Breeding and Seed Reproduction Techniques of a New Tetraploid Common Buckwheat Variety‘Pintianqiao 3’

[Objectives]This study was conducted to actively carry out the breeding of new tetraploid common buckwheat varieties and its supporting breeding techniques.[Methods]Pintianqiao 3 is a new tetraploid common buckwheat variety developed by College of Agriculture of Shanxi Agricultural University and Agricultural Genetic Resources Center of Shanxi Agricultural University,using‘Pintianqiao 1’as the parent,through mutation treatment with 0.2%colchicine aqueous solution,grain selection,plant selection,isolation and identification,variety comparison,regional test and field investigation.The variety has chromosomes 2n=4X=32,and shows a spring sowing period of 101 d and a summer sowing period of 80 d,large flowers and seeds(with a 1000-grain weight of 41.4 g),and good resistance to lodging.[Results]From 2021 to 2022,Pintianqiao 3 participated in the independent joint regional test of common buckwheat varieties in Shanxi Province,and the average yield in 10 test positions was 1.8 kg,equivalent to 1800 kg/hm^(2),which was 8.4%higher than the control.It passed the field investigation conducted by Shanxi provincial expert group for identification of non-major crop varieties in Dongyang and Kelan experimental sites on September 2-3,2022.On January 4,2024,it passed the preliminary examination of Shanxi Provincial Crop Variety Approval Committee.The seed reproduction technique of Pintianqiao 3 including land selection,preparation before sowing,sowing,field management and timely harvesting has been developed.[Conclusions]This study provides technical support for the demonstration and popularization of this new variety.

The lectin gene TRpL1 of tetraploid Robinia pseudoacacia L.response to salt stress

Lectins are natural proteins in animals,plants,and microorganisms and can be divided into 12 families.These lectins play important roles in various environmental stresses.Some polyploid plants show tolerance to environmental stresses and to insect pests.However,the mechanism of stress tolerance is unclear.Tetraploid Robinia pseudoacacia(4×)under salt stress showed higher tolerance than diploid R.pseudoacacia(2×).As lectin can improve stress tolerance,it was questioned whether the stress resistance of polyploid plants was related to the lectin protein.In this study,salt resistance of lectin gene TRpL1 was verified by its over-expression in plants.In addition,salt resistance of lectin protein by E.coli strains was detected.The data revealed that the over-expression transgenic plants of TRpL1 showed better salt tolerance than control plants under salt stress,and the TRpL1-expressing strain also grew better in the medium with added NaCl.Therefore,tetraploid plants can resist salt stress through TRpL1 protein regulation.

Comparative Analysis of the Transcriptome and Metabolome in Leaves of Diploid and Tetraploid Fagopyrum tataricum

Tartary buckwheat(Fagopyrum tataricum)is a dual-purpose medicinal and food crop grown for its high contents of functional compounds and abundant nutrients.Although studies have shown the differences of total flavonoid content in Tartary buckwheat at different ploidy levels,the composition of flavonoid and its regulatory mechanisms are largely unknown.In this study,the leaf metabolome and transcriptome of diploid and tetraploid accessions of Tartary buckwheat were analyzed to gain insight into the impact of polyploidization on comparative secondary metabolite composition and molecular regulatory mechanism.Based on a widely targeted metabolomics analysis,a total of 792 metabolites were identified,including 127 flavonoids.The accumulation of 127 metabolites and expression of 3871 genes differed significantly between diploid and tetraploid Tartary buckwheat.Integrated metabolomics and transcriptome analysis revealed that chromosome doubling up-regulated the expression of upstream genes in the flavonoid biosynthesis pathway to promote the accumulation of flavonoids.The present results contribute to elucidation of the molecular mechanism of phenotypic variation associated with polyploidy in Tartary buckwheat.The findings provide a reference for further studies on phenotypic traits in polyploid Tartary buckwheat,the cloning of crucial regulatory genes,and utilization of genetic engineering technologies in the breeding of Tartary buckwheat.

Construction and Analysis of Asexual Mutant Population of Tetraploid Potato Longshu 7 Induced by EMS

[Objective] This study was conducted to detect the variation induction ability of ethyl methanesulfonate （EMS）, to construct a mutant population. [Method] 0.6%, 1.2% and 2.4% EMS solutions were used for inducing 2 400 stem segments of test-tube plantlets of tetraploid potato Longshu 7, and an asexual mutant population including leaf, plant type, stem, stolon, glandular hair traits was constructed. [Result] Twenty two mutation types, i.e. etiolated plantlet, albino plantlet, leaf-color gradient, etiolated vein, wrapped bud, lobed leaf, crinkled leaf, deformed compound leaf, cluster, branch, top enlargement, top bifurcation, succulent, stolon distortion, stolon shift, early tuberization, hair stolon, stem enlargement, vine stem, foliage stem, glandular hair and whorled leaf types were observed in total. There were 52 mutant single plants in total, with a mutation rate of 21.67‰. [Conclusion] The glandular hair, clustered short-stem, branched stem and early tuberization types of mutants have higher application value, and are anticipated to be used in potato functional genome research, gene improvement and germplasm innovation.

Study on Relation between Endogenous Phytohormones and Ovule Abortion in Tetraploid Robinia pseudoacacia

This study was conducted to investigate ovule abortion of tetraploid Robinia pseudoacacia and its reasons. It was found that in the ovule development period of tetraploid R. pseudoacacia from 15 d after coronal opening, the levels of growth-promoting endogenous hormones in abortive ovules were lower than those in normal ovules, and the level of a growth-inhibiting hormone was always higher than those in normal ovules, indicating that ovule abortion of tetraploid R. pseudoacacia might be related to abnormal endogenous phytohormones in ovules. This study laid a foundation for further study on highly sterile phenomenon of tetraploid R. pseudoacacia seeds.

Research on Pollen Grains of Tetraploid Lines and Diploid Control Line of Chrysanthemum cinerariifolium (Trev.) Vis.

[Objective] The aim was to study characters of pollen grains of tetraploid lines and diploid control line of Chrysanthemum cinerariifolium (Trev.) Vis.,morphological characters,fertility of pollen grain and germination percentage of seeds. [Method] Pollen grains were prepared by sulphuric acid-acetyl oxide decomposition method. The lengths of polar axis and equatorial axis of pollen grains were determined with general optical microscope. The morphology of pollen grains was observed with SEM (scanning electron microscope) and the typical visual fields of 2 500× (or 2 000×),7 000× were taken pictures. [Result] Comparing with the diploid control line,the pollen grains of five tetraploid lines which were tested were different from the diploid line in morphology,sculpture,etc.. 4 of the 5 tested samples were significant larger than the diploid line in size and one was similar to the diploid line. [Conclusion] This research provided references for breeding tetraploid improved varieties of Chrysanthemum cinerariifolium (Trev.) Vis. with good fertility and high germination percentage.

The Quadraplex Tetraploids Hybrids and Duplex Tetraploids Hybrids Are Responsible for Heterosis and Inbreeding Depression in Maize

The maize quadraplex tetraploids and duplex tetraploids were developed using Kato’s protocols. The phenotype ofheterosis and inbreeding depression over generations in their parents and progenies of F1, F2 and F3 were investigated.The results indicated that different duplex tetraploids have different genetic backgrounds, but they acquire maximumheterosis at same traits, such as the leaf length, leaf width, culm circumference and days to flowering. P.N. rises muchfaster from the F2 to F3 segment than the A.W. does for the plant height in duplex tetraploids. In comparing duplex andquadraplex over a generation the quadriplex is showing the greatest heterosis in plant height, leaf height, leaf width anddays to flowering. Most of the examples achieve the maximum heterosis at Qu F2, with the exception of culm circumference,which achieves greatest heterosis at PNAW F1. Meanwhile, this experiment shows that quadraplex tetraploids has distinctadditional favorable alleles that are not contained in duplex tetraploid, this is demonstrated by the heterosis found incrosses between the two duplex tetraploid. This finding helps explain quadraploids superiority and unique breedingbehavior, in which, the progressive heterosis and inbreeding depression in maize are due mainly to linkage disequilibrium.The severe inbreeding depression in duplex tetraploids is due mainly to the rapid loss of complementary chromosomes orgenes interactions in the first few generation of inbreeding. Correspondingly, the progressive heterosis in quadraplextetraploids is due mainly to a progressive increase in complementarities of homologous chromosomes or genes interactions.Greater complementarities of homologous chromosomes or genes interactions in tetraploids maize alse helps explainrecent molecular biology research indicating that some of traits in quadraplex tetraploids are more responsive to geneticdiversity than in duplex tetraploids. In addition, the dosage effect of polyploid in relation to the genetic basis of heterosisand inbreeding depression were discussed also.

Comparison Between a Tetraploid Turnip and Its Diploid Progenitor(Brassica rapa L.):The Adaptation to Salinity Stress

Polyploidy is pursued in plant breeding programs due mainly to its ability to yield larger vegetative or reproductive organs. In controlled growth chamber experiments, a tetraploid turnip （cv. Aijiaohuang, 4n） and its diploid progenitor （cv. Aijiaohuang, 2n） were evaluated for their tolerance to salinity stress via investigations on a group of physiological parameters. The results indicate that the tetraploid turnip exhibit better adaptation to a high concentration salt medium （200 mmol L-1）, as evidenced by a less-affected germination rate and a healthier morphological appearance at the seedling stage. Furthermore, an extension of salinity stress up to a certain period of time at the 5-7-leaf stage shows differences between the tetraploid turnip and its diploid progenitor. The former had a higher K＋/Na＋ ratio in the roots, higher glutathione concentration and antioxidant activities in the leaves, and smaller reductions in photosynthetic capacity in terms of leaf chlorophyll content. Studies on the differences between an autopolyploid and its respective relative, from which the autopolyploid originated, in terms of their tolerance to salinity and/or other abiotic stresses, have remained rather limited. The comparison is interesting due to a homogenous genetic background.

Mapping QTL underlying tuber starch content and plant maturity in tetraploid potato

Tuber starch content and plant maturity are two important agronomic traits of potato. To investigate the complex genetic basis of these traits in the cultivated potato, as well as the relationship between them, we developed a linkage map in a tetraploid population of 192 clones derived from the cross Longshu 8 × Zaodabai and mapped quantitative trait loci(QTL) for tuber starch content and plant maturity using data collected in three diverse environments over two years. We detected eleven QTL for tuber starch content distributed on seven chromosomes, of which four, on chromosomes I, II, and VIII, were expressed in at least three environments. For plant maturity, we identified six QTL on chromosomes II, IV,V, VII, and XI, one of which, on chromosome V, showed LOD peaks ranging from 45.2 to 62.5 cM and explained 21.6%–26.6% of phenotypic variation was expressed in five of the six environments. Because the reproducible QTL for plant maturity and tuber starch content mapped to different chromosomes and neither overlapping QTL, nor any genetic interaction between QTL were detected, we infer that tuber starch content and plant maturity are controlled by independent genetic loci. This inference supports the prospect of breeding potato for both early maturity and high starch content.

Analysis of three types of triterpenoids in tetraploid white birches(Betula platyphylla Suk.) and selection of plus trees

Betulin, oleanolic acid, and betulinic acid are naturally occurring pentacyclic triterpenoids that have significant medicinal value. Considerable amounts of these triterpenoids are available in the outer bark of white birch. In this study, we used ultrasound-assisted extraction （UAE） to extract triterpenoids from birch bark rapidly and with high efficiency. Using high performance liquid chro- matography （HPLC）, three types of triterpenoids were separated and detected. We examined the differences among triterpenoids extracted from diploid versus tetra- ploid white birch. Then, we used factor analysis to screen out tetraploid white birches with comprehensively excel- lent performance. The results indicate that the optimum conditions for extraction include the use of ethanol as an extraction solvent, a solid-to-liquid ratio of 0.1 g/10 ml, ultrasonic power set at 100 W, a temperature of 60 ℃ and an extraction time of 15 min. A reversed-phase C18 col- umn （4.6 mm × 250 mm × 5 μm） with a column tem- perature of 30 ℃ and the mobile phase composed of A （acetonitrile） and B （0.1% aqueous phosphoric acid, v/v） at a flow rate of 0.5 ml/min were used, and the detection wavelength was 195 nm. No significant difference wasobserved between diploid and tetraploid white birch in terms of the content of three types of triterpenoids （at a confidence level of 0.05）. As triterpenoid content, height, and DBH （diameter at breast height） are strongly interre- lated, we used factor analysis to evaluate all individuals, and we screened out six plus trees with excellent com- prehensive characters.

Effects of different supplements on tetraploid black locust (Robinia pseudoacacia L.) silage

Preparation of silage is a common method to preserve green forage. It plays an important role in improving forage utilization, solving the problem of forage provision and meeting the nutritional needs of livestock in winter and spring. The effects of various supplements on tetraploid black locust （Robinia pseudoacacia L.） silage were studied by analyzing its color, odor, texture, pH value, the composition and amount of organic acids, the ammonia-N/total-N ratio, crude protein, neutral detergent fibers and acid detergent fibers. Our results show that the silage quality of wilted tetraploid R. pseudoacacia preserved alone is acceptable, while adding brown sugar and Yishengkang to tetraploid R. pseudoacacia foliage improved its quality. The treatments consisting of the tet- raploid R. pseudoacacia mixed with 33% and 50% corn stalk achieved the best silage quality in terms ofpH value （p 〈 0.01）, amount of lactic acid （p 〈 0.01）, the ammonia-N/total-N ratio （p 〈 0.01） and the fermentation quality. These treatments also resulted in a high content of crude protein and lower amounts of acid detergent fibers. The treatment consisting of the tetraploid R. pseudoacacia mixed with 20% corn stalk was also good in the quality of its fermentation.

High density genetic map and quantitative trait loci(QTLs)associated with petal number and flower diameter identified in tetraploid rose

Rose is one of the most important ornamental and economic plants in the world.Modern rose cultivars are primarily tetraploid,and during meiosis,they may exhibit double reduction or preferential chromosome pairing.Therefore,the construction of a high density genetic map of tetraploid rose is both challenging and instructive.In this study,a tetraploid rose population was used to conduct a genetic analysis using genome sequencing.A total of 17382 single nucleotide polymorphism(SNP)markers were selected from 2308042 detected SNPs.Combined with 440 previously developed simple sequence repeats(SSR)and amplified fragment length polymorphism(AFLP)markers,a marker dosage of 6885 high quality markers was successfully assigned by GATK software in the tetraploid model.These markers were used in the construction of a high density genetic map,containing the expected seven linkage groups with 6842 markers,a total map length of 1158.9 c M,and an average inter-marker distance of 0.18 c M.Quantitative trait locus(QTL)analysis was subsequently performed to characterize the genetic architecture of petal number and flower diameter.One major QTL(qpnum-3-1)was detected for petal number in three consecutive years,which explained 20.18–22.11%of the variation in petal number.Four QTLs were detected for flower diameter;the main locus,qfdia-2-2,was identified in two consecutive years.Our results will benefit the molecular marker-assisted breeding of modern rose cultivars.In addition,this study provides a guide for the genetic and QTL analysis of autotetraploid plants using sequencing-based genotyping methods.

Agronomic characterization and genetic analysis of the supernume-r ary spikelet in tetraploid wheat(Triticum turgidum L.)

The supernumerary spikelets（SS）characters of tetraploid wheat（Triticum turgidum L.）resulting in more spikelets and kernels per spike,thus enhancing sink capacity may contribute to potential wheat yield improvement.In order to investigate the effect of different SS types on agronomic characters and understand the genetic base of SS phenotype in tetraploid wheat,near isogenic lines（NILs）,bh-50 with normal spikelets（NS）,bh-51 with four-rowed spikelets（FRS）,bh-52 with short-ramified spikelets（SRS）,and bh-53 with long-ramified spikelets（LRS）in a Triticum durum cv.ZY1286 genetic background were developed by continuous backcrossing.Agronomic characters showed that the SS phenotype lines,bh-51,bh-52and bh-53 have significant increase in the number of spikelets and grains per spike compared with the NS phenotype line bh-50（P〈0.05）,and bh-53 line showed much more increase than those of bh-51 and bh-52.However,bh-53 had the lowest grain weight and the longest spike development stage than those of other spike phenotypes.These results indicated that the different SS types have different effects on the agronomic and spike characters.Genetic analysis through bh-50/bh-51and bh-51/bh-53 F2 populations showed that a recessive major gene controlled the spike architecture to transform from NS to FRS,and a dominant major gene determined the change of spike phenotype from FRS to RS.DNA sequences of Tt BH/WFZP ortholog on chromosome 2AS revealed that a single nucleotide polymorphism（SNP）substitution happened in the open reading frame（ORF）region of all the SS tetraploid wheat accessions,which may lead to the generation of lateral meristems between glume and lemma during the immature spike development.While the fates of the lateral meristems,developing into lateral spikelets or branched spikelets,may be determined by another major gene.Our results presented here may advance our understanding and knowledge of the genes and genetic pathways determining the spike architecture development in wheat.

Expression of the Gene Encoding the Tetraploid of Carboxyl-terminal Peptide of β-hCG Containing Thirtyseven Amino Acid Residues in E.coli

This study was carried out to investigate the possible enhancement of im- munogenicity of the carboxyl-terminal peptide of β-hCG which is made up of 37 amino acid residues (109-145) and contains the specific epitope (antigenic determinant) of hCG. Materials &. Methods hCGβ-CTP37 tetraploid cDNA was constructed by linking four hCGβ-CTP37 cDNAs together. The product was then subcloned into the E. coli expres- sion vector pQE60 to construct the expression vector pQE60/ (hCGβ-CTP37)4. Recom- binant (hCGβ-CTP37)4 was expressed in E. colt-X-blue. Results Western blot analysis showed that the tetraploid of hCGβ-CTP37 had an ap- parent molecular weight of 20 kD and had relatively stronger anti-hCG antibody-bind- ing activity compared with the diploid from. Conclusion The tetraploid of hCGβ-CTP37 may be a more potent immunogen for raising anti-hCG vaccines for fertility regulation or suppression of tumor.

Transcript and Sequence Analysis of S-RNases in Self-Compatible Tetraploid Chinese Cherry (Prunus pseudocerasus L.)

Chinese cherry （Prunus pseudocerasus L.） is an allotetraploid species and exhibits natural self-compatibility.However,the full-length cDNA sequences,functional analysis and the transcripts of S-RNase alleles in Chinese cherry cultivars are not known.In the two cultivars Taixiaohongying and Laiyang Short Cherry with S1S2S3S4 genotypes,two S-RNases were transcribed in Northern blotting,and the two full-length cDNAs of S-RNase were cloned and analyzed.As the result,the transcribed S-RNases were S1-RNase and S2-RNase.The two complete cDNA sequences of S1-RNase and S2-RNase were registered as EU073938 and EU073939,respectively,and had characteristic structure of rosaceous S-RNases based on their sequences indicating that they had normal function for S-RNase in the style.The S3-RNase and S4-RNase were not transcribed in the style and were nonfunctional for S-RNase,so S3m and S4m could be used to represent the nonfunctional S3-RNase and S4-RNase.The phylogenetic analysis implied that the S-RNases of Prunus,including Chinese cherry,had lower intra-specific similarity and diverged earlier than the divergence of species in Prunus.

Tetraploid induced by physical and chemical methods in Jinjiang oyster（Crassostrea rivularis）

In order to induce tetraploid into Jinjiang oyster (Crassostrea rivularis), embryos were treated with physical (cold or heat shock) and chemical (chlorpromazinum or traditional Chinese medicine)methods in the first 3 min before the cleavage of zygotes or at the onset of the first cleavage of one or two eggs. Measurements show that the induction rates of tetraploid in the embryos inspected are 28%, 30%, 28. 4% and 35. 8% for heat-shock (39℃, 3 min),cold-shock (10℃, 3min),chlorpromazinum (0. 375 mg/dm3, 10 min) and traditional Chinese medicine (50 g/dm3 )extract (57 cm3, 10 min),respectively. The tetraploid induction rate in the larvae treated with traditional Chinese medicine is 32. 5%.It is evident that the induction rate of tetraploid in embryos insected at an early stage is the most remarkable when treated. with traditional Chinese medicine.

Cytogenetic Mechanism for the Aneuploidy and Mosaicism Foundin Tetraploid Pacific Oyster Crassostrea gigas （Thunberg）

Chromosome constitution was investigated in adult tetraploid Pacific oyster produced by blocking the first polar body oftriploid eggs which were fertilized with haploid sperms. A high incidence of aneuploid and heteroploid mosaics were found amongthe offspring. Of 20 individuals identified, only 9 （45%） were eutetraploid which contained 40 chromosomes; 2 （10%） were ane-uploid （hypotetraploid）, which contained 39 and 38 chromosomes, respectively; and 9 （45%） were heteroploid mosaics. One mosaicwas consisted of cells containing 40 and 39 chromosomes, respectiovely （1：1 in cell number）, while the other 8 were consisted ofcells containing chromosomes varying between tetraploid and triploid. It was also interesting to note that 3 mosaics even containedsome diploid cells with 20 chromosomes. A certain number of cells of 2 tetraploids and 8 mosaics spread with 32-37 well-scatteredand some clumped chromosomes at metaphase. The percentage of aneuploid cells with chromosomes varying between triploid andtetraploid correlated significantly with that of heteroploid mosaics cells with clumping chromosomes （P〈0.05）. Our findings sug-gested that reversion existed in both tetraploid and triploid oyster and chromosome clumping may underline the chromosome elimi-nation in tetraploid oyster. It seems that the reversing cells, at least some of them, continuously eliminate their chromosomes until themost stable diploid state is established.

TETRAPLOID INDUCTION BY INHIBITING MITOSIS I IN SCALLOP CHLAMYS FARRERI

Tetraploid induction was carried out by inhibiting mitosis I in fertilized eggs ofChlamys farreri. Mitosis I was blocked with cold shock (5–7°C), Cytochalasin B (0.75 mg/L) and 6-dimethylaminopurine (6-DMAP) (60–75 mg/L) when 60% fertilized eggs released polar body II at 20°C. At 4-cells embryo stage, the ploidy was determined by counting chromosome number. In control groups, most embryos were diploids (72.22%) and aneuploids (24.78%). In Cytochalasin B, cold shock and 6-DMAP treated groups, tetraploids were respectively 10.51%, 4.08%, and 13.34%; aneuploids were 43.10%, 35.93% and 29.16%, and triploids were 7.84%, 8.52% and 18.33%. At D-larva stage, ploidy was determined by flow cytometry (FCM). The ploidy analysis of day 2 larvae showed diploids in control group and also in three treated groups. Juvenile scallops (0.2–0.3cm) which were harvested in two control groups and two CB treated groups were all diploids through checking ploidy individually by FCM.

Primary Study on Experiment of Tetraploid Induction in Haliotis discus hannai with PEG

The experiment was performed on induction tetraploid of Haliotis discus hannai at two-cell stage through cell fusion with PEG treatment. In this paper, the orthogonal experiment of three factors and three levels [L9 （3^4）] was used. Three factors and three levels were molecular weight of PEG： 8 000, 6 000, 4 000 mol ·g^-1; PEG concentration： 45%, 50%, 55%; treatment duration time： 1, 2, 3 min, respectively. The results showed that the optimal pattern of three factors and three levels on inducing tetraploid of Haliotis discus hannai at two-cell stage through using PEG treatment were： molecular weight 4 000, concentration 55%, treatment duration time： 2 min. The highest tetraploid induction rate was 10.8% at embryo period. The three factors treatment sequence was treatment duration time→concentration→molecular weight.

Analysis of Seed Phenotypic and Metabolic Characteristics of Diploid and Tetraploid Tartary Buckwheat

Polyploid plants grow well,are stress tolerant,and are rich in nutrients and bioactive compounds.Thus,they are useful for improving crop quality and yield.In this study,we compared the seed characteristics and metabolite profiles of diploid and tetraploid tartary buckwheat,which was developed via an artificially induced chromosome doubling event.The length,width,area,and thousand-grain weight were greater for the tetraploid seeds than for the diploid seeds.However,the germination rate decreased for the tetraploid seeds.Additionally,there was a gap between the shell and kernel of the tetraploid seeds.Moreover,the water absorption rate was higher for the tetraploid than for the diploid seeds.Chromosome doubling increased the seed total flavonoid content and deepened the seed color.A principal component analysis of the ultrahigh-pressure liquid chromatography-high resolution mass spectrometry data revealed the clear separation between the diploid and tetraploid samples.An orthogonal partial least squares-discriminant analysis and other multivariate statistical analyses identified 83 differentially abundant compounds,with most of the flavonoid metabolites more abundant in the tetraploid than in the diploid seeds.Research on tartary buckwheat polyploidy may result in enhanced germplasm resources and may clarify the mechanism underlying the biosynthesis of bioactive compounds.