| Loquat(Eriobotrya japonica(Thunb.)Lindl.),named after its Pipa-like shape,belongs to the Rosaceae Eriobotrya Lindl and is one of the main economic tree species and one of the most famous fruit trees in the south of China.The fruit of loquat has good-looking color and appearance,juicy and soft fruit pulp,delicious taste,as well as rich nutrition.At the same time,the fruit can also clear lung heat and harmonize the stomach,relieve hiccup and dissolve phlegm,relieve cough,engender liquid and moisten the lung,promote appetite and help digestion.It is widely popular among customers for its health-giving benefits.With the development of the industry and the change of market demands,varietal problems have become increasingly prominent in loquat production.The fruit of red-fleshed loquat is relatively large,however,with a pale flavor and poor taste,while the fruit of white-fleshed loquat has fine fruit pulp and excellent taste,however,with a small size and poor storability.Therefore,the demand of characteristic resources in loquat industry cannot be met for such breeding problems.Therefore,accelerating the breeding of new cultivars of high quality has been an important guarantee and effective measure for the sustainabledevelopment of loquat industry in China.Fruit quality is the most important factor of determining the commodity of loquat.There are great differences between the fruit quality of the two type loquats,of which the underlying mechanism is very complex.Therefore,new insights into the formation mechanism of loquat fruit quality can provide a theoretical basis for regulation of the loquat fruit quality.SNP scan of loquat allows us to obtain genetic linkage markers of important trait genes,which can greatly shorten the breeding cycle.In the present study,a mutant plant that was actually derived from a mutant bud in the middle trunk of Dongting loquat(red-fleshed fruit)found in Mao County,Aba prefecture was used as the material.The wild type(TBY)was of yellowished fresh and the mutant type(TBW)was of white-fleshed fruit.Field biology,indoor physiological and biochemical tests and molecular methods were used to clarify the biological characteristics and the differences in fruit quality between TBW and TBY samples,three polymorphic bands were generated and the CDS sequences of TBW,TBY and 9 other loquat cultivars were cloned.The main results are as follows:1.The differences in phenophase,leaf and fruit quality between TBW and TBY were identified through field investigation and physiological and biochemical analysis.There was no significant difference in shooting period,early flowering season,profuse flowering season and final flowering season between the mutant and wild types,while the initial fruit maturation phase of the TBW was about 7d later than that of the TBY.Morphological indexes of TBW and TBY,including the density and depth of leaf dents,the shape of leaf apex,leaf cross-section shape and the green degree of leaf surface were very similar,while the mutant type was found with longer leaf and petiole length but thinner leaf thickness compared with the wild type.There were significant differences in appearance qualities,including fruit shape,the color of fruit peel and pulp,between TBW and TBY:TBW fruit was nearly round-shaped with yellow peel and whitish pulp,while the TBY fruit was oval-shaped with orange peel and orange-red pulp.There were some differences between the TBW and TBY in fruit pulp texture:the texture of TBW fruit pulp was very tender,while the texture of wild fruit pulp was loose;the crude fiber content of TBW pulp was 12.5%lower than that of the TBY pulp and the fruit edible rate was 5%lower than that of the TBY type fruit.Fructose,glucose and sucrose were the main components of the soluble sugar in TBW and TBY fruits.The fructose and sucrose content of TBW fruit were 14.9%and 90.9%higher than those of the TBY fruit,respectively.And the relative sweetness of the TBW fruit was 24.5%higher than the TBY.The contents of vitamin C in mutant and wild fruits were the same,which were both 4.53mg/100g.The content of beta-carotene in TBW pulp was significantly lower than that of the TBY,only 7.55%of that in the wild type pulp.In addition,TBW variation character was stable,which was verified by the analysis of fruit quality for grafted plant from TBY and TBW.2.We evaluated the fruit quality and polymorphic bands between wild type and mutant genome by ISSR,SSR and SRAP markers.No polymorphism was detected in all the bands amplified by 116 pairs of SSR primers and by 9 x 11 primer combinations in the TBY and TBW samples.Among 95 pairs of ISSR primers,40 pairs produced good amplified bands.Each primer could amplify 5?12 DNA fragments,of which the average number was 8.2.There was slightly polymorphism between the wild type and mutant samples for 7 ISSR primers(UBC813,UBC834,UBC842,UBC807,UBC854,UBC880 and UBC881),of which the bands were dim and unstable.No significant difference was observed among the other ISSR primers.The results of SSR,ISSR and SRAP markers detection showed that the genome backgrounds were almost identical between the mutant and wild types with few polymorphic fragments,which confirmed that the TBW plant was derived from a mutant bud of the TBY at the molecular level.3.Two polymorphic bands with 1369bp(assigned as TB1)and 535bp(TB2)were obtained from TBY,and they were both absent in TBW by 1SSR markers UBC854 and UBC813,respectively.In addition,the marker OPH-01/1800bp that linked with yellowish orange flesh color was also assayed in this study.The sequence of OPH-01/1800bp in TBY was 1719bp(TB3),which was also absent in TBW.A homology search against the NCBI database using a translated nucleotide query(tblastx)was performed,and Tbl might be the production of nonspecific amplification,TB2 sequences were highly related to NAD-dependent sorbitol dehydrogenase in Eriobotrya japonica,1-aminocyclopropane-l-carboxylate synthase in Pyrus and Malus domestica.TB3 had highly homologous to genes sequences with F-box proteins,S-RNase,and MdSFBB9-alpha and beta genes in Malus and Pyrus pyrifolia.4.The CDS sequences of the NAD+-SDH gene were cloned in the TBW,TBY,'Dawuxing','Huangfeng','Maomu','Dabaili','Baisha','Guifei','Xiangzhong 11','Wanzhong 518'and the 'Xinbai 8'.Three SNPs were found in the CDS sequence of the NAD+-SDH gene in the TBW pulp,compared with the TBY and the other 9 loquat cultivars,resulting in amino acid changed at all points.The function of the NAD+-SDH gene affected by the variations of the three SNPs and their coding amino acids would be explored in the future.5.The enzymic activity of NAD-SDH and the contents of sorbitol and fructose were analyzed.Results showed that,from the fruits' color change to maturity,the NAD-SDH enzyme activity in TBW loquat showed a low-high-low trend,compared with the TBY;the content of sorbitol showed a high-high-low trend;and the fructose content was always higher than that of the TBY.Real-time fluorescent quantitative PCR analysis showed that the expression level of NAD+-SDH gene in mature TBW pulp was about 5 times lower than that of TBY;NAD-SDH enzyme promoted the conversion from sorbitol to fructose in loquat fruit.Therefore,we speculated that the 3 SNPs might over activate the NAD-SDH enzyme-promoted conversion inmature TBW fruit,leading to elevated fructose content.The mRNA expression abundance decreased after transcription.NAD+-SDH gene may play an important role in the variation of the fruit color and the sugar contents in TBW loquat. |
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