RNA-Seq analysis and development of SSR and KASP markers in lentil(Lens culinaris Medikus subsp. culinaris)

Lentil(Lens culinaris Medikus subsp.culinaris,2 n=14)is a cool-season legume with high production potential for multiple uses.However,limited molecular research has been conducted in this species owing to its large genome,which impedes the generation of genome sequences and the development of molecular markers.In this study,more than 1.37 billion filtered clean reads were collected by RNA-Seq of six diverse lentil accessions and217,836 transcripts and 161,095 unigenes were de novo assembled,yielding respectively 257.1 and 240.6 million nucleotides.The mean transcript length was 1180 bp and the N50 and N90 lengths were respectively 2075 and 479 bp.The mean length of the unigenes was 1494 bp and their N50 and N90 values were respectively 2203 and 714 bp.The unigenes were annotated against seven databases.The FLOWERING LOCUS T(FT)gene homolog in lentil showed high protein sequence similarity to the FT gene homologs of pea and alfalfa.On the basis of the RNA-Seq analysis,26,449 EST-SSR markers were designed in silico,and 276 preliminarily screened markers were selected to evaluate polymorphism in 94 diverse lentil accessions.In total,125(45.29%)of 276 EST-SSR markers were found to be polymorphic.A total of 130,073 SNP loci were detected and 78(61.41%)of 127 SNPs were successfully converted to KASP markers.Population genetic analyses of the lentil accessions with EST-SSR and KASP markers revealed similar genetic structures,suggesting that the RNA-Seq-generated resources and the developed markers are reliable for use in molecular marker-assisted breeding of lentil.

Establishment and Operation of Crop Genebank in Shandong Province

Shandong Province has abundant and diverse crop germplasm resources, but it is in lack of unified and effective preservation and management, leading to the decline and even extinction of many rare germplasm resources. In this study, the necessity for the establishment of crop genebank in Shandong Province was briefly described. In addition, the type, functions and technical indicators, as well as the operation of the crop genebank were introduced comprehensively to provide a reference for the establishment of genebank by other units or organizations.

Effects of Seed Viability and Number of Generations on Genetic Integrity of Soybean Germplasm Zhonghuang 18 by AFLP Markers

The seeds of a soybean cultivar Zhonghuang 18 were subjected to accelerated aging for 0(population G_0-1), 112(population G_0-2), 154(population G_0-3) and 196 d(population G_0-4), whose germination percentage was found to be 98.0%, 95.0%, 81.0%, and 79.0%, respectively. Then, the four populations were regenerated twice in the field. The first descendant populations were marked as G_1-1, G_1-2, G_1-3 and G_1-4, and the second were marked as G_2-1, G_2-2, G_2-3 and G_2-4, respectively. The genetic variation between the control population(G_0-1) and the experimental populations was analyzed using 12 AFLP primer combinations. The results showed that there was no significant difference in genetic similarity between the 11 experimental populations and the control population G_0-1. The genetic similarity between population G_2-4 and G_0-1 was still as high as 0.933 3, indicating that the F_2 generation of the population whose germination percentage was only 79.0% still had a high genetic similarity to the control population. The results of t-tests revealed that the populations G_1-1, G_2-1, G_1-2 and G_2-2 showed no significant difference from the control population G_0-1 in effective number of alleles per locus(Ae), genetic diversity index(H) and Shannon's diversity index(I), while these indices of populations G_0-3, G_0-4, G_1-3, G_1-4, G_2-3 and G_2-4 were significantly reduced. χ~2 tests indicated that the populations G_1-1 and G_2-1 showed little difference, and the populations G_0-2, G_0-3, G_0-4, G_1-2, G_1-3, G_1-4, G_2-2, G_2-3, and G_2-4 showed great difference in allele frequency distribution from the control population G_0-1, and the difference was greater when the seed viability was lower. Compared with the control population G_0-1, the number of rare alleles(Nr) of the populations G_0-2, G_1-1, G_2-1, G_1-2 and G_2-2 showed no significant difference, while that of the populations G_0-3, G_0-4, G_1-3, G_1-4, G_2-3 and G_2-4 declined obviously. These results revealed that compared with the control population, the genetic diversity and Nr for the descendant populations of the populations with 98.0% and 95.0% germination percentages did not change significantly, but declined greatly for the descendant populations of the populations with 81.0.% and 79.0% percentages. The results suggested that the decline in seed viability has a greater impact than the number of generations on genetic structure of soybean germplasm. So, it is suggested that soybean seed with an initial germination percentage of 98.0% should be regenerated before its germination percentage declines to 81.0%.

Effects of Seed Vitality and Regeneration on Genetic Integrity in Soybean by SSR Markers

The seeds of Zhonghuang 18 were selected as a test material,and subjected to artificial aging treatment(0,112,154 and 196 d),obtaining four 4 populations,i.e.,G_0-1,G_0-2,G_0-3 and G_0-4,the germination rates of which were 98.0%,95.0%,81.0%and 79.0%,respectively.The four populations were reproduced twice in field,giving four populations of the first reproduced generation G_1-1,G_1-2,G_1-3 and G_1-4 and four populations of the second reproduced generation G_2-1,G_2-2,G_2-3 and G_2-4.The results showed that the number of alleles(Ae)per locus and genetic identity of all the treatment populations did not change significantly compared with the control population G_0-1,and population G_0-4 still shared 0.996 2 genetic identity with the control population,indicating that the genetic identity between the population with a germination rate of 79.0%and the control population was still high.The results of t test showed that populations G_0-2,G_1-1 and G_2-1 showed number of alleles per locus(A),genetic diversity index(H)and Shannon index without significantly differences from the control population G_0-1;populations G_1-2 and G_2-2 had the number of alleles per locus(A)significantly decreased;and the above genetic diversity parameters of populations G_0-3,G_0-4,G_1-3,G_1-4,G_2-3 and G_2-4 decreased significantly or very significantly.The results ofχ~2test showed that there were almost no differences in the allelic frequency distribution between populations G_0-2,G_1-1 and G_2-1 and the control populaiton G_0-1;and populations G_0-3,G_0-4,G_1-2,G_1-3,G_1-4,G_2-2,G_2-3and G_2-4 differed in allele frequency distribution,and the lower the vitality level,the greater the differences.Compared with the control population G_0-1,populations G_0-2,G_1-1 and G_2-1 had no significant changes in number of rare alleles,while populations G_0-3,G_0-4,G_1-2,G_2-2,G_1-3,G_1-4,G_2-3 and G_2-4 decreased significantly in number of rare alleles.The above results showed that compared with the control population,the progeny populations reproduced from the population with a germination rate of 98.0%had significant changes in genetic diversity and number of rare alleles,while the values of the progeny populations reproduced from populations having germination rates of 81.0%and 79.0%,respectively,decreased significantly,and the number of alleles per locus and number of rare alleles of the progeny populations reproduced from the population with a germination rate of 95.0%began to decrease.The decline in viability has a greater effect on the genetic structure of soybean germplasm populations than reproduction generation.It is recommended that the germination rate standard for regeneration of soybean germplasm with an initial germination rate of 98.0%should not be lower than 81.0%.

Investigation and Analysis of Crop Germplasm Resources in Coastal Areas of Shandong Province

This study focused on the investigation of crop germplasm resources in coastal areas of Shandong Province, including132 villages in 82 towns of 34 counties. The survey collected local varieties and wild resources of grain crops, economic crops,vegetables and fruit trees, and a total of 848 samples were collected, belonging to 54 species of 39 genera in 15 families. In this study, the current situation and growth and decline conditions of crop germplasm resources were investigated, and their botanical classification and utilization importance were analyzed. Furthermore, the conservation, development and utilization of crop germplasm resources in coastal areas of Shandong Province were also discussed in this paper.

Genetic Diversity of Clover by SRAP

In the present study,genetic diversity and genetic relationship of 11materials including red clover ( Trifolium pretense L.),white clover ( T.repen L.) and alsike clover ( T.hybridum L.) and leaf-type variants of white clover were investigated by SRAP (sequence related amplified polymorphism).Forty SRAP primers were screened,and 792 reliable bands were amplified,among which 426 (53.8%) were polymorphic.The number of polymorphic bands per pair of primers ranged from 3 to 38 with an average of 10.6.SRAP cluster analysis showed that the similarity coefficients between white clover materials were from 0.465 to 0.997 with an average of 0.812,indicating that there was certain genetic diversity among white clover.Specific bands appeared in white clover variants,indicating that there were certain differences in DNA sequence between normal while clover plants and their leaf-type variants.The similarity coefficients between red clover materials were from 0.457 to 0.827 with an average of 0.597,indicating that the different red clover germplasms had genetic diversity at the DNA molecular level.The results also showed that the genetic relationship of the alsike clover with the red clover was closer than that with the white clover.This study shows that SRAP technique can be effectively used for the analysis on interspecific and intraspecific relationship,germplasm resource identification and genetic diversity of clover.

Experimental Insights on the Structural Patterns and Their Formation Mechanisms of the Xujiaweizi Fault Depression in the Songliao Basin

The Xujiaweizi （XJWZ） fault depression is a primary area for deep-seated gas explo- ration in Daqing. Through seismic profile interpretation, the main sag-controlling fault and struc- tural characteristics in the XJWZ fault depression were analyzed based on folds and faults analysis. A three-dimensional geological model of the XJWZ fault depression was also set up to enable fur- ther discussion by means of structural physical modeling. According to our research, the basic structural framework of the XJWZ fault depression was the result of regional extension. Its forma- tion and evolution were mainly controlled by the Xuxi fault. The structural framework of S-N blocking was due to changes in the fracture attitude. The Xuzhong uplift and the depressions on both sides belong to a normal-fault-related fold and were formed by controlling of the ramp/flat normal fault in the same tectonic movement period.

Gene mapping of a new coat mutation in mouse

Gene mapping of a mouse coat mutation has been investigated. First, 100 10-bp random primers were used to amplify DNA, but the mutation could not be located by this method because there were no correlation between the amplified products and coat phenotypes. Second, by using ldh1 , Car2, Mup1 , Pgb1 , Hbb, Es1O, Es1, Mod1, Gdc1 , Ce2, Es3 as genetic markers, linkage test crosses (two-point test) consisting of intercrossing uncovered BALB/c mice (homozygotes) to CBA/N and C57BL/6 mice with normal hair and backcrossing the heterozygotes of the F1 to the uncovered BALB/c mice were made. It was soon evident that the mutation was linked to Es3 on chromosome 11. Furthermore, three-point test was made by using Es3 and D11 Mit8 (a microsatellite DNA) as genetic markers. The result showed that the mutation was linked to Es3 with the percentage recombination of (7.89 ± 2.19) %, and linked to D11Mit8 with the percentage recombination of (26.30 ± 3.57)%. The percentage recombination between Es3 and D11Mit8 was