Rapid and high-precision marker assisted backcrossing to introgress the <Emphasis Type="Italic">SUB1</Emphasis> QTL into BR11, the rainfed lowland rice mega variety of Bangladesh

Flooding is one of the major hazards of rice production for the rainfed lowland rice ecosystem, and tolerant cultivars are urgently needed to help protect farmers from submergence damage. A quick and efficient strategy was implemented to introgress SUB1, a major QTL for submergence tolerance, into a rainfed lowland mega variety BR11 of Bangladesh by only two backcrosses and one selfing generation. In marker-assisted backcrossing (MABC), one tightly-linked simple sequence repeat (SSR) and two gene-based markers, four flanking SSR and 116 background SSR markers were used for foreground, recombinant and background selection, respectively, in backcrosses between a SUB1 donor IR40931-33-1-3-2 and BR11. BR11-Sub1, identified in a BC₂F₂ plant, possessed BR11 type SSR alleles on all fragments analyzed except the SUB1 QTL. The introgression size in BR11-Sub1 was 800 Kb indicating approximately 99.8% identity to BR11. BR11-Sub1 along with other introgression lines showed submergence tolerance similar to the tolerant parent. Yield, yield-component parameters and grain physico-chemical properties showed successful recovery of the BR11 traits in BR11-Sub1, with yield potential ranging from 5.2 to 5.6 t/ha, not significantly different from the recurrent parent mega variety BR11. Producing a large number (~1000) of backcross F₁ plants was considered essential to achieve recombination on both sides of the gene, limiting linkage drag with only two backcrosses. A large number of background markers ensured proper recovery of the recurrent parent genome in the BC₂F₂ generation. The study demonstrates a rapid and highly precise strategy to introgress a major QTL by BC₂F₂ generation into a modern rice variety using an unadapted donor. The variety can replace BR11 on more than 2 million of ha in Bangladesh and provide major increases in rice production.     

Mapping three new interspecific hybrid sterile loci between Oryza sativa and O. glaberrima

Hybrid sterility hinders the transfer of useful traits between Oryza sativa and O. glaberrima. In order to further understand the nature of interspecific hybrid sterility between these two species, a strategy of multi-donors was used to elucidate the range of interspecific hybrid sterility in this study. Fifty-nine accessions of O. glaberrima were used as female parents for hybridization with japonica cultivar Dianjingyou 1, after several backcrossings using Dianjingyou 1 as the recurrent parent and 135 BC₆F₁ sterile plants were selected for genotyping and deducing hybrid sterility QTLs. BC₆F₁ plants containing heterozygous target markers were selected and used to raise BC₇F₁ mapping populations for QTL confirmation and as a result, one locus for gamete elimination on chromosome 1 and two loci for pollen sterility on chromosome 4 and 12, which were distinguished from previous reports, were confirmed and designated as S37(t), S38(t) and S39(t), respectively. These results will be valuable for understanding the range of interspecific hybrid sterility, cloning these genes and improving rice breeding through gene introgression.     

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer’s fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F₁ hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC₁ obtained from B. juncea × F₁ hybrid, and this BC₁ exhibited a plant type like that of B. juncea. Most markers were deleted in BC₂ and BC₃ plants, with only two markers persisting in the BC₃. These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.     

Rice tungro spherical virus resistance into photoperiod-insensitive japonica rice by marker-assisted selection

Rice tungro disease (RTD) is one of the destructive and prevalent diseases in the tropical region. RTD is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. Cultivation of japonica rice (Oryza sativa L. ssp japonica) in tropical Asia has often been restricted because most japonica cultivars are sensitive to short photoperiod, which is characteristic of tropical conditions. Japonica1, a rice variety bred for tropical conditions, is photoperiod-insensitive, has a high yield potential, but is susceptible to RTD and has poor grain quality. To transfer RTD resistance into Japonica1, we made two backcrosses (BC) and 8 three-way crosses (3-WC) among Japonica1 and RTSV-resistant cultivars. Among 8,876 BC₁F₂ and 3-WCF₂ plants, 342 were selected for photoperiod-insensitivity and good grain quality. Photoperiod-insensitive progenies were evaluated for RTSV resistance by a bioassay and marker-assisted selection (MAS), and 22 BC₁F₇ and 3-WCF₇ lines were selected based on the results of an observational yield trial. The results demonstrated that conventional selection for photoperiod-insensitivity and MAS for RTSV resistance can greatly facilitate the development of japonica rice that is suitable for cultivation in tropical Asia.     

Marker assisted backcrossing for introgression of Fusarium wilt resistance gene into melon

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis is a common vascular wilt fungal disease in melon across the world. The resistance gene to race 1 of this causal agent, Fom-2, has been previously cloned and its sequence is available. The objective of this research was the introgression of Fom-2 from one resistant (Isabelle) genotype into two susceptible cultivars (Garmak and Tile-torogh) via marker assisted backcrossing. First, the leucine-rich repeats (LRR) domain of Fom-2 from resistant and susceptible genotypes was sequenced to develop functional markers. A length of 1274 bp of the 3′ end of this gene was isolated, cloned and sequenced. The difference between resistant and susceptible genotypes in this region was 28 nucleotide substitutions. Two allele specific primer pairs, Fom2-R409 and Fom2-S253, were designed based on nucleotide substitutions to amplify resistant and susceptible alleles, respectively. For introgression of the gene, donor (Isabelle) and recurrent (Garmak and Tile-torogh) parents were crossed. Resistant plants in BC₁F₁ and BC₂F₁ generations were first detected using artificial pathogen inoculation and later the plants were genotyped by functional markers to validate their resistance. The resistant plants were also selected phenotypically in each generation for background genome recovery, which conduced to high similarity of BC₃ generation with the recurrent parents. It was proved the developed markers are more precise and efficient than inoculation trial and could be used as confident tools for screening of resistant melon genotypes to Fusarium wilt.     

利用染色体片段代换系定位陆地棉株高QTL

以陆地棉中棉所36为轮回亲本和海岛棉海1为供体亲本, 构建染色体片段代换系。为了能检测到稳定的株高QTL,将三个代换系群体(BC₅F₃, BC₅F_3:4和BC₅F_3:5)在5个环境中种植,2009年和2010年分别在河南安阳种植BC₅F₃单株、BC₅F_3:4株行, 2011年分别在河南安阳、辽宁辽阳和新疆石河子种植BC₅F_3:4株系。结果表明,在不同群体环境中株高的超亲比例为53.43%~88.97%。从早期构建的总图距为5088.28 cM, 含有2280个SSR标记位点,覆盖26条染色体的遗传连锁图谱中筛选标记,对408个单株进行的SSR鉴定,结果检测到16个株高QTL,分布在10条染色体上。单个QTL解释的表型变异为7.35%~13.17%。有7个QTL在2个以上环境被检测到。与标记MUSS563紧密连锁的qPH-15-19在一个环境中被检测到,在前人的研究中也有报道。这些结果为进一步精细定位QTL、基因克隆、分子辅助选择等研究奠定基础。     

Molecular tagging and marker-assisted selection of fiber quality traits using chromosome segment introgression lines (CSILs) in cotton

Gossypium barbadense L is an important genetic resource to improve fiber quality of Gossypium hirsutum L., but breeders have generally encountered difficulties in introgression following whole genome crosses primarily due to genomic incompatibility, complex genetic basis and low efficiency of phenotypic evaluation and selection on fiber quality. Chromosome segment substitution lines (CSILs) are a powerful tool to dissect and introgress alien alleles while minimizing negative effects from alleles on other chromosome segments of the donor parent. In the present study, using a CSIL+F₂ mapping strategy, three QTLs each for fiber length (FL), fiber strength (FS) and micronaire value (MIC) were identified on chromosome 11 and 1, explaining 6.23–10.73 % of the phenotypic variation in the F_2:3 population. In addition, through marker-assisted backcrossing, the G. barbadense alleles of these QTLs were incorporated into two elite commerical Upland cotton cultivars, Lumianyan28 (L28) and Shannongmian6 (SNM6). Field evaluation indicated that 80 % of the BC₂F₃ lines containing the qFL-c11-1 and qFS-c11-1 from Hai7124 had significantly higher FL and FS, while only 14.5 % BC₂F₃ lines containing the qMIC-c1-1 in SNM6 genetic background showed significant decrease in MIC. Some BC₂F₃ lines with improved target fiber quality traits and without remarkable deviations in non-target lint yield components were obtained.     

Marker‐assisted backcrossing of a null allele of the α‐subunit of Soybean (Glycine max) β‐conglycinin with a Chinese soybean cultivar (a). The development of improved lines

The development of soybean varieties that lack the β‐conglycinin α‐subunit is an attractive goal because the β‐conglycinin α‐subunit negatively influences the nutrition and gelation of tofu and is a major allergen. To remove this undesirable allergen and simultaneously improve the seed nutritional value and food‐processing quality, marker‐assisted background selection (MABS) was used in backcross breeding to incorporate cgy‐2, a null phenotype version of the gene encoding the β‐conglycinin α‐subunit, from the donor line ‘RiB’ into the genetic background of the Chinese cultivar ‘Dongnong47’ (DN47), a popular high‐oil superfine seed soybean cultivar from Heilongjiang Province, China. In each F₂ (F₂, BC_nF₂) generation of the breeding programme, the offspring that carried the introgressed cgy‐2 were identified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and rescreened by MABS using simple sequence repeat markers to accelerate recurrent parent genome recovery. Of the 49 advanced backcrossing breeding lines (ABLs), the three best lines, ABL1, ABL2 and ABL3, were selected from the BC₁, BC₂ and BC₃ populations, respectively. The ABLs were evaluated for desirable agronomic characteristics, yield‐related traits, amino acid composition, free amino acid composition and tofu‐processing quality in the mature seeds. All of the ABLs lacked the α‐subunit but grew and reproduced normally without deleterious effects on physiological processes such as seed development and germination. The free amino acid content of ABL1 was significantly higher than that of ‘DN47’, with arginine (Arg) being particularly enriched. Compared to the recurrent parent ‘DN47’, the total protein content of the three ABLs was higher, the amino acid composition of the seed proteins was markedly modified and the yield and hardness of the tofu that was made from the ABLs were significantly increased. MABS combined with stringent phenotypic selection in a backcross breeding programme is a feasible strategy for the genetic engineering of seed protein components to produce allergenic subunit‐deficient variant alleles.     

Breeding cowpea for resistance to Striga gesnerioides in the Nigerian dry savannas using marker‐assisted selection

Historically, conventional breeding has been the primary strategy used to develop a number of Striga‐resistant varieties currently grown in the Sahel of Western Africa. In this study, we have successfully developed and applied a marker‐assisted selection strategy that employs a single backcross programme to introgress Striga resistance into farmer preferred varieties of cowpea for the Nigeria savannas. In this strategy, we have introduced the Striga resistance gene from the donor parent IT97K‐499‐35 into an elite farmer preferred cowpea cultivar ‘Borno Brown’. The selected 47 BC₁F₂ populations confirmed the recombinants with desirable progeny having Striga resistance gene(s). The 28 lines selected in the BC₁F_2:4 generation with large seed size, brown seed coat colour and carrying marker alleles were evaluated in the field for resistance to Striga resistance. This led to the selection of a number of desirable improved lines that were immune to Striga having local genetic background with higher yield than those of their parents and standard varieties.     

Modes of inheritance of two apomixis components,diplospory and parthenogenesis,in Chinese chive (Allium ramosum) revealed by analysis of the segregating population generated by back-crossing between amphimictic and apomictic diploids

To investigate the mode of inheritance of apomixis in Chinese chive, the degrees of diplospory and parthenogenesis were evaluated in F₁ and BC₁ progenies derived from crosses between amphimictic and apomictic diploids (2n = 16, 2x). The F₁ population was generated by crossing three amphimictic diploids 94Mo13, 94Mo49 and 94Mo50 with an apomictic diploid KaD2 and comprised 110 diploids and 773 triploids. All the diploid F₁ plants examined were completely or highly eusporous and completely syngamic. All the triploid F₁ plants examined were highly diplosporous and highly parthenogenetic. KaD2 could not transmit its high level of apomixis via monoploid pollen grains. The BC₁ population, generated by crossing 94Mo49 with apomictic triploids found in the F₁ offspring, exhibited heteroploidy; it comprised haploid, diploid, triploid, tetraploid and various aneuploid individuals. In this generation, clear segregation was observed between diplospory and parthenogenesis. Analysis of the BC₁ population suggests that diplospory and parthenogenesis are each controlled by single dominant genes, D and P, respectively. However, all the BC₁ plants characterized as parthenogenetic were diplosporous. The absence of phenotypically eusporous parthenogenetic plants can be explained by assuming that the presence of diplospory gene is a prerequisite for the parthenogenesis gene expression in Chinese chive.     

Identification of qRL7, a major quantitative trait locus associated with rice root length in hydroponic conditions

Root system development is an important target for improving yield in rice. Active roots that can take up nutrients more efficiently are essential for improving grain yield. In this study, we performed quantitative trait locus (QTL) analyses using 215 recombinant inbred lines derived from a cross between Xieqingzao B (XB), a maintainer line with short roots and R9308, a restorer line with long roots. Only a QTLs associated with root length were mapped on chromosomes 7. The QTL, named qRL7, was located between markers RM3859 and RM214 on chromosome 7 and explained 18.14–18.36% of the total phenotypic variance evaluated across two years. Fine mapping of qRL7 using eight BC₃F₃ recombinant lines mapped the QTL to between markers InDel11 and InDel17, which delimit a 657.35 kb interval in the reference cultivar Nipponbare. To determine the genotype classes for the target QTL in these BC₃F₃ recombinants, the root lengths of their BC₃F₄ progeny were investigated, and the result showed that qRL7 plays a crucial role in root length. The results of this study will increase our understanding of the genetic factors controlling root architecture, which will help rice breeders to breed varieties with deep, strong and vigorous root systems.     

Characterization of donor genome contents of backcross progenies detected by SSR markers in rice

Backcrossing is a trait introgression method of renewed importance in crops. The evolution of introgressed or substituted segments through backcross generations has been analyzed theoretically using simulations. In this study, the content of donor genomes, including donor segment number (DSN), donor segment length (DSL), and donor genome size (DGS), were directly analyzed in six crosses over three successive backcrosses using a set of single sequence repeat (SSR) markers covering the entire rice genome. The results of this analysis demonstrated that the average DSN in each genome was 8.39 in BC₂F₁, 4.13 in BC₃F₁, and 2.41 in BC₄F₁, decreasing nearly by half with each backcrossing. The average DSL was 33.43 cM (centiMorgans) in BC₂F₁, 29.04 cM in BC₃F₁, and 25.07 cM in BC₄F₁, display a progressive decrease slightly greater than 10% in each additional backcross generation. Meanwhile the average DGS was 280.51 cM in BC₂F₁, 119.97 cM in BC₃F₁, and 60.53 cM in BC₄F₁, decreasing 57.2% from BC₂F₁ to BC₃F₁ and 50.4% from BC₃F₁ to BC₄F₁. This revealed that the reduction in DGS was approximately 50% with each backcrossing. These results provide a guide for introgression or substitution of target chromosome segments from donors into recipients in backcross programs. Zhang-Ying Xi and Feng-Hua He contributed equally to this work.     

Improving bacterial blight resistance of'6078', an elite restorer line of hybrid rice, by molecular marker-assisted selection

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (X00), is one of the most devastating diseases of rice world‐wide; it is also a serious problem of hybrid rice production in China. In this study, a molecular marker‐assisted introgression of Xa21, a gene highly resistant to a broad spectrum of Xoo strains, from ‘IRBB21’ was performed to improve the BB resistance of‘6078′, a new restorer line with high yielding potential. The entire process took one generation of crossing followed by three generations of backcrossing and one generation of selfing. The presence of Xa21 in each generation was determined by both polymerase chain reaction (PCR) and pathogen inoculation. Recombinations between Xa21 and flanking markers were identified by PCR analysis. Background selection was conducted in BC₁F₁ and BC₂F₁ using amplified fragment length polymorphism (AFLP) markers detecting a total of 129 polymorphic bands between‘6078’ and ‘IRBB21′. The individual selected in BC₃F₂, or‘6078′(Xa21), carried a fragment of less than 3.8 cM from the donor line in the Xa21 region on chromosome 11, and about 98.8% of the genetic background from the recurrent parent. The results showed that‘6078′(Xa21) had the same level and spectrum of BB resistance as the donor parent ‘IRBB21′, while maintaining the agronomic performance and combining ability of the original 6078. A significant increase in BB resistance was also achieved in the hybrid using 6078(Xa21) as the restorer line.     

Genetic architecture of plant height in maize phenotype‐selected introgression families

This study aimed at developing, characterizing and evaluating two maize phenotypic‐selected introgression libraries for a collection of dominant plant height (PHT)‐increasing alleles by introgressing donor chromosome segments (DCS) from Germplasm Enhancement of Maize (GEM) accessions into elite inbred lines: PHB47 and PHZ51. Different backcross generations (BC₁‐BC₄) were developed and the tallest 23 phenotype‐selected introgression families (PIFs) from each introgression library (PHB47 or PHZ51) were selected for single nucleotide polymorphism genotyping to localize DCS underlying PHT. The result shows that most PIFs carrying DCS were significantly (α = 0.01) taller than the respective recurrent parent. In addition, they contained larger donor genome proportions than expected in the absence of selection or random mating across all BC generations. The DCS were distributed over the whole genome, indicating a complex genetic nature underlying PHT. We conclude that our PIFs are enriched for favourable PHT‐increasing alleles. These two libraries offer opportunities for future PHT gene isolation and allele characterization and for breeding purposes, such as novel cultivars for biofuel production.     

抗水稻纹枯病qSB-9Tq基因效应及作用方式分析

水稻第9染色体上存在1个抗纹枯病QTL,被命名为qSB-9,水稻品种特青在该QTL上携带抗性等位基因qSB-9^Tq,而Lemont携带相对感病等位基因qSB-9^Le。为精确地评价qSB-9^Tq的抗病效应,分析其作用方式,利用分子标记进行前景选择和背景选择,从轮回亲本Lemont与特青回交后代群体中筛选到1个目标单株。连续3年对该单株的扩繁后代(BC₆F₂)及随后获得的近等基因系采用嵌入法进行接种鉴定试验。田间试验采取2种不同的设计。第一种是完全随机试验,即从BC₆F₂分离群体中筛选出目标区间为qSB-9^TqTq纯合型、qSB-9^LeLe纯合型和qSB-9^TqLe杂合型个体,并对3种基因型个体间的病级平均数差异进行统计分析。第二种设计为随机区组设计,即在BC₆F₃和BC₆F₄代,分别对上述3种基因型的近等基因系群体,按3次重复的随机区组设计进行移栽和接种鉴定试验。结果表明,3年的试验结果表现出一致的趋势,即qSB-9^Tq存在于分子标记RM242~Y92.5之间,可减轻病级1.0级(0~9级病情分级系统)左右,且其抗性表现为几乎完全的显性特征。本研究的结果为qSB-9^Tq的精细定位和育种利用奠定了基础。     

Bulk pollen pollination in maize for efficient construction of introgression populations with high genome coverage

Introgression populations consist of a set of introgression lines or families, constructed by continuous backcrossing to the recurrent parent, while carrying a limited number of chromosome segments from a donor parent in their genomes. Increasing the genome coverage is an important aim when constructing introgression population. In this study, we proposed bulk pollen pollination (BPP) method and used it to increase the genome coverage of a maize introgression population. The results showed that the genome coverage of the introgression population constructed using BPP method reached 100% at BC₃ generation, which accorded with the simulation result. The BPP‐based BC₃F_1:2 population could identify most quantitative trait loci (QTL) detected using the F_2:3 population, especially major QTL. Simulation analysis showed that the genome coverage of introgression population increased with the increase of population size and the number of bulked plants, and decreased with the increase of backcross generation. Our results proved the reliability of the BPP‐based introgression population in increasing genome coverage and detecting QTL, and provided references for constructing high‐coverage introgression populations.     

Transfer of architectural traits from perennial Helianthus mollis Lam. to sunflower (H. annuus L.) and localisation of introgression

Common sunflower (i.e., Helianthus annuus) is occasionally crossed with other Helianthus species to gain favourable agronomic traits. An interspecific cross between H. annuus and Helianthus mollis was used to examine events that occurred during the five first generations. First generation hybrid plants were verified using GISH, and F₁ hybrid (#17) was back-crossed to common sunflower to obtain BC₁ (#4) and BC₂ (#21). Some BC₂ progeny possessed new traits, i.e., compact (Compact plant architecture is due to short inter-node length, short petiole length and short leaf lamina length) architecture without and with corrugated leaves, which were not observed in the parents. These traits were selected for and fixed after the BC₂-S₃ (#18) to produce a H. mollis introgression line (namely HM374). Although the size of the population was small, we tentatively construct a genetic map using data from the BC₂ generation. Two linkage groups were identified carrying H. mollis specific AFLP markers that were associated with compact plant architecture, and one unlinked marker was shown to explain variation for the corrugated leaves. Introgression line HM374 was used to test the inheritance of compact plant architecture in a cross with an elite high oleic line, OL75HO. Microsatellite (SSR) markers were used to genotype F₁ and F₂ offspring’s. Our data suggested that the line HM374 was still heterozygous for introgression after eight generations. HM374 displays new architecture traits (i.e. compact architecture with or without corrugated leaves) and carries H. mollis fragments anchored on LG1 and LG11 of sunflower public map.     

Parent genome contribution and its relationship with grain yield in backcross-derived lines of soybean (Glycine max)

Introgression of yellow mosaic disease (YMD) resistance and effect of recurrent parent genome (RPG) on grain yield was studied in 84 soybean genotypes from four populations namely, F_2:7, BC₁F₆, BC₂F₅ and BC₃F₄ derived from cross JS335 x SL525. It was observed that in F_2:7, BC₁F₆, BC₂F₅ and BC₃F₄ derived lines, RPG contribution was 42.5%, 54.9%, 66.4% and 77.6%, respectively, which is significantly less than expected values. Linkage drag from donor parent with YMD resistance gene may be a possible reason for such deviations. Average grain yield per plant in F_2:7, BC₁F₆, BC₂F₅ and BC₃F₄ generations was observed as 13.0, 14.3, 14.9 and 16.1 g, respectively. It was observed that genotypes with more than 80% RPG observed to have both YMD resistance and good yield potential. Graphical genotyping (GGT) analysis revealed that maximum RPG was recovered in chromosomes 8 and 10 and maximum introgression occurred in chromosomes 6 and 19. Our results demonstrated that RPG was positively associated with yield as evident from yield increase with increase in RPG.     

一个水稻落粒性基因SH1的SSR标记定位

以籼稻品种93-11为轮回亲本,与粳稻品种日本晴杂交并回交的高世代分离群体为研究材料,选用104个多态性的SSR标记对水稻的落粒性基因进行定位。结果表明,在BC₄F₂群体中,6个标记的基因型来自于日本晴;在BC₄F₃定位群体中,难落粒植株数与易落粒植株数的分离比例为3:1,落粒性受1对显性基因控制,命名为SH1;分子标记与落粒性共分离分析将SH1定位在SSR标记RM5389和RM1068、RM1387之间,与3个标记的遗传距离分别为0.7cM、5.5cM和13.1cM,此结果为该基因的分子标记辅助选择奠定了基础。     

Comparing mating designs to restore seed production of interspecific hybrids between Trifolium repens (white clover) and Trifolium uniflorum

Interspecific hybrids between Trifolium uniflorum and cultivated white clover (Trifolium repens) have highly useful characteristics for temperate pastoral systems derived from both parent species. However, the early hybrids (F₁ and BC₁) also have unacceptably poor seed production for commerce. This study analysed the basis for the poor seed production and investigated breeding strategies for overcoming the problem. The BC₁F₁ generation produced lower‐than‐expected numbers of heads per plant and seeds per floret. Backcrossing of selected hybrids to white clover corrected these deficiencies and created new variation. Seed numbers were also returned to near target levels by recurrent selection within the BC₁ generation. Thus, it was possible to retain a theoretical average of 25% of T. uniflorum genome and still achieve high seed production per plant. The BC₁F₂ and BC₂F₁ generations produced high seed numbers per plant, along with reasonable variation. Both of these second‐generation hybrid forms have high reproductive potential and should be the focus for the selection of the desired combinations of agronomic and seed production traits.