26 categories according to the types of pathways in KEGG.According to the scatter plot of enrichment of differentially expressed gene KEGG pathway,the 7 metabolic pathways with larger enrichment factors,that was,the most significant and reliable,and the most relevant to mutation were vitamin B6 metabolism,starch and sucrose metabolism,glycerophospholipid metabolism,galactose metabolism,ether lipid metabolism,glucosinolate biosynthesis and cyanoamino acid metabolism in turn,the ID of the corresponding differential genes was c54791.graph＿c0;c60505.graph＿c0;c61088.graph＿c0,etc.8.The phenophase results of mutant and control blueberry showed that from flower bud expansion stage to fruit maturity stage,mutant 1 and 2 had earlier stages than the other four varieties,flower bud expansion stage was 10-16 days earlier than the other four varieties,and fruit maturity stage was 6-19 days earlier than the other four varieties.Therefore,variant strains 1 and 2 are presumed to be early-maturing variants.9.Comprehensive membership analysis results of growth indexes(length of main branch,number of vegetative branches,average leaf area,etc.)of mutant and control blueberry showed that the growth potentials of Variant 1(0.328)and Variant 2(0.460)were moderate,the growth potentials of both were weaker than Berkeley(0.734)and brigitta(0.654),and stronger than Lanfeng(0.061).10.The comprehensive membership function results of the fruiting indexes(number of fruiting branches,number of long fruiting branches,number of short fruiting branches,etc.)of mutant and control blueberries showed that mutant 1(0.783)and mutant 2(0.769)had better fruiting characteristics than control blueberries,Berkeley(0.677)and brigitta(0.416)had moderate fruiting characteristics,and Lanfeng had the worst fruiting characteristics(0.067).11.After analyzing the comprehensive membership function values of the mutant and the control group in and out of quality characteristics(soluble solids,sugar-acid ratio,soluble protein,anthocyanin,total phenol,flavonoid,fruit shape index,fruit powder thickness,fruit uniformity,etc.),it can be seen that the comprehensive quality of variant 2 was the best,with the comprehensive membership function value of 0.774.Variation 1 had the worst comprehensive quality(0.351).12.The comparison results of anatomical structure differences between mutant and control blueberry leaves showed that Variant 1 had the largest palisade tissue thickness,palisade-sea ratio and tissue structure tightness,which were 114.761μm,1.514 and 0.498 respectively.13.The results of SRAP polymorphism analysis of leaf DNA of mutant and control varieties showed that 2 of the 10 pairs of primers,namely primers ME7/EM7 and ME8/EM8,amplified polymorphic bands.There were 47 polymorphic bands,including 33 polymorphic bands.On average,there were 23.5 bands amplified by one pair of primers and 16.5polymorphic bands.The percentage of polymorphism was 65% ～ 74.07%.Both primer combinations can reveal DNA polymorphism.Among them,primer ME8/EM8 amplified fewer than 13 polymorphic bands and the polymorphism ratio was 65%,while primer ME7/EM7 amplified 20 polymorphic bands and the polymorphism ratio was 74.07%.SRAP amplification maps of genomic DNA of mutant and control blueberries showed that none of the varieties in control blueberries had the same band type and position as variant1 or 2,and the bands of variant 1 and variant 2 were also different.Therefore,combining with the phenological observation results,variant 1 and variant 2 were early-maturing variants of a blueberry.In addition,the variation range of genetic similarity coefficient between mutant and control blueberry was 0.0865 ～ 0.875,and the genetic relationship among variant 1,Lanfeng,Berkeley and brigitta was close.The genetic relationship between Variant 1 and brigitta was the closest,with the highest genetic similarity coefficient of 0.875,followed by Variant 1 and Lanfeng,with the genetic similarity coefficient of 0.625.Lan Feng was also closely related to brigitta and Berkeley,with genetic similarity coefficients of 0.75 and 0.7 respectively.The genetic relationship between Variant 2 and control blueberry was general,with the closest genetic relationship with Lekszi,and the genetic coefficient is 0.5715. |
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