| Black locust(Robinia pseudoacacia L.)belongs to Robinia genus of Legume family.It is a broadleaved tree species,with resistance to drought,poor site coditions,and mild alkali.Particularly it has a strong natural regeneration ability,making it the pioneer species for afforestation in barren hills,and it is good for water and soil conservation.It is suitable for a variety of soil and climate conditions,with high values for uses as ornamental,timber production,ecological protection,honey source and fodder.Robinia pseudoacacia L.is an exotic species,which was first introduced to Qingdao by Germen in the late 19 th century.It has been cultivated for nearly 120 years,gradually expanding from Qingdao to 27 provinces(municipalities and autonomous regions in China).The ecological characteristics,growth habit,adaptability(drought and salt resistance),asexual and sexual reproduction,etc.,have always been the focus of attention by Chinese as well as foreign researchers.This study was intended to analyze genetic diversity of the genetic resources in China.But its genetic background information is relatively scarce,and its genetic basis research is weak.China's regional culture source of black locust clones have no detailed informations,without clear genetic relationship.And its genetic diversity leval is also still unclear.Meanwhile synonyms phenomenon occurs frequently.Therefore,exhaustiv surveys on genetic diversity and construction of fingerprint maps for black locust germplasm are of importance and will lay a solid foundation for the furture breeding,genetic improvement and variety identification.In this study,the main purpose is to clarify the genetic diversity and build fingerprint maps for a large germplasm diversity panel of black locust collected in China and globally by use of a set of thorough phenotypic surveys and SSR markers.As well,RNA-sequencing was applied here to expaned the resource of SSR marker in black locust.The results of this study are as follows:(1)An comprehensive full-year-spaning surveys on phenotypes,including flowering phenology,legume phenology and morphology of leaves,flowers,fruit and thorns were conducted for 75 clones from the United States,Shandong and Liaoning,.Phenotypic variations among individual clones and geographic levels were analized,and showed that no significant relationships existing between geographic index and phenotypic variations,while most of the indexes within clones were significant.The positive correlation of seed setting rate and legume index and flowering phenology is greater than that of fruit growth.We infer that flowering period has important effects on fruit development.Growth period traits negatively related to flowers and the plump seed rate,showing that the clones growing fast after sprouting,especially the earlier flowering clones,has obvious advantages in terms of amount of flowers and seed setting rate.Through the analysis of average thorn length we found that the longest average stem thorn length was in Shandong,while the order of average branch thorn length of was American > Shandong > Liaoning.The largest variation coefficient of branch thorns was 84.82%,in the U.S.region,and the relative minimum stem thorn variation was 43.63%,in Liaoning.Correlation analysis was carried out by comparing the correlation between branch thorns and stem thorns,the results are all significant at the 0.01 level.Flowering traits variation coefficient analysis of three years from 2014 to 2016 showed that the variation coefficient of the same characters changed obviously in different years,which is likely to be affected by environmental factors such as rainfall and temperature.(2)Transcriptome sequencing and gene function annotation were conducted obtaining 12.2 Gb transcriptome data in inflorescences and leaves,gotting 141 948 Unigenes,the average length was 1209 bp,N50 was 1997 bp.These unigenes were compared to 4 databases,Nt,Swiss-Prot,KOG and KO,respectively.72% unigenes were compared to Nt.35423 EST-SSR loci were detected in the 141 948 Unigenes,the detection rate 24.95%.There was a SSR locus in average 4.84 kb.Two nucleotides,three nucleotides and five nucleotide were advantage repeats.The distribution law of SSR initiation site was analysed,focused on 0~2 000 bp.Microsatellite length ranged of 12~84bp,with an average of 16.5 bp,concentrated in a relatively short sequence.The second and third nucleotide repeats were the main kinds of motif,GA/CT and GGC/CCG respectively.Only two and three nucleotides repeated microsatellite repeats were negatively related between primitive abundance and length.A significant negative correlation was showed on SSR frequency and length of the SSR series,the correlation coefficient was-0.500.(3)25 882 specific primers were developed,the successful developing primer rate was 73.07%.30 primers were pick out,and the research on the effectiveness and generality of primers were conducted.The successfull amplification rate was 80%.9 of them were highly polymorphic primers,mostly in UTR region.The molecular identification rate of 385 clones was 95.32%.The commonality detection results showed that 9 of primers used can distinguish three species of Robinia,and has obvious polymorphism.(4)A total of 68 alleles in 383 clones were identified using 8 pairs of SSR primers,with a mean of 8.5 alleles per locus,ranging from 4~17.The value of PIC(polymorphism informative of content)in different loci ranged from 0.146 to 0.898,with the average of 0.527 9.(5)A total of 66 alleles in 9 different regions were identified using 8 pairs of SSR primers,with a mean of 8.25 alleles per locus,ranging from 4~16.The average number of alleles of 9 different regions was 5.617,the average value of eight domestic regions is 5.597,6.00 in Shandong.The actually observed heterozygosity(Ho)varied between 0.479(Liaoning)and 0.555(Shandong),with a mean of 0.519,and the Ho value in Shandong was slightly higher than the domestic eight regional average of 0.514.Average expected heterozygosity(He)varied from 0.509(Liaoning)to 0.553(USA),the He average value is 0.533,and the He in Shandong is 0.548,slightly higher than the domestic eight regional average of 0.533 as well.Nine regional He values were greater than 0.5,showing that genetic diversity are relatively abundant.Shandong and shanxi Ho/He was greater than 1,showing that the clones in the two regions had higher heterozygosity.The above data indicate that the genetic diversity level of Shandong clones is slightly higher than the domestic mean level,and the genetic diversity is relatively abundent.Shandong,Gansu and other regions have private alleles,so these clones can be selected as parent when cross-breeding;Introduction can be appropriately carried through between these different provinces,in order to increase genetic diversity within the region.Genetic variation analysis of 318 clones in nine regions showed that only 2% variation comes among regions,8% comes among the clones,90% within the clones.Different regional principal coordinates analysis and the related analysis results are basically identical.Shandong,the United States,Liaoning,Henan and Shanxi are close relatives,get together for a big group;Gansu and Hebei are close relatives,get together for a group;Shanxi and Beijing are close relatives,get together for another group.The minimum genetic distance is 0.011,between Shandong and the United States,illustrating the two smaller regional differences of clonal genetic differentiation.(6)Clustering results based on SSRs and phenotypic differences of 75 clones from Shandong,the United States and Liaoning,showed that phenotypic data and SSR data were not relevant.(7)A total of 51 alleles in 49 clones from Shandong Province were identified using 8 pairs of SSR primers,with a mean of 6.375 alleles per locus,ranging from 2~15.The mean value of PIC was 0.509 8,ranging from 0.092~0.879.The results of clustering analysis showed that 49 clones did not cluster together strictly in accordance with cultivation areas.The molecular identification rate of Primers Rply109 and rops16 for 49 clones was 91.84%,so they can be used as efficient molecular markers in fingerprint construction and molecular identification.(8)Molecular identification system for black locust clones was established based on the technology of SSR markers.Here,378 clones can be identifed by the 18 primers,and the molecular identification rate was 96.36%.The NJ analysis results of the collected 383 clones,Using the 18 primers showed that all species did not strictly cluster together according to the different geographic origins,but it has a certain regularity.(9)The results showed that the fluorescent SSR capillary electrophoresis detection technology is a anvenu of simple,rapid and efficient method for the preliminary identification of variaties ether polyploidy or not.Here,293 clones with three to six allelic variations could be detected by primers RP13.The polyploid detection rate is as high as 76.1%.So RP13 was determined to be an efficient molecular markers for fingerprint construction and molecular identification,especially for the polyploid detection of black locust.(10)At last,building fingerprints of 385 clones using the 18 fluorescent SSR primers gives an unexpected results that 343 clones had one or more locus(the maximum is 7)with 3 ~ 6 polymophic alleles,and the polyploid rate was as high as 89.09%. |
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