Tomato Microrna Analysis and Variety Identification Using Dna Markers.pdf

1、MicroRNA analysis as well as plant variety and cultivar identification are two broad and important aspects in fundamental and applied agricultural research respectively.The need for deeper understanding of how genes enco

2、ding morphological characters and plant responses to environmental stimuli are regulated led to the discovery microRNAs whose study has grown to become a hot topic in plant science.On the other hand, the large number of

3、varieties or landraces among crop plants has made it difficult to identify and characterize varieties solely on the basis of morphological characters because they are non stable and originate due to environmental and cli

4、matic conditions, and therefore phenotypic plasticity is an outcome of adaptation.This study was conceptualized in two sections with the first dealing with microRNA research and focusing on tomato as a model crop (Chapte

5、rs 2, 3 and 4).The second section sheds light on plant variety and cultivar identification through a critical review and practical applications of some of the techniques reviewed (Chapters 5, 6 and 7).The general introdu

6、ction to the entire study is expounded in Chapter l.With regards to miRNA analysis, we endeavored to review the current status of plant miRNA focusing on the advancement of techniques and principles so as to identify res

7、earch gaps and appreciate the wealth of information generated.Using the same pattern we zeroed in on tomato (Solanum lycopersicon) as a model crop and sought to find out why there is less information on sly-miRNAs despit

8、e the apparent importance of this plant species.Furthermore, we undertook a global analysis of the tomato miRNAs deposited in miRbase in order to reveal their conservartion characteristics, target genes as well as expres

9、sion profiles in different organs tomato.We established that although most of these miRNA are conserved in the plant kingdom, a few are unique to tomato and could not be found in any of the 56 other plant species assayed

10、.In addition the degree of conservation varied between sly-miRNAs and also between similar miRNAs in other plant species whereby miR160a, miR171a, miR171c and miR172a were the most conserved whilemiR1916, miR1917, miR437

11、6 and miR5303 were unique to tomato.The target genes for the miRNAs varied with some genes being targeted by multiple miRNAs (mostly from the same family) while in some instances one miRNA targeted a number of genes as e

12、xemplified by miR159 and miR482 both of which were predicted to target many genes.Expression analysis by qRT-PCR revealed that while some miRNAs were ubiquitous in all tomato tissues, others were tissue specific which is

13、 a pointer towards target specificity.All the miRNAs were confirmed in tomato ‘Jiangshu14’ by the presence of the target bands after 3'and 5 race amplification and expression analysis.The findings of this study add to th

14、e progress in sly-miRNA investigation and provide valuable information on this important subject.Plant variety and cultivar identification is an important aspect in agricultural production and it is plagued by many probl

15、ems.To mitigate some of the drawbacks in identification of plant varieties and cultivars using morphological descriptors, scientists have developed and employed statistical tests, software and molecular markers in identi

16、fying and characterizing the required plant cultivars,varieties or other genotypes for cultivation, breeding programs as well as for cultivar-right-protection.The current status of plant variety and cultivar identificati

17、on was critically analysed, where an attempt was made to describe the principles and application of the different strategies available for this critical exercise.It was found that although the establishment of genome and

18、 transcriptome sequencing projects for many horticultural crops has led to generation of a huge wealth of sequence information and generation of an array of suitable molecular markers for a wide range of crops, there is

19、dearth of simple ways of making both morphological descriptors and molecular markers as well as statistical software and databases easy, referable and practical to use in plant identification although there are ongoing a

20、ttempts at making this possible.Limitations that present challenges in the development and utilization of modern scientific methods in variety or cultivar identification in many important crops are outlinedand the future

21、 prospects detailed.Following the results of the above analysis, practical application of the manual cultivar identification diagram (MCID) developed and used in our laboratory and which has been found to make DNA makers

22、 more usable in the identification of genotyped plant individuals was tested on tomato varieties.Based on this MCID strategy, RAPD and EST-SSR markers were used to identify a total of 42 tomato varieties from different g

23、eographical origins, with all the cultivars being clearly separated and individually identified by reproducible fingerprints of only 6 RAPD or 7 EST-SSR primers.The tomato CIDs generated in this case are both referable a

24、nd workable in the identification of any two or more tomato varieties among those studied.The information on these first versions of the tomato MCID can be enriched through identification and incorporation of more variet

25、ies and adaptation to other molecular markers so as to provide a much more comprehensive tomato variety identification service in the horticultural industry.To enrich the the information generated by the MCIDs, there was

26、 need to show the genetic closeness among the tomato varieties through examination of genetic relationships based on SSR marker polymorphism.Twenty nine tomato EST-SSR markers could clearly separate the tomato varieties

27、and the clustering outline of most cultivars was in accordance with their lineage.The average gene diversity and polymorphism information content (PIC) were 0.49 and 0.45, respectively indicating a fairly high diversity

28、and wide genetic background for cultivated tomato which is basically known to have low genetic diversity.Five variety groups were obtained using UPGMA cluster and PCA methods, with the first 3 components in the PCA scatt

29、er plot accounting for most of the variation.Only Suhong2003 and Jiahongl were uniquely classified while the rest of the accessions were grouped together in two major clusters in both the UPGMA cluster and PCA plot.There

30、 were no clear differences in geographic distribution characteristics among the 42 varieties studied.There is still a huge need for deeper analysis to compare more SSR and other markers in more varieties and crop species

31、 before drawing a final conclusion and recommendation for use in variety identification and genetic diversity analysis as well as in other studies like genetic mapping, marker assisted selection and gene discovery.In gen

32、eral, the objectives of the study were met and it is believed that the information generated adds to the increasing repertoire of knowledge on the two broad fields of agricultural research covered.There were some challen

33、ges encountered in execution of these studies and the measures instituted to mitigate them provided for interesting learning opportunities in these research areas.It is recommended that research on tomato microRNAs and t

34、heir target genes and well as their interaction mechanisms be accelerated as it appears to lag behind compared to other equally important model crop species.In addition, concerted efforts need to be placed so as to ensur