Improvement of three popular Indian groundnut varieties for foliar disease resistance and high oleic acid using SSR markers and SNP array in marker-assisted backcrossing

Foliar fungal diseases(rust and late leaf spot)incur large yield losses,in addition to the deterioration of fodder quality in groundnut worldwide.High oleic acid has emerged as a key market trait in groundnut,as it increases the shelf life of the produce/products in addition to providing health benefits to consumers.Marker-assisted backcrossing(MABC)is the most successful approach to introgressing or pyramiding one or more traits using traitlinked markers.We used MABC to improve three popular Indian cultivars(GJG 9,GG 20,and GJGHPS 1)for foliar disease resistance(FDR)and high oleic acid content.A total of 22 BC3F4 and 30 BC2F4 introgression lines(ILs)for FDR and 46 BC3F4 and 41 BC2F4 ILs for high oleic acid were developed.Recurrent parent genome analysis using the 58 K Axiom_Arachis array identified several lines showing upto 94%of genome recovery among second and third backcross progenies.Phenotyping of these ILs revealed FDR scores comparable to the resistant parent,GPBD 4,and ILs with high(~80%)oleic acid in addition to high genome recovery.These ILs provide further opportunities for pyramiding FDR and high oleic acid in all three genetic backgrounds as well as for conducting multi-location yield trials for further evaluation and release for cultivation in target regions of India.

Development of New Submergence Tolerant Rice Variety for Bangladesh Using Marker-Assisted Backcrossing

Submergence tolerant high yielding rice variety was developed using BR11 as a recipient parent applying foreground, phenotypic and background selection approaches. Recombinant selection was found essential to minimize linkage drag by BC2F2 generation. Without recombinant selection, the introgression size in the backcross recombinant lines （BRLs） was approximately 15 Mb on the carrier chromosome. The BRLs were found submergence tolerance compared to the check varieties under complete submergence for two weeks at Bangladesh Rice Research Institute, and produced higher yield compared to the isogenic Subl-line under controlled submerged condition. The BRL IR85260-66-654-Gaz2 was released as BRRI dhan52 in 2010, which was the first high yielding submergence tolerant variety in Bangladesh. BRRI dhan52 produced grain yield ranging from 4.2 to 5.2 t/hm2 under different flash flood prone areas of Bangladesh in three consecutive seasons. The study demonstrated the efficiency of recombinant selection and better adaptability of the newly released submergence tolerant high yielding variety in flash flood prone different areas of the country with respect to submergence tolerance and yield potential.

Transfer of Lysozyme Gene into indica Parents of Hybrid Rice by Backcrossing

A lysozyme gene resistant to rice blast was transferred from the donor transgenic japonica rice Zhonghua 9 （D2-1-2） into a sterile line Pei＇ai 64S（PA 64S） and restorer line 9311 of the two-line hybrid rice Liangyoupeijiu, and the restorer line Minghui 63 （MH 63） of three-line hybrid rice Shanyou 63 by successive backcrossing. The PCR analysis confirmed that foreign lysozyme gene was segregated at ratio of 1 ： 1 in backcross generations of B39311, B3MH63 and B2PA64S, and at ratio of 3 ： 1 in selfed generations of B2F2 9311, B2F2 MH63 and B1F2 PA64S, indicating that the foreign gene was stably inherited over successive generations as a dominant single copy gene. The resistance against rice blast in backcross or selfed generations and corresponding testcross combinations were investigated in 2003 and 2004. The results showed that the resistance of the transgenic rice to blast had a greater improvement than that of the corresponding recurrent parents or the corresponding check hybrid combinations. The resistance of the advanced backcross and selfed generations to rice blast is much stronger than that of the early generations. The study confirmed that transferring the lysozyme gene into hybrid parents by backcrossing was a simple and effective approach to develop new hybrid rice resistant to rice blast.

Application of Marker Assisted Backcrossing to Introgress the Submergence Tolerance QTL <i>SUB</i>1 into the Vietnam Elite Rice Variety-AS996

The result of the study contributes to enhancing and sustaining future livelihoods and food security in Vietnam vis-a-vis climate change. An innovative strategy based on marker-assisted backcrossing (MABC) was used to transform popular rice variety AS996 into the one can tolerate submergence while maintaining its original characteristics preferred by farmers and consumers. The submergence tolerance QTL SUB1 counts for up to 70% of the submergence tolerant and provides a marked improvement of submergence tolerance in all genetic backgrounds and environments tested so far. Parental diversity was carried out with 460 markers. Of which, 53 polymorphic markers were used for assessment on BC1F1, BC2F1 and BC3F1 generations. The best BC1F1 plant was P422 with highest recipient allele was 87.5%, while the additional chosen plants were P412, P428, P215 and P39 (81% - 84%). All these plants were used to develop BC2F1 generation. The six BC2F1 plants were used to develop BC3F1 and BC2F2 were the plants number P422-11 and P422-14 having 93.75% recipient alleles and P422-12, P422-3, P39-17, P39-25 having 92.25% recipient alleles. Total of 445 BC3F1 plants were confirmed the introgresion of SUB1 using ART5 and SC3. After three generations of backcrossing, application of MABC resulted in the best BC3F1 individual P422-14-177 with 100% of recipient alleles based on the number of 53 markers used with only the introgression size of SUB1 was 0.3Mb between ART5 and SC3. Phenotyping was carried out on BC3F1 and BC2F2 of the selected lines. The survival ratio of these selected lines and IR64SUB1 were the same. It convinced the successfully introgress SUB1 into AS996 rice variety. The breeding line BC4F1 having 100% genetic background of donor variety is ready for develop new submergence tolerant rice variety ASS996-SUB1 to cope with climate change.

Creating Wheat Germplasm for High Quality Breeding by Monosomic Backcrossing

By crossing bread wheat cultlvar GC8901 with the 1D monosonlc line of Xiaoyan No. 6 and backcrosslng the offsprlng with the Xlaoyan No. 6 1D monosonlc llne for 5 years, high-molecular-welght glutenin subunlts 1Dx5＋1Dy10 from GC8901 have been transferred Into wheat cultivar Xiaoyan No. 6. The BC5F1 offspring lines had been detected by using methods of cytology, marker, molecular marker and six elite single plants with high molecular-welght glutenin subunlts： lAx1, 1Bx14＋1 By15, 1Dx5＋1 Dy10 were Identified. Those lines have high-yleld potential with better agronomic characters and have been used In high quality wheat breeding processes as well.

Genome-wide recombination variation in biparental segregating and reciprocal backcross populations provides information for introgression breeding in Brassica napus

Variation in patterns of recombination in plant genomes provides information about species evolution,genetic diversity and crop improvement. We investigated meiotic crossovers generated in biparental segregating and reciprocal backcross populations of the allopolyploid genome of rapeseed(Brassica napus)(AACC, 2n = 38). A structured set of 1445 intercrossed lines was derived from two homozygous de novo genome-assembled parents that represented the major genetic clusters of semi-winter Chinese and winter European rapeseeds, and was used to increase QTL resolution and achieve genomic reciprocal introgression. A high-density genetic map constructed with 6161 genetic bins and anchored centromere regions was used to establish the pattern of recombination variation in each chromosome. Around 93%of the genome contained crossovers at a mean rate of 3.8 c M Mb^(-1), with the remaining 7% attributed to centromeres or low marker density. Recombination hotspots predominated in the A genome, including two-thirds of those associated with breeding introgression from B. rapa. Genetic background might affect recombination variation. Introgression of genetic diversity from European winter to Chinese semi-winter rapeseed showed an increase in crossover rate under the semi-winter environment. Evidence for an elevated recombination rate having historically contributed to selective trait improvement includes accumulation of favorable alleles for seed oil content on hotspots of chromosome A10. Conversely, strong artificial selection may affect recombination rate variation, as appears to be the case with a coldspot resulting from strong selection for glucosinolate alleles on A09. But the cold region would be promptly reactivated by crossing design indicated by the pedigree analysis. Knowledge of recombination hotspots and coldspots associated with QTL for 22 traits can guide selection strategies for introgression breeding between the two gene pools. These results and rich genomic resources broaden our understanding of recombination behavior in allopolyploids and may advance rapeseed genetic improvement.

Saline-Alkali Tolerance in Rice: Physiological Response, Molecular Mechanism, and QTL Identification and Application to Breeding

Salinity-alkalinity is incipient abiotic stress that impairs plant growth and development.Rice(Oryza sativa)is a major food crop greatly affected by soil salinity and alkalinity,requiring tolerant varieties in the saline-alkali prone areas.Understanding the molecular and physiological mechanisms of saline-alkali tolerance paves the base for improving saline-alkali tolerance in rice and leads to progress in breeding.This review illustrated the physiological consequences,and molecular mechanisms especially signaling and function of regulating genes for saline-alkali tolerance in rice plants.We also discussed QTLs regarding saline-alkali tolerance accordingly and ways of deployment for improvement.More efforts are needed to identify and utilize the identified QTLs for saline-alkali tolerance in rice.

Introgression the Salinity Tolerance QTLs <i>Saltol</i>into AS996, the Elite Rice Variety of Vietnam

This study focus on developing new salinity tolerance and high yielding rice lines, using markers assisted backrossing (MABC). Total of 500 SSR markers on 12 rice chromosomes were screened for parental polymorphic markers. Of which, 52 primers in the Saltol region were checked with the two parents varieties to identify polymorphic primers for screening the Saltol region of the breeding populations. For each backcross generation of ASS996/FL478, approx. 500 plants were screened with 63 polymorphic markers distributed on 12 chromosomes. The two BC1F1 plants P284 and P307 which had the highest recipient alleles up to 89.06% and 86.36%, were chosen for the next backcrossing. Three BC2F1 plants with the recipient alleles up to 94.03% and 93.18% were used to develop BC3F1 generation. The best BC3F1 plant was P284-112-209 with all the recipient alleles and Saltol region. The four plants P307-305-21, P284-112-195, P284-112-198, P284-112-213 were the second ranking with only one loci heterozygous (applied 63 markers covered on 12 chromosomes). These five plants were chosen as the breeding lines for result of Saltol-AS996 introgression. The breeding line BC4F1 having 100% genetic background of donor variety is ready for develop new salinity tolerant variety ASS996-Saltol to cope with climate change.

Marker-Assisted Selection of Xa21 Conferring Resistance to Bacterial Leaf Blight in indica Rice Cultivar LT2

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive diseases in Asian rice fields. A high-quality rice variety, LT2, was used as the recipient parent.IRBB21, which carries the Xa21 gene, was used as the donor parent. The resistance gene Xa21 was introduced into LT2 by marker-assisted backcrossing. Three Xoo races were used to inoculate the improved lines following the clipping method. Eleven BC_3F_3 lines carrying Xa21 were obtained based on molecular markers and agronomic performance. The 11 lines were then inoculated with the three Xoo races. All the 11 improved lines showed better resistance to BLB than the recipient parent LT2. Based on the level of resistance to BLB and their agronomic performance, five lines (BC_3F_3 5.1.5.1, BC_3F_3 5.1.5.12, BC_3F_3 8.5.6.44, BC_3F_3 9.5.4.1 and BC_3F_3 9.5.4.23) were selected as the most promising for commercial release. These improved lines could contribute to rice production in terms of food security.

Utilization of eui Gene from a Recessive Tall Rice Mutant 02428h in Breeding

In order to improve the panicle extrusion of photo- and thermo-sensitive sterile line ‘Pei＇ai 64S＇ by using elongated uppermost internode （eul） gene of the wide compatibility rice mutant ‘02428h＇, a new photo- and thermo-sensitive sterile line ‘P8hS＇ characterized with elongated uppermost internode was developed by transferring the eui gene into Pei＇ai 64S through three successive backcrossing, Compared with Pei＇ai 64S, the plant height of P8hS was 35.6 cm higher resulted from the elongation of the uppermost and the second internodes from the top. The panicle extrusion of Pei＇ai 64S was completely improved and positive effects were found on the main economic characters of P8hS and its hybrids by introducing euigene into Pei＇ai 64S.

Introgression of Gene for Non-Pollen Type Thermo-Sensitive Genic Male Sterility to Thai Rice Cultivars

For the two-line hybrid rice system, pol en sterility is regulated by recessive gene that responds to temperature. The recessive gene controlling thermo-sensitive genetic male sterility （TGMS） is expressed when the plants are grown in conditions with higher or lower critical temperatures. To transfer tgms gene（s） control ing TGMS to Thai rice cultivars by backcross breeding method, a male sterile line was used as a donor parent while Thai rice cultivars ChaiNat 1, PathumThani 1, and SuphanBuri 1 were used as recurrent parents. The BC2F2 lines were developed from backcrossing and selfing. Moreover, the simple sequence repeat （SSR） markers were developed for identifying tgms gene and the linked marker was used for assisting selection in backcrossing. The identification lines were confirmed by pol en observation. The results showed the success of introgression of the tgms gene into Thai rice cultivars. These lines will be tested for combining ability and used as female parent in hybrid rice production in Thailand.

Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)

A set of 240 introgression lines derived from the advanced backcross population of a cross between a japonica cultivar,Xiushui 09,and an indica breeding line,IR2061,was developed to dissect QTLs affecting cold tolerance （CT） at seedling stage and heat tolerance （HT） at anthesis.Survival rate of seedlings （SRS） and spikelet fertility （SF）,the index traits of CT and HT,showed significant differences between the two parents under stresses.A total of four QTLs （qSRS1,qSRS7,qSRS11a and qSRS11b） for CT were identified on chromosomes 1,7,11,and the Xiushui 09 alleles increased SRS at all loci except qSRS7.Four QTLs for SF were identified on chromosomes 4,5,6,and 11.These QTLs could be classified into two major types based on their behaviors under normal and stress conditions.The first was QTL expressed only under normal condition;and the second QTL was apparently stress induced and only expressed under stress.Among them,two QTLs （qSF4 and qSF6） which reduced the trait difference between heat stress and normal conditions must have contributed to HT because of their obvious contribution to trait stability,and the IR2061 allele at the qSF6 and the Xiushui 09 allele at the qSF4 improved HT,respectively.No similar QTL was found between CT at seedling stage and HT at anthesis.Therefore,it is possible to breed a new variety with CT and HT by pyramiding the favorable CT-and HT-improved alleles at above loci from Xiushui 09 and IR2061,respectively,through marker-assisted selection （MAS）.

Current Status of Conventional and Molecular Interventions for Blast Resistance in Rice

Pyricularia oryzae anamorph of Magnaporthe oryzae is one of the most notorious fungal pathogens causing severe economic loss in rice production worldwide. Various methods, viz. cultural, biological and molecular approaches, are utilized to counteract this pathogen. Moreover, some tolerant or resistant rice varieties have been developed with the help of breeding programmes. Isolation and molecular characterization of different blast resistance genes now open the gate for new possibilities to elucidate the actual allelic variants of these genes via various molecular breeding and transgenic approaches. However, the behavioral pattern of this fungus breakups the resistance barriers in the resistant or tolerant rice varieties. This host-pathogen barrier will be possibly countered in future research by comparative genomics data from available genome sequence data of rice and M. oryzae for durable resistance. Present review emphasized fascinating recent updates, new molecular breeding approaches, transgenic and genomics approaches(i.e. mi RNA and genome editing) for the management of blast disease in rice. The updated information will be helpful for the durable, resistance breeding programme in rice against blast pathogen.

Use of Major Quantitative Trait Loci to Improve Grain Yield of Rice

Further improvement of rice productivity remains a challenge. Breeding is perceived as an important option to increase rice yield. However, the genetic progress of grain yield in most rice breeding programs was slow in the last decades. Although great progress in rice genomics and molecular biology has been achieved, the effect of such technological innovations on rice breeding is far small. Marker-assisted selection （MAS） for a few target quantitative trait loci （QTLs） has significant effects in improving qualitative traits, such as disease resistance. The success of MAS has therefore motivated breeders to identify and use major QTLs for yield and yield component traits. In this review, we summarized the recent methods in QTL identification, including novel statistical methods for linkage and association mapping, special population types, and whole-genome sequencing. We reviewed the successful application of marker-assisted gene introgression and gene pyramiding to improve grain yield and discussed the design of efficient MAS schemes to further increase the success rate of breeding programs. The use of well-characterized major QTLs through introgression and gene pyramiding is proven effective in improving grain yield, particularly yield under abiotic stress. Major QTLs that are stable across genetic background and growing environments are often found in less adapted germplasms, such as landraces and wild relatives. Advanced backcross QTL analysis and introgression lines, which integrate QTL discovery and utilization, are important methods for exploiting major QTLs contained in such germplasms. Next-generation sequencing substantially increases mapping resolution and accelerates the identification of casual genes underlying major QTLs. Practical guidelines derived from theoretical and empirical studies are given to guide the design of efficient marker-assisted gene introgression and pyramiding schemes.

Effect of HMW-GS 5+10 on Quality Parameters in Four Leading Wheat Cultivars

HMW-GS 5 + 10 was introduced to four major wheat cultivars Kehan9, Kefeng3, Longmai20 and Kenda4 through 5 or 6 consecutive backcrosses. No significant difference in protein content and dry gluten content was observed between cultivars containing 5 + 10 and 2 + 12 or 3 + 12 (P>0.1). In cultivars containing HMW-GS 5 + 10, the ratio of wet gluten content to dry gluten content was 2. 9 - 5.0% (P<0. 01) lower than the ratio from the cultivars containing 2 + 12 or 3 + 12, the ratio of Zeleny sedimentation volume to dry gluten content was 4. 5 - 13. 4% (P<0.05) higher. The degree of softening in cultivars having 5 + 10 was 15 -25 FU (P<0. 01) lower, while the maximum resistance was 82 - 193 EU (P<0. 05) higher. Backcrosses with biochemical marker assisted selection in the improvement of quality in wheat cultivars was discussed.

Breeding by selective introgression: Theory, practices, and lessons learned from rice

Future demands for increased productivity and resilience to abiotic/biotic stresses of major crops require new technologies of breeding by design(BBD)built on massive information from functional and population genomics research.A novel strategy of breeding by selective introgression(BBSI)has been proposed and practiced for simultaneous improvement,genetic dissection and allele mining of complex traits to realize BBD.BBSI has three phases:a)developing large numbers of trait-specific introgression lines(ILs)using backcross breeding in elite genetic backgrounds as the material platform of BBD;b)efficiently identifying genes or quantitative trait loci(QTL)and mining desirable alleles affecting different target traits from diverse donors as the information platform of BBD;and c)developing superior cultivars by BBD using designed QTL pyramiding or marker-assisted recurrent selection.Phase(a)has been implemented massively in rice by many Chinese research institutions and IRRI,resulting in the development of many new green super rice cultivars plus large numbers of ILs in 30+elite genetic backgrounds.Phase(b)has been demonstrated in a series of proof-of-concept studies of high-efficiency genetic dissection of rice yield and tolerance to abiotic stresses using ILs and DNA markers.Phase(c)has also been implemented by designed QTL pyramiding,resulting in a prototype of BBD in several successful cases.The BBSI strategy can be easily extended for simultaneous trait improvement,efficient gene and QTL discovery and allele mining of complex traits using advanced breeding lines from crosses between a common"backbone"parent and a set of elite parents in conventional pedigree breeding programs.BBSI can be relatively easily adopted by breeding programs with small budgets,but the BBSI-based BBD strategy can be fully and more efficiently implemented by large seed companies with sufficient capacity.

Selection efficiencies for improving drought/salt tolerances and yield using introgression breeding in rice(Oryza sativa L.)

The backcross(BC) breeding strategy has been increasingly used for developing high yielding varieties with improved abiotic stress tolerances in rice. In this study, 189Huang-Hua-Zhan(HHZ) introgression lines(ILs) developed from three different selection schemes were evaluated for yield related traits under drought stress and non-stress conditions in the target and off-season winter nursery environments to assess the selection efficiency of BC breeding for improving different complex traits, and led us to five important results. The first result indicated that the primary target traits should be selected first in the target environments(TEs) in order to achieve the maximum genetic gain. Secondly, BC breeding for drought tolerance(DT) in rice was almost equally effective by strong phenotypic selection in the main target environments and in the winter-season of Hainan.Thirdly, exploiting genetic diversity in the subspecific gene pools is of great importance for future genetic improvement of complex traits in rice. Fourthly, considerable genetic gain can be effectively achieved by selection for secondary target traits among the ILs with the primary traits. Finally, the developed ILs provide useful materials for future genetic/genomic dissection and molecular breeding of complex traits.

Identification and QTL mapping of Z550, a rice backcrossed inbred line with increased grains per panicle

An elite backcrossed inbred line Z550 with increased grains per panicle was identified from advanced backcrosses between Nipponbare and Xihui 18 by simple sequence repeat(SSR) marker-assisted selection(MAS). Z550 carries 13 substitution segments distributed on chromosomes 1, 6, 7, 8, 9, 10, and 12, with an average substitution length of 1.68 Mb. Compared with the Nipponbare parental line, plant height, panicle length, spikelets per panicle, grains per panicle, and grain weight for Z550 were significantly increased. While the grain width of Z550 was significantly narrower, and the seed setting ratio(81.43%) was significantly lower than that of Nipponbare, it is still sufficient for breeding purposes. Quantitative trait loci(QTLs) mapping for important agronomic traits was conducted with the F_2 population derived from Nipponbare crossed with Z550 using the restricted maximum likelihood(REML) method. A total of 16, including 12 previously unreported QTLs were detected, with contribution rates ranging from 1.46 to 10.49%. Grains per panicle was controlled by 8 QTLs, 5 of which increased number of grains whereas 3 decreased it. qGPP-1, with the largest contribution(10.49%), was estimated to increase grains per panicle by 30.67, while q GPP-9, with the minimum contribution rate(2.47%), had an effect of increasing grains per panicle by 15.79. These results will be useful for further development of single segment substitution lines with major QTLs, and for research of their molecular functions via QTL cloning.

Dissecting Genetic Basis of Deep Rooting in Dongxiang Wild Rice

Deep rooting is an important trait in rice drought resistance.Genetic resources of deep-rooting varieties are valuable in breeding of water-saving and drought-resistant rice.In the present study,234BC2F7 backcross introgression lines were derived from a cross of Dongye 80(an accession of Dongxiang wild rice as the donor parent)and R974(an indica restorer line as the recurrent parent).A genetic linkage map containing 1977 bin markers was constructed by ddRADSeq for QTL analysis.Thirty-one QTLs for four root traits(the number of deep roots,the number of shallow roots,the total number of deep roots and the ratio of deep roots)were assessed on six rice chromosomes in two environments(2020 Shanghai and 2021 Hainan).Two of the QTLs,qDR5.1 and qTR5.2,were located on chromosome 5 in a 70-kb interval.They were detected in both environments.qDR5.1 explained 13.35%of the phenotypic variance in 2020 Shanghai and 12.01%of the phenotypic variance in 2021 Hainan.qTR5.2 accounted for 10.88%and 10.93%of the phenotypic variance,respectively.One QTL(qRDR2.2)for the ratio of deep roots was detected on chromosome 2 in a 210-kb interval and accounted for 6.72%of the phenotypic variance in 2020.The positive effects of these three QTLs were all from Dongxiang wild rice.Furthermore,nine and four putative candidate genes were identified in qRDR2.2 and qDR5.1/qTR5.2,respectively.These findings added to our knowledge of the genetic control of root traits in rice.In addition,this study will facilitate the future isolation of candidate genes of the deep-rooting trait and the utilization of Dongxiang wild rice in the improvement of rice drought resistance.

Evaluation of Rice Seedling Tolerance to Constant and Intermittent Low Temperature Stress

M202, IR50 and advanced backcross （BC4F6） lines carrying the cold tolerance QTLs qCTS4 and qCTS12 were evaluated using three low temperature stress assays （constant, intermittent and constant with recovery period）. Under constant stress, two BC4F6 lines （MIb 4853-9 and MIb 6885-2） exhibited differences in seedling growth and accumulation of stress-related compounds although both carry the two QTLs in the IRS0 background. Differences between these lines were also observed in the constant with recovery period assay, although both of them performed comparably under intermittent stress, MIb 6885-2 performed more similarly to M202 in the constant and constant with recovery period assays, suggesting that this line contains an introgressed region（s） not present in MIb 4853-9. The three assays were also applied to assess variations in cold tolerance in a set of diverse germplasms. Performance metrics and visual ratings were comparable for evaluating tolerance to constant stress. However, differences in response to intermittent stress were more evident from growth and total chlorophyll measurements than from visual ratings. With regard to the constant with recovery period assay, about 15% of the germplasm accessions did not recover. However, some lines which exhibited chilling injuries of comparable severity were able to overcome them. This recovery phenotype may improve rice performance in the field and warrant further investigation.