Study On DUS Test Techniques And Molecular Mechanism Of Stress Resistance Of Common Vetch (Vicia Sativa L.) Varieties

The DUS(Distinctness,Uniformity,and Stability)test has dramatically protected the original innovation of breeding and improved the enthusiasm of breeders in variety selection.Since China joined UPOV(International Union for the protection of new varieties of plants)to implement the plant variety protection system,more than 200DUS test guidelines were developed,but the DUS test guidelines of common vetch(Vicia sativa L.)has not been developed.As an excellent forage,common vetch suitable for cultivation in temperate regions,and plays an important role in the grassland agricultural system of China.In recent years,with the continuous promotion of the common vetch breeding progress,it is critical to developing a China situated DUS test guideline of common vetch.Thus,a total of 48 common vetch varieties were collected from worldwide as experimental material to study the graded and genetic diversity of34 characteristics in common vetch.Correspondingly,the core technology of the common vetch DUS test,including characteristics selection and reference varieties determination were studied,and the DNA fingerprint was constructed by molecular technology.In addition,according to the drought tolerance of common vetch,and the cold resistance characteristics of the breeding status in China,transcriptome sequencing technology was used to study the mechanism of common vetch adapt to drought and cold stresses.The main results are as follows:1.Two years of field experiment was conducted,a total of 34 characteristics of48 common vetch varieties were measured,graded,and analyzed genetic diversity.According to the UPOV technical document TG/1/3,four qualitative characteristic(QL),five pseudo-qualitative characteristic(PQ)and nine quantitative characteristic(QN)were graded according to their expression status.The least significant difference method(LSD)was used to divide 16 measured QNs into 1,3,5,7,and 9 continuous grade ranges.The variation coefficient of QL and PQ was 18.52⁵5.49%,the average value was 34.94%.The distribution frequency of the characteristics indicated that the varieties of common vetch mainly consisted of violet flowers(92.31%),shattering rate(86.21%),gray-brown cotyledon color(79.41%),concave leaf apex(55.77%)and oval seeds(52.94%).The 25 QNs were divided into 9 visual characteristics and 16 measured characteristics.The variation coefficient of the visual QNs range from 19.31%to55.49%,with an average value of 41.79%;the variation coefficients of the measured QNs between two years are 11.50%to 35.43%and 11.80%to 35.40%;the average values are:21.09%and 21.36%.The correlation analysis of the measured characteristics showed that a significant positive correlation between the leaf width and ratio length/width of leaflet;width of second primary leaf and ratio length/width of the second primary leaf;natural height and absolute height;natural height and stem thickness;absolute height and stem thickness;time of beginning of flowering and pod length;pod length and pod width,pod length and length of beak(P<0.001).While,a significant negative correlation between leaf length and leaf width;length of second primary leaf and width of second primary leaf;natural height and lodging index(p<0.001).The results of principal component analysis showed that the first four principal components could reflect 78%of the variety information.2.The TG/1/3,TG/32/7 and other technical documents were used as guidelines to study the core technical content of the common vetch DUS test,which includes test characteristics and reference varieties.We evaluated 31 test characteristics,including23 UPOV common vetch DUS test characteristics,and eight new characteristics representative of the distinctness feature.The expression status and the observation period/method/number of each characteristic was scientifically graded and accurately described.Finally,a total of 32 reference varieties(21 international varieties and 11varieties from China)corresponding to 123 expression states were screened.We further complemented the expression states of“ground color of testa”and“seed shape”in the UPOV common vetch DUS test guideline.3.The potential transcription factor(TF)genes of common vetch were identified using the transcriptome sequence data.Two hundred and eight pairs of SSR primers were designed from 1,816 TF sequences,and 35 pairs of primers were screened out with a high polymorphism.The 35 pairs of SSR primers amplified 276 alleles in 30common vetch varieties,with an average of 7.91 alleles per marker;the polymorphic information content(PIC)ranged from 0.06 to 0.90,with an average of 0.68.Both of the STRUCTURE and UPGMA clustering analysis showed that the same geographical origin of common vetch varieties usually shared a high genetic similarity.Through the efficiency of primer identification analyze,VsTFSSR-157,VsTFSSR-111,and VsTFSSR-101 were chosen as core primers.Based on three pairs of core primers,the fingerprints of common vetch were constructed,and a unique fingerprint code was established for each variety,and a QR code was also formed.This study can provide a technical basis for variety approval,similar variety selection and genetic background analysis of common vetch.4.Through Pacific Biosciences and Illumina RNA-Seq sequencing technology,a total of 39,709 non-redundant full-length transcripts were obtained,and the first common vetch three-generation full-length transcript database was constructed.Under drought stress treatment,a total of 30,427 full-length transcripts were identified,with an average length of 2,278.89 bp,annotation ratio of 98.01%.A total of 3,464 and 3,062differentially expressed genes(DEGs)were identified in the leaves and roots,respectively.GO enrichment analyses identified that the dehydrin and?1-pyrroline-5-carboxylate synthase genes were induced for the biosynthesis of proline and water conservation.The KEGG enrichment analysis results indicated that the DEGs were significantly enriched in hormone signal transduction,starch and sucrose metabolism,and arginine and proline metabolism pathways.Abscisic acid(ABA;the AREB/ABF-SnRK2 pathway)regulates the activity of AMY3 and BAM1 to induce starch degradation in leaves and increase carbon export to roots,which may be associated with the drought stress responses in common vetch.In transgenic yeast,the overexpression of ATNAC3(F01.PB8282),ERF107(F01.PB3413),ANAC019(F01.PB9565)and ATMYB59(F01.PB10800)increased yeast tolerance to osmotic stresses.5.Under cold stress treatment,a total of 30,525 full-length transcripts were identified,with an annotation ratio of 98.09%,the average length of 2,291.56 bp.Among these transcripts,7,689 and 3,415 DEGs were identified in leaves and roots,respectively.The GO enrichment results displayed that the DEGs were significantly enriched in the photosynthetic related categories in leaves.Correspondingly,the“Response to cold”category was enriched considerably in roots,and most of them were upregulated.The KEGG enrichment showed that Ca²⁺,redox,hormonal signaling,circadian clock,and photosynthesis-antenna proteins play an important role through CBF cold-response dependent or independent transcriptional pathways to response cold stress in common vetch.Besides,TFs,chlorophyll AB binding protein,CTP synthetase N-terminal protein,dehydratin and caspase inhibitor protein were extensively induced in cold stress.Heterologous expression of ATCYS6(F01.PB10156),CYSB(F01.PB322),RCI2B(F01.PB5382),ATWRKY33(F01.PB13794),BHLH130(F01.PB9425)and RAP2.1(F01.PB372)can improve transgenic yeast cold stresses tolerance,suggested that these DEGs play a vital role in response to low-temperature stress in common vetch.