Pyramiding of multiple genes generates rapeseed introgression lines with clubroot and herbicide resistance, high oleic acid content, and early maturity

Clubroot and herbicide resistance,high oleic acid(OA)content,and early maturity are targets of rapeseed(Brassica napus L.)breeding.The objective of this study was to develop new male-fertility restorer lines by pyramiding favorable genes to improve these traits simultaneously.Seven elite alleles for the four traits were introduced into the restorer line 621R by speed breeding with marker-assisted and phenotypic selection.Six introgression lines(ILs)were developed with four-to seven-gene combinations and crossed with two elite parents to develop hybrids.All ILs and their corresponding hybrids displayed high resistance to both clubroot pathotype 4 and sulfonylurea herbicides.Three ILs and their hybrids showed large increases in OA contents and four showed earlier maturity.These new ILs may be useful in rapeseed hybrid breeding for the target traits.

Stimulation Study of Gene Pyramiding inAnimals by Marker-Assisted Selection

This gene pyramiding strategy is based on the idea of efficiently pyramiding genes of interest by crosses and selection to obtain a population with favorable alleles from different breeds or lines, which is called an ideal population. We investigate impacts of some factors on the pyramiding efficiencies by simulation. These factors include selection strategies （the breeding value selection, the molecular scores selection and the index selection）, proportion selected （2, 10 and 20%）, recombination rates between adjacent target genes （0.1, 0.3 and 0.5） and different mating types （the random mating and the positive assortative mating avoiding sib mating）. The results show that： （1） The more recombination rate and the lower proportion male selected, the better pyramiding efficiency; （2） the ideal population is obtained via various selection strategies, while different selection strategies are suitable for different breeding objectives. From the perspective of pyramiding target genes merely, the molecular scores selection is the best one, for the purpose of pyramiding target genes and recovering genetic background of the target trait, the index selection is the best one, while from the saving cost point of view, the breeding value selection is the best one; （3） the positive assortative mating is more efficient for gene pyramiding compared with the random mating in the terms of the number of generations of intercross for getting the ideal population.

Marker-assisted pyramiding of soybean resistance genes R_(SC4),R_(SC8),and R_(SC14Q) to soybean mosaic virus

Soybean mosaic virus （SMV） is one of the major viral pathogens affecting soybean crops worldwide. Three SMV resistance genes, Rsc4, Rsc8, and Rsc14Q, have been identified and mapped on soybean chromosomes 14, 2, and 13 from Dabaima, Kefeng 1, and Qihuang 1 cultivars, respectively. Soybean cultivar Nannong 1138-2 is widely grown in the Yangtze River Valley of China. In this study, crosses were made between Qihuang l^Kefeng 1 and DabaimaxNannong 1138-2. Ten simple sequence repeat （SSR） markers linked to three resistance loci （Rsc4, Rsc8, and Rsc^4Q） were used to assist pyramided breeding. Pyramided families containing three resistance loci （Rsc4, Rsc8, and Rsc14Q） were evaluated by inoculating them with 21 SMV strains from China. Results indicated that the 10 markers can be used effectively to assist the selection of resistant individuals containing Rsc4, Rsc8, and Rsc14Q. A total of 53 F6 plants were confirmed to contain three homozygous alleles conferring resistance to SMV. Five F7 homozygous pyramided families exhibited resistance to 21 strains of SMV and showed desirable agronomic traits using dual selection. The strategy of pyramiding resistance gene derived from different varieties has practical breeding value in providing broad-spectrum resistance against the existing strains of SMV in China.

Genetic Improvement of Japonica Rice Variety Wuyujing 3 for Stripe Disease Resistance and Eating Quality by Pyramiding Stv-b^i and Wx-mq

Japonica rice variety Kanto 194 as the donor of resistance gene Stv-b^i and Iow-amylose content gene Wx-mq was used to improve the resistance and eating quality of Wuyujing 3 by the breeding strategy of backcross. In continuous backcross and selfcross generations, the related molecular markers with Stvobi and Wx-mq genes were utilized for genotypic detection by associated with resistance identification of rice stripe disease and agronomic traits selection. Finally, 10 improved lines with homozygous genotype Stv-b^i Stv-b^iWx-mqWx-mq were obtained from BC3F4 generation. The results of comparative analysis indicated that most characters of these lines were consistent with those of recipient parent Wuyujing 3, except for the improved resistance, appearance and eating quality. By evaluation of the comprehensive performance of them, two excellent lines K01 and K04 were selected for further experiments.

Simulation of gene pyramiding in Drosophila melanogaster

Gene pyramiding has been successfully practiced in plant breeding for developing new breeds or lines in which favorable genes from several different lines were integrated. But it has not been used in animal breeding, and some theoretical investigation and simulation analysis with respect to its strategies, feasibility and efficiency are needed before it can be implemented in animals. In this study, we used four different pure lines of Drosophila melanogaster, each of which is homozygous at a specific mutant gene with a visible effect on phenotype, to simulate the gene pyramiding process and analyze the duration and population size required in different pyramiding strategies. We finally got the ideal individuals, which are homozygous at the four target genes simultaneously. This study demonstrates that gene pyramiding is feasible in animal breeding and the interaction between genes may affect the final results.

Application of Molecular Marker Assisted Selection in Gene Pyramiding and Selection of New Cultivars

The feasibility of molecule markers＇ application in gene pyramiding has been proved,and obvious progresses in crop breeding have been made till now.Furthermore,different QTLs or molecular markers linked tightly to yield,quality or resistance may be used for marker assisted selection.MAS will be applied widely in crop breeding due to the development of more gene-based markers and efficient quantitative trait locus（QTL） as well as lower cost marking systems.

Marker-Assisted Pyramiding of Genes Conferring ResistanceAgainst Bacterial Blight and Blast Diseases into Indian RiceVariety MTU1010

Two major bacterial blight （BB） resistance genes （Xa21 and xa13） and a major gene for blastresistance （Pi54） were introgressed into an Indian rice variety MTU1010 through marker-assistedbackcross breeding. Improved Samba Mahsuri （possessing Xa21 and xa13） and NLR145 （possessingPi54） were used as donor parents. Marker-assisted backcrossing was continued till BC2 generationwherein PCR based functional markers specific for the resistance genes were used for foregroundselection and a set of parental polymorphic microsatellite markers were used for background selectionat each stage of backcrossing. Selected BC2F1 plants from both crosses, having the highest recoveriesof MTU1010 genome （90% and 92%, respectively）, were intercrossed to obtain intercross F1 （ICF1） plants,which were then selfed to generate 880 ICF2 plants possessing different combinations of the BB andblast resistance genes. Among the ICF2 plants, seven triple homozygous plants （xa13xa13Xa21Xa21Pi54Pi54）with recurrent parent genome recovery ranging from 82% to 92% were identified. All the seven ICF2plants showed high resistance against the bacterial blight disease with a lesion lengths of only 0.53–2.28 cm, 1%–5% disease leaf areas and disease scoring values of ‘1’ or ‘3’. The seven ICF2 plants wereselfed to generate ICF3, which were then screened for blast resistance, and all were observed to behighly resistant to the diseases. Several ICF3 lines possessing high level of resistance against BB andblast, coupled with yield, grain quality and plant type on par with MTU1010 were identified and advanced forfurther selection and evaluation.

Development and identification of Verticillium wilt-resistant upland cotton accessions by pyramiding QTL related to resistance

Cotton Verticillium wilt is a serious soil-borne disease that leads to significant losses in fiber yield and quality worldwide. Currently, the most effective way to increase Verticillium wilt resistance is to develop new resistant cotton varieties. Lines 5026 and 60182 are two Verticillium wilt-resistant upland cotton accessions. We previously identified a total of 25 quantitative trait loci（QTLs） related to Verticillium wilt resistance from 5026 and 60182 by assembling segregating populations from hybridization with susceptible parents. In the current study, using 13 microsatellite markers flanking QTLs related to Verticillium wilt resistance, we developed 155 cotton inbred lines by pyramiding different QTLs related to Verticillium wilt resistance from a filial generation produced by crossing 5026 and 60182. By examining each allele＇s effect and performing multiple comparison analysis, we detected four elite QTLs/alleles（q-5/NAU905-2, q-6/NAU2754-2, q-8/NAU3053-1 and q-13/NAU6598-1） significant for Verticillium wilt resistance, pyramiding these elite alleles increased the disease resistance of inbred lines. Furthermore, we selected 34 elite inbred lines, including five lines simultaneously performing elite fiber quality, high yield and resistance to V. dahliae, 14 lines with elite fiber quality and disease resistance, three lines with high yield and disease resistance, and 12 lines with resistance to V. dahliae. No correlation between Verticillium wilt resistance and fiber quality traits/yield and its components was detected in the 155 developed inbred lines. Our results provide candidate markers for disease resistance for use in marker-assisted breeding（MAS）, as well as elite germplasms for improving important agronomic traits via modern cotton breeding.

Pyramiding disease resistance genes in elite winter wheat germplasm for Western Canada

We report on pyramiding different disease resistance genes against fungal pathogens in Canadian winter wheat germplasm based on available DNA markers and gene sequences.Genetic resistance represents a safe, economical and ecological method for protecting plants, growers and the health of consumers. Major diseases of wheat on the Canadian Prairies are common bunt, rusts(leaf, stem and stripe) and Fusarium head blight. Over the years markers for resistance genes against these diseases have been identified and used by the international wheat community. We describe markers that we have used to pyramid different resistance genes and indicate their presence in Canadian winter wheat cultivars issued from the winter wheat breeding program at the Agriculture and Agri-Food Canada,Lethbridge Research and Development Centre, the only winter wheat breeding program in Western Canada actively delivering new varieties for all regions of the Canadian Prairies.The sources of resistance and identities of PCR primers and amplification conditions are indicated to enable the transfer and pyramiding of different resistance(R) genes to breeding lines. We conclude by reviewing new tools for identifying R genes in wheat and indicate how mutagenesis and gene editing can help future efforts to extend the protection offered by known R genes.

Improvement of Upland Rice Variety by Pyramiding Drought Tolerance QTL with Two Major Blast Resistance Genes for Sustainable Rice Production

Varalu is an early maturing rice variety widely grown in the rainfed ecosystem preferred for its grain type and cooking quality.However,the yield of Varalu is substantially low since it is being affected by reproductive drought stress along with the blast disease.The genetic improvement of Varalu was done by introgressing a major yield QTL,qDTY_(12.1),along with two major blast resistance genes i.e.Pi54 and Pi1 through marker-assisted backcross breeding.Both traits were transferred till BC_(2) generation and intercrossing was followed to pyramid the two traits.Stringent foreground selection was carried out using linked markers as well as peak markers(RM28099,RM28130,RM511 and RM28163)for the targeted QTL(qDTY_(12.1)),RM206 for Pi54 and RM224 for Pi1.Extensive background selection was done using genome-wide SSR markers.Six best lines(MSM-36,MSM-49,MSM-53,MSM-57,MSM-60 and MSM-63)having qDTY_(12.1) and two blast resistance genes in homozygous condition with recurrent parent genome of 95.0%-96.5% having minimal linkage drag of about 0.1 to 0.7 Mb were identified.These lines showed yield advantage under drought stress as well as irrigated conditions.MSM-36 showed better performance in the national coordinated trials conducted across India,which indicated that improved lines of Varalu expected to replace Varalu and may have an important role in sustaining rice production.The present study demonstrated the successful marker-assisted pyramiding strategy for introgression of genes/QTLs conferring biotic stress resistance and yield under abiotic stress in rice.

Improvement of Rice Resistance to Sheath Blight by Pyramiding QTLs Conditioning Disease Resistance and Tiller Angle

Rice resistance to sheath blight （SB） is controlled by polygenes or quantitative trait loci （QTLs） and affected by plant morphological traits, qSB-9^TQ and TAG1^TQ, which control SB resistance and tiller angle （TA）, respectively, were employed to test whether the combination of the two genes will further improve rice SB resistance and reduce yield loss rather than only one of them or neither. Using two pairs of near isogenic lines （NILs）, TAC1^TQ was confirmed to contribute to SB resistance. However, its effect was less considerable than that of qSB-9^TQ. Subsequently, the two genes were introduced into two commercial rice varieties to develop a series of NILs. The NILs carrying both TAG1^TQ and qSB-9^TQ showed more resistance than the NILs containing only one of them. Comparing the grain yield per plant （GYP） under the three different SB disease conditions, namely slight, moderate and severe conditions, NILs carrying both genes apparently lost lower GYP than the NILs without the two genes and the NILs carrying only TAC1^TQ. Under slight disease condition, no significant differences were found on morphology, development and GYP associated traits except for TA between the NILs carrying both genes and either of them, indicating that the two genes have no inferior effect on rice agronomic traits. Results indicated that mAC1^TQ and qSB-9^TQ have high breeding potential, and pyramiding SB resistance QTL and morphological trait QTL is a potential approach in improving rice SB resistance.

Pyramiding Breeding by Marker-Assisted Recurrent Selection in Upland Cotton: Selected Effects on Resistance to Helicoverpa armigera

The coincidence rates were more than 96% among the instar-weighted average of bioassaysin the lab, the percentage of resistance to Km in the field and the percentage of plantscontaining Bt gene. So, the performance of resistance to Km in the field can be used torepresent the transgenic Bt gene for selecting the resistance to bollworm. The instar-weighted averages were 30.585, 24.182, 16.615, 10.601, 10.123, 7.440 and 7.215 for theC0, P1, M1, M2, MP1, P2 and MP2 populations, respectively. The variance analysisindicated that the instar-weighted average in C0 was greatly significantly higher thanthat in all other populations, i.e., the performance of resistance to bollworm in C0 washighly significantly lower than all other populations. And the resistance in P1 wasgreatly lower than that of M1, M2, MP1, P2 and MP2, and M1 greatly lower than that of M2,MP1, P2 and MP2. There were no significant differences among M2, MP1, P2 and MP2. Withinthe populations of the first cycle selection, MP1 and M1 were greatly significantlyhigher than P1, and MP1 significantly higher than M1. The populations of the second cycleselection were significantly higher than their initial population M1, but no significantdifference among them. The boll size, seed index, the percent of the first harvest yield,fiber length, strength and elongation of the resistant plants to bollworm were significantlylower than that of sensitive plants to bollworm. And the yield of seed and lint cottonof the resistant plant to bollworm were lower than that of the sensitive to bollworm, butno significant difference between them. The boll numbers per plant, lint percent andmicronaire of the resistant plants to bollworm were significantly higher than that of thesensitive plant to bollworm.

Pyramiding the disease resistant genes to southern rust and stalk rot in maize(Zea mays L.) with marker-assisted selection

Southern corn rust(SCR) caused by Puccinia polysora Underw and maize stalk rot caused by Pythium inflatum Matthews(MSR-2) are two destructive diseases of maize(Zea mays L.) in China.Our previous studies indicated that maize inbred line Qi319 is highly resistant to SCR but susceptible to MSR-2,while inbred line 1145 is highly resistant to MSR-2 but susceptible to SCR.The SCR resistant gene(RppQ) in Qi319 and MSR-2 resistant gene(Rpi1) in 1145 have been mapped on chromosome 10 and 4 respectively.In this research,through marker-assisted selection(MAS) with the molecular markers,bnlg1937 tightly linked to Rpi1 and phi041 tightly linked to RppQ,pyramid breeding of the two kinds of disease resistant genes were carried out from the year of 2003 to 2007.Two homozygotic inbred lines of F5 generation,DR94-1-1-1 and DR36-1-1-1 were identified.MAS result suggested DR94-1-1-1 and DR36-1-1-1 contained the two resistance genes RppQ and Rpi1.Field inoculation tests confirmed their high resistance to the two diseases.In addition,field investigation indicated that the two selected inbred lines,particularly DR94-1-1-1,had excellent agronomic traits such as plant height,ear height and yield-relating traits including ear length,ear diameter,ear weight,kernels per ear,kernels per row and kernel weight per ear.The two selected inbred lines DR94-1-1-1 and DR36-1-1-1 can either be directly developed into commercial variety or used as immediate donors of SCR and MSR resistance breeding programs in maize.

Breeding of Rice Restorer Line Haihui 818 of Multi-resistant Gene Pyramiding

Haihui 818 is a new restorer line by cross breeding with Hua 23 S and Huahui 8131.Good plant type,high tillering ability,fine grain quality,strong restoring ability and high yield were shown in hybrid production.After artificial inoculation and field stress and molecular market-assisted selection,the results showed that rice-blast resistance genes Pi1,BB resistance genes Xa7,and brown planthopper resistance genes Bph3,Bph14 and Bph15 were pyramided together.Among its hybrids,Bo II You 818(Bo II A/Haihui 818)was approved for commercial production by Hainan Provincial Crop Variety Approval Committee in 2015.

Insect resistance management in Bacillus thuringiensis cotton by MGPS(multiple genes pyramiding and silencing)

The introduction of Bacillus thuringiensis(Bt)cotton has reduced the burden of pests without harming the environment and human health.However,the efficacy of Bt cotton has decreased due to field-evolved resistance in insect pests over time.In this review,we have discussed various factors that facilitate the evolution of resistance in cotton pests.Currently,different strategies like pyramided cotton expressing two or more distinct Bt toxin genes,refuge strategy,releasing of sterile insects,and gene silencing by RNAi are being used to control insect pests.Pyramided cotton has shown resistance against different cotton pests.The multiple genes pyramiding and silencing(MGPS)approach has been proposed for the management of cotton pests.The genome information of cotton pests is necessary for the development of MGPS-based cotton.The expression cassettes against various essential genes involved in defense,detoxification,digestion,and development of cotton pests will successfully obtain favorable agronomic characters for crop protection and production.The MGPS involves the construction of transformable artificial chromosomes,that can express multiple distinct Bt toxins and RNAi to knockdown various essential target genes to control pests.The evolution of resistance in cotton pests will be delayed or blocked by the synergistic action of high dose of Bt toxins and RNAi as well as compliance of refuge requirement.

Pyramiding blast,bacterial blight and brown planthopper resistance genes in rice restorer lines

Rice blast, bacterial blight （BB） and brown planthopper （BPH） are the three main pests of rice. This study investigated pyr-amiding genes resistant to blast, BB and BPH to develop restorer lines. Ten new lines with blast, BB and/or BPH resistance genes were developed using marker-assisted selection （MAS） technique and agronomic trait selection （ATS） method. Only HR13 with resistance genes to blast, BB and BPH was obtained. In addition to blast and BB resistance, four lines （HR39, HR41, HR42, HR43） demonstrated moderate resistance to BPH, but MAS for BPH resistance genes were not conducted in developing these four lines. These data suggested that there were unknown elite BPH resistance genes in the Zhongzu 14 donor parent. A more effective defense was demonstrated in the lines withPi1 andPi2 genes although the weather in 2012 was favorable to disease incidence. Blast resistance of the lines with a single resistance gene,Pita, was easily inlfuenced by the weather. Overal, the information obtained through pyramiding multiple resistance genes on developing the restorer lines is helpful for rice resistance breeding.

Regulation of specific abnormal calcium signals in the hippocampal CA1 and primary cortex M1 alleviates the progression of temporal lobe epilepsy

Temporal lobe epilepsy is a multifactorial neurological dysfunction syndrome that is refractory,resistant to antiepileptic drugs,and has a high recurrence rate.The pathogenesis of temporal lobe epilepsy is complex and is not fully understood.Intracellular calcium dynamics have been implicated in temporal lobe epilepsy.However,the effect of fluctuating calcium activity in CA1 pyramidal neurons on temporal lobe epilepsy is unknown,and no longitudinal studies have investigated calcium activity in pyramidal neurons in the hippocampal CA1 and primary motor cortex M1 of freely moving mice.In this study,we used a multichannel fiber photometry system to continuously record calcium signals in CA1 and M1 during the temporal lobe epilepsy process.We found that calcium signals varied according to the grade of temporal lobe epilepsy episodes.In particular,cortical spreading depression,which has recently been frequently used to represent the continuously and substantially increased calcium signals,was found to correspond to complex and severe behavioral characteristics of temporal lobe epilepsy ranging from gradeⅡto gradeⅤ.However,vigorous calcium oscillations and highly synchronized calcium signals in CA1 and M1 were strongly related to convulsive motor seizures.Chemogenetic inhibition of pyramidal neurons in CA1 significantly attenuated the amplitudes of the calcium signals corresponding to gradeⅠepisodes.In addition,the latency of cortical spreading depression was prolonged,and the above-mentioned abnormal calcium signals in CA1 and M1 were also significantly reduced.Intriguingly,it was possible to rescue the altered intracellular calcium dynamics.Via simultaneous analysis of calcium signals and epileptic behaviors,we found that the progression of temporal lobe epilepsy was alleviated when specific calcium signals were reduced,and that the end-point behaviors of temporal lobe epilepsy were improved.Our results indicate that the calcium dynamic between CA1 and M1 may reflect specific epileptic behaviors corresponding to different grades.Furthermore,the selective regulation of abnormal calcium signals in CA1 pyramidal neurons appears to effectively alleviate temporal lobe epilepsy,thereby providing a potential molecular mechanism for a new temporal lobe epilepsy diagnosis and treatment strategy.

An anti-aliasing filtering of quantum images in spatial domain using a pyramid structure

As a part of quantum image processing,quantum image filtering is a crucial technology in the development of quantum computing.Low-pass filtering can effectively achieve anti-aliasing effects on images.Currently,most quantum image filterings are based on classical domains and grayscale images,and there are relatively fewer studies on anti-aliasing in the quantum domain.This paper proposes a scheme for anti-aliasing filtering based on quantum grayscale and color image scaling in the spatial domain.It achieves the effect of anti-aliasing filtering on quantum images during the scaling process.First,we use the novel enhanced quantum representation(NEQR)and the improved quantum representation of color images(INCQI)to represent classical images.Since aliasing phenomena are more pronounced when images are scaled down,this paper focuses only on the anti-aliasing effects in the case of reduction.Subsequently,we perform anti-aliasing filtering on the quantum representation of the original image and then use bilinear interpolation to scale down the image,achieving the anti-aliasing effect.The constructed pyramid model is then used to select an appropriate image for upscaling to the original image size.Finally,the complexity of the circuit is analyzed.Compared to the images experiencing aliasing effects solely due to scaling,applying anti-aliasing filtering to the images results in smoother and clearer outputs.Additionally,the anti-aliasing filtering allows for manual intervention to select the desired level of image smoothness.

Control of GaN inverted pyramids growth on c-plane patterned sapphire substrates

Growth of gallium nitride(GaN)inverted pyramids on c-plane sapphire substrates is benefit for fabricating novel devices as it forms the semipolar facets.In this work,GaN inverted pyramids are directly grown on c-plane patterned sapphire substrates(PSS)by metal organic vapor phase epitaxy(MOVPE).The influences of growth conditions on the surface morphol-ogy are experimentally studied and explained by Wulff constructions.The competition of growth rate among{0001},{1011},and{1122}facets results in the various surface morphologies of GaN.A higher growth temperature of 985°C and a lowerⅤ/Ⅲratio of 25 can expand the area of{}facets in GaN inverted pyramids.On the other hand,GaN inverted pyramids with almost pure{}facets are obtained by using a lower growth temperature of 930℃,a higherⅤ/Ⅲratio of 100,and PSS with pattern arrangement perpendicular to the substrate primary flat.

NFHP-RN:AMethod of Few-Shot Network Attack Detection Based on the Network Flow Holographic Picture-ResNet

Due to the rapid evolution of Advanced Persistent Threats(APTs)attacks,the emergence of new and rare attack samples,and even those never seen before,make it challenging for traditional rule-based detection methods to extract universal rules for effective detection.With the progress in techniques such as transfer learning and meta-learning,few-shot network attack detection has progressed.However,challenges in few-shot network attack detection arise from the inability of time sequence flow features to adapt to the fixed length input requirement of deep learning,difficulties in capturing rich information from original flow in the case of insufficient samples,and the challenge of high-level abstract representation.To address these challenges,a few-shot network attack detection based on NFHP(Network Flow Holographic Picture)-RN(ResNet)is proposed.Specifically,leveraging inherent properties of images such as translation invariance,rotation invariance,scale invariance,and illumination invariance,network attack traffic features and contextual relationships are intuitively represented in NFHP.In addition,an improved RN network model is employed for high-level abstract feature extraction,ensuring that the extracted high-level abstract features maintain the detailed characteristics of the original traffic behavior,regardless of changes in background traffic.Finally,a meta-learning model based on the self-attention mechanism is constructed,achieving the detection of novel APT few-shot network attacks through the empirical generalization of high-level abstract feature representations of known-class network attack behaviors.Experimental results demonstrate that the proposed method can learn high-level abstract features of network attacks across different traffic detail granularities.Comparedwith state-of-the-artmethods,it achieves favorable accuracy,precision,recall,and F1 scores for the identification of unknown-class network attacks through cross-validation onmultiple datasets.