Discrimination of haploid and diploid maize kernels via multispectral imaging

The use of doubled haploids (DHs) in maize has become ubiquitous in maize breeding programmes as it allows breeders to go from cross to evaluation in as little as 2 years. Two important aspects of the in vivo DH system used in maize are as follows: (i) the identification of haploid progeny and (ii) doubling of the haploid genome to produce fertile inbred lines. This study is focused on the first step. Currently, identification of maize haploid progeny is performed manually using the R1‐nj seed colour marker. This is a labour‐intensive and time‐consuming process; a method for automated sorting of haploids would increase the efficiency of DH line development. In this study, six inbred lines were crossed with the maternal haploid inducer ‘RWS/RWK‐76’ and a sample of seed was sorted manually for each line. Using the VideometerLab 3 system, spectral imaging techniques were applied to discriminate between haploids and hybrids. Using DNA markers to confirm the haploid/diploid state of the tested seed, for the majority of genotypes haploid identification was possible with over 50% accuracy.     

Classification approaches for sorting maize (Zea mays subsp. mays) haploids using single-kernel near-infrared spectroscopy

Doubled haploids (DHs) are an important breeding tool for creating maize inbred lines. One bottleneck in the DH process is the manual separation of haploids from among the much larger pool of hybrid siblings in a haploid induction cross. Here, we demonstrate the ability of single-kernel near-infrared reflectance spectroscopy (skNIR) to identify haploid kernels. The skNIR is a high-throughput device that acquires an NIR spectrum to predict individual kernel traits. We collected skNIR data from haploid and hybrid kernels in 15 haploid induction crosses and found significant differences in multiple traits such as percent oil, seed weight, or volume, within each cross. The two kernel classes were separated by their NIR profile using Partial Least Squares Linear Discriminant Analysis (PLS-LDA). A general classification model, in which all induction crosses were used in the discrimination model, and a specific model, in which only kernels within a specific induction cross, were compared. Specific models outperformed the general model and were able to enrich a haploid selection pool to above 50% haploids. Applications for the instrument are discussed.     

Germplasm enhancement of maize: a look into haploid induction and chromosomal doubling of haploids from temperate‐adapted tropical sources

The allelic diversity (AD) project of the Germplasm Enhancement of Maize (GEM) programme utilized the double haploid (DH) breeding method to expedite development and release of lines derived from 300 exotic maize races. Using 18 races in this study, differential effects on haploid induction rates (HIRs) and doubling rates (DRs) by the recurrent parents PHB47 and PHZ51, the elevation that the race is traditionally grown at, and by the race itself were addressed in this study. Races from the AD project were grouped by elevation of their origin, high, middle or low altitude. Six races per elevation were randomly selected and backcrossed using both recurrent parents to generate 36 populations. Ten replications were randomized in a complete randomized design for two growing seasons. The recurrent parent effect was significant, with PHB47 having a higher HIR than PHZ51. Effect of elevation was significant with higher HIR associated with low‐elevation origin, and race also proved to be significant. Effects of elevation, recurrent parent and race were not significant for DR.     

Early spontaneous diploidization of maternal maize haploids generated by in vivo haploid induction

The production of doubled haploid (DH) lines has become a key technology in maize (Zea mays L.) research and breeding. However, most of the haploid plants are sterile and in many cases artificial chromosome doubling involves the use of costly and toxic chemicals. Here, we report a special kind of doubled haploid named the early doubled haploid (EH) that was generated directly by in vivo haploid induction. We found 83 EH plants induced from the hybrid Zhengdan958, 55 families of its F_2:3 population and the parental lines, all of which were confirmed to be homozygous diploids via flow cytometry and 104 SSR markers. The progeny of EH₀ (EH₁) behaved in the same manner and showed the same potentialities as the parents of Zheng58 and Chang7-2. EH plants were also detected in other genetic backgrounds at a frequency of 1–3.5 % based on the total number of haploid plants. Because the EH lines exhibited completely fertility and were obtained from induction directly in one step, they could be used in DH breeding as a new breeding strategy. According to our observations, it is likely that spontaneous doubling in EH occurred during embryo development when haploid induction. The possible mechanism of EH is also discussed.     

Agronomic performance of anther‐derived doubled haploid and single seed descent lines in crosses between primary and secondary winter triticale

Octoploid (8x) and hexaploid (6x) primary triticales (xTriticosecale Wittm.) can be used as crossing parents with secondary 6x triticales to enlarge the genetic basis of a breeding programme or introgress traits. Doubled haploid (DH) production permits to develop homozygous lines more rapidly from a segregating generation than other breeding methods such as single seed descent (SSD). Both anther‐derived DH and SSD lines were produced from reciprocal cross‐combinations between 8x and 6x primary and 6x secondary triticales. Field experiments of DH and SSD lines were conducted in three environments as two‐replicate lattices to measure seven agronomic traits. A tendency for higher grain yield, taller plants and a higher 1000‐kernel weight of SSD lines compared with DH lines was found. Significant genetic variation for all traits in both breeding methods was revealed, indicating their suitability to select superior genotypes. Hexaploid and even more so 8x primary triticales can profitably be included as crossing parents in a commercial breeding programme. In such crosses, the primary triticales should be used as the male parents if followed by DH method.     

Conditions favouring doubled haploid breeding over conventional breeding of self-fertilizing crops

Summary Monte Carlo computer simulation was used to investigate the conditions favouring doubled haploid breeding over conventional breeding of self-fertilizing crops. Two different systems of doubled haploid breeding and three systems of conventional breeding were compared for two criterion parameters, i.e., the probability of obtaining desirable genotypes and the expected genetic advance of selected lines. It was inferred that the efficiency of production of haploid and doubled haploid plants primarily determines the success of the doubled haploid breeding method. In doubled haploid breeding, about 1/5, hopefully 1/2 as many test plants need to be raised as in conventional breeding to achieve the same level of success. With this condition begin satisfied, the doubled haploid breeding method can efficiently be used when one or more of the following conditions are met: (i) a relatively small number of loci, presumably ten of less, is involved with the breeding objective concerned, (ii) desirable alleles are recessive to undesirable ones at most, if not all, of the segregating loci, and (iii) the genes are not strongly linked. It was confirmed that the doubling of haploids can better be applied to selected F₂ plants rather than to F₁ plants.     

Marker-assisted increase of genetic diversity in a double-low seed quality winter oilseed rape genetic background

Generation of novel genetic diversity for maximization of heterosis in hybrid production is a significant goal in winter oilseed rape breeding. Here, we demonstrate that doubled haploid (DH) production using microspore cultivation can simultaneously introgress favourable alleles for double‐low seed quality (low erucic acid and low‐glucosinolate content) into a genetically diverse Brassica napus genetic background. The DH lines were derived from a cross between a double‐low quality winter rapeseed variety and a genetically diverse semisynthetic B. napus line with high erucic acid and high glucosinolates (++ quality). Twenty‐three low‐glucosinolate lines were identified with a genome component of 50–67% derived from the ++ parent. Four of these lines, with a genome component of 50–55% derived from the ++ parent, also contained low erucic acid. Heterosis for seed yield was confirmed in test‐crosses using these genetically diverse lines as pollinator. The results demonstrate the potential of marker‐assisted identification of novel genetic pools for breeding of double‐low quality winter oilseed rape hybrids.     

Low relative humidity increases haploid production in durum wheat X maize crosses

Haploidization is a useful tool for genetic analysis and plant breeding, but a consistent and satisfactory protocol for haploid production has been difficult to achieve in durum wheat. The objective of this study was to analyze the influence of the relative humidity of the environment, when culturing detached tillers during the production of haploids plants in durum wheat by the maize method. Thirty‐eight F3 lines from eight crosses of durum wheat were pollinated with bulked pollen from three commercial maize hybrids. A mixture of 2‐4D and dicamba was used as a hormone treatment. The numbers of caryopses, embryos and haploids plants were scored. When 65‐85% (light‐dark) humidity was substituted for 55‐65% the number of haploids per spike increased notably. This increased frequency was largely attributed to a rise in the production of generated caryopses. On average, 15.2 vs. 9.3 caryopses, 5.0 vs. 2.8 embryos, and 3.1 vs. 0.6 haploid plants, per spike, were produced under low and high humidity regimes, respectively.     

Rye doubled haploids as a research and breeding tool – a practical point of view

Doubled haploid (DH) plants were produced using anther culture from out‐crossing rye, including breeders’ lines, cultivars and F₁ plants with DH parents, to examine the feasibility of using the DH technique for breeding and specifically for developing mapping populations. Only 10–36% of green regenerants produced via anther culture were suitable for research or breeding purposes because of low survival rate or low fertility. Spontaneously arising DH regenerants were more often fertile compared with the colchicine‐treated ones. The fertility of spontaneous DHs varied from sterile to half that found in a normal rye population, which has implications for the design of a crossing scheme and subsequent anther culture. In the reciprocal crosses within one DH population, fertility was the lowest observed, probably because of self‐incompatibility factors, whereas in the DH crosses with normal heterozygous cultivars fertility was the highest. Two mapping populations using DHs were established, the first for out‐crossing rye it would seem. These populations will be used for mapping two important traits, the semi‐dwarf growth habit and preharvest sprouting resistance in rye.     

Genetic dissection of haploid male fertility in maize (Zea mays L.)

Haploid genome doubling is a key limiting step of haploid breeding in maize. Spontaneous restoration of haploid male fertility (HMF) provides a more promising method than the artificial doubling process. To reveal the genetic basis of HMF, haploids were obtained from the offspring of 285 F_2:3 families, derived from the cross Zheng58 × K22. The F_2:3 families were used as the female donor and Yu high inducer No. 1 (YHI‐1) as the male inducer line. The rates of HMF from each family line were evaluated at two field sites over two planting seasons. HMF displayed incomplete dominance. Transgressive segregation of haploids from F_2:3 families was observed relative to haploids derived from the two parents of the mapping population. A total of nine quantitative trait loci (QTL) were detected, which were distributed on chromosomes 1, 3, 4, 7 and 8. Three major QTL, qHMF3b, qHMF7a and qHMF7b were detected in both locations, respectively. These QTL could be useful to predict the ability of spontaneous haploid genome doubling, and to accelerate the haploid breeding process by introgression or aggregation of those QTL.     

Identification of the fourth allele of the ANS (anthocyanidin synthase) gene and its effect on red color intensity in onions (Allium cepa)

Bulb color in onions (Allium cepa) is an important trait, and homogenous red coloration is desirable in red onion cultivars. The gene encoding anthocyanin synthase (ANS) is required for anthocyanin biosynthesis in onions. We have previously described three different alleles of the ANS gene. Here we report identification of the fourth allele of ANS, ANS-h1, found in a dark red doubled haploid line. ANS-h1 is similar to a non-functional allele found in Brazilian yellow cultivars except that it has several point mutations and indels throughout the promoter and coding regions, none of which are predicted to inactivate enzymatic activity. F₂ and backcross populations originating from the crosses between wild-type (ANS-L) allele-containing red and pink (ANS-p) allele-containing white or yellow parents show a discrete segregation ratio of 3 red to 1 light pink, indicating that the wild-type allele is completely dominant over the pink allele. In contrast, segregating populations derived from the crosses between ANS-h1 allele-containing red and the same white or yellow parents show a gradient of red intensity from light pink to dark red, suggesting that other genetic factors may affect expression of ANS-h1. A newly developed PCR-based marker and two previously developed markers for allelic selection of the ANS gene were used to examine allele composition in fifty-six breeding lines and commercial cultivars. Most lines are heterogeneous for the ANS gene with two or three alleles detected. The frequency of the pink allele is low in red breeding lines, but it is predominant in white and yellow lines.     

Advances on Plant Cell Engineering Technology Application in Rapeseed Breeding

综述了近20年来国内外利用植物细胞工程技术进行油菜育种所取得的成就。器官、组织和单细胞培养不仅可用于繁殖和保存种质，而且已用来诱导体细胞变异，产生突变体。花药、小孢子体系的建立，促进了单倍体育种的进行，加速了作物育种进程。子房、胚珠和胚培养以及体细胞杂交可用于克服远缘杂交的不亲和性和杂种的不育性，有利于拓宽种质资源。通过分析提出了在今后的油菜育种中，植物细胞工程技术将是一种重要的辅助育种手段。     

Effect of thidiazuron on microspore embryogenesis and plantlet regeneration in Chinese flowering cabbage (Brassica rapa. var. parachinenis)

Traditional breeding methods require more than 6 years to obtain homozygous inbred lines, while isolated microspore culture (IMC) is an effective way to cultivate double haploid homozygous lines in only 2 years. However, low embryogenesis induction frequency in Chinese flowering cabbage remains a key obstacle to the practical application of this technique. Thidiazuron was added at different concentrations to NLN‐13 medium to estimate its effects on microspore embryogenesis and plantlet regeneration. Results showed that three genotypes responded positively. Optimum thidiazuron concentrations produced embryo yields of up to 14.67 embryos per bud and increased microspore embryogenesis frequency with up to 100% survival. Plantlet regeneration rates were up to 81.67%, and the treatment groups showed lower callus formation. We obtained up to 552 diploid plants from the tested genotypes, and the percentage of doubled haploid at different TDZ concentrations showed slight differences, and doubled haploid rates in the three genotypes were above 70%. They showed a high uniformity and can be directly used for hybrid breeding. This method accelerates microspore application in Chinese flowering cabbage hybrid breeding.     

玉米生物诱导孤雌生殖单倍体影响因素研究进展

玉米单倍体育种技术能显著缩短育种周期,提高育种效率,在现代商业化育种中占有重要的地位。规模化高效单倍体的产生是单倍体育种技术应用的前提条件。总结了父本诱导系、母本的遗传背景、诱导的环境、授粉方式等影响玉米生物诱导孤雌生殖单倍体产生的因素,通过文献综合分析得出了获得大量的孤雌生殖单倍体除了选择合适的高频率孤雌生殖诱导系外,同时要考虑其他诱导因素的影响。并就高效生产孤雌生殖单倍体的未来研究及发展方向进行了讨论,以期促进单倍育种技术的规模化应用。     

林木基因工程研究进展

林木生长周期长,采用常规育种技术进行新品种选育所需时间长、见效慢,同时还存在基因源缺乏和杂交不亲和等制约因素。基因工程是生物技术的核心,为林木遗传改良开辟了一条新的途径。因此依靠现代基因工程与常规育种技术相结合,可极大地缩短林木育种周期,加速育种进程,创造新种质,选育新品种,对营造优质人工林,缓解木材供需矛盾,保护生态环境具有重要意义。近年来,一些新的技术和方法的应用,如体胚转基因系统和超声波辅助根癌农杆菌介导法,以及很多有用目的基因的克隆促使林木基因工程取得了可喜的进展。本文就应用于林木基因工程的新技术和新方法,以及林木木质素改良、缩短林木育种周期和促进开花、林木生长性状改良和植物修复(林木抗环境污染)等基因工程方面所取得的进展进行了概述。     

玉米生物诱导孤雌生殖单倍体影响因素研究进展

玉米单倍体育种技术能显著缩短育种周期,提高育种效率,在现代商业化育种中占有重要的地位。规模化高效单倍体的产生是单倍体育种技术应用的前提条件。总结了父本诱导系、母本的遗传背景、诱导的环境、授粉方式等影响玉米生物诱导孤雌生殖单倍体产生的因素,通过文献综合分析得出了获得大量的孤雌生殖单倍体除了选择合适的高频率孤雌生殖诱导系外,同时要考虑其他诱导因素的影响。并就高效生产孤雌生殖单倍体的未来研究及发展方向进行了讨论,以期促进单倍育种技术的规模化应用。

     

单倍体育种技术的应用进展

为了给今后西番莲等热带果树的单倍体育种研究提供理论指导,文章归纳了人工诱导孤雄生殖、人工诱导孤雌生殖、染色体加倍等不同单倍体育种培养技术的技术要点及发展现状,总结了单倍体育种技术在禾谷类作物、花卉、林木和果树育种方面的应用进展。同时指出,单倍体育种技术的发展有利于建立作物品种纯合系,推进遗传学研究,形成新的基因型,加快热带果树的育种进程。     

Tuber yield and quality of 4x-2x (FDR) potato progenies derived from the wild diploid species Solanum berthaultii and Solanum tarijense

Field experiments were carried out in order to evaluate 4x‐2x families derived from crosses between elite 4x potato cultivars and 2x Tuberosum‐Solanum tarijense and 2x Tuberosum‐Solanum berthaultii clones. Three traits were assessed: total tuber yield (TTY), haulm maturity (HM) and general tuber appearance (GTA). The degree of heterosis of these hybrid families was evaluated by comparison with the respective 2x and 4x parents as well as with seven 4x cultivars. The parental haploid species hybrids derived from S. berthaultii and S. tarijense combined two or more positive horticultural characteristics. Expressed as yield percentage of the 4x parents, the TTY of the families ranged from 53% to 246%. For TTY, the best 4x‐2x hybrid family ranked better than seven out of nine elite 4x cultivars. Some families had GTA scores in the range of the highly selected 4x cultivars. The families, however, were generally later maturing than the 4x parent group. Specific combining ability for TTY and GTA were the only two significant sources of variation observed in this genetic material. Parent‐offspring correlation coefficients were low for all traits, and indicated that parental performance would not be informative at either ploidy level. These results parallel previous investigations with distinct haploid species hybrids where a 4x‐2x breeding scheme was found to be an effective strategy for increasing progeny TTY over the 4x parents. However, the high degree of heterosis for TTY along with good GTA scores observed in certain cross combinations derived from these unadapted 2x species was a somewhat surprising result. Thus far, the importance of these two South American wild potato species, from the potato‐breeding standpoint, has been limited to the fact that they are natural reservoirs of major genes controlling resistance against important diseases and insects. However, the level of performance of some 4x‐2x families in comparative assays with elite cultivars suggests the unanticipated possibility of introgressing genetic factors from S. berthaultii and S. tarijense with positive effects on quantitative traits of horticultural importance along with these major resistance genes.     

Quantitative trait locus analysis of seed germination,seedling vigour and seedling‐regulated hormones in Brassica napus

Good germination and seedling vigour are major breeding targets in winter oilseed rape (Brassica napus), because seedling vigour and prewinter crop establishment are closely associated with postwinter growth and yield. Here, we identified quantitative trait loci (QTL) related to germination, seedling vigour and seedling‐regulated hormones in a doubled haploid (DH) mapping population from a cross between winter oilseed rape parents with high vigour (Express 617) and low vigour (1012‐98). By phenotyping in a climate‐controlled glasshouse, we identified a total of 13 QTL on nine chromosomes for germination and seedling‐related traits at 7 and 14 days after sowing (DAS), explaining up to 11.2% of the phenotypic variation for seedling vigour. Forty‐seven metabolic QTL on 15 chromosomes were identified for auxin, abscisic acid (ABA) and dihydrophaseic acid (DPA) at 5 and 12 DAS, explaining up to 49.4% of phenotypic variation in seedling hormone composition. Multitrait QTL hot spots contribute to our understanding of the genetics and metabolomics of germination and seeding vigour in B. napus, and represent potential targets to breed high‐vigour cultivars.     

同一基础材料的玉米双单倍体(DH)系配合力的分析

利用高频玉米单倍体诱导系,对选系材料M35/F35的F1代进行诱导获得大量单倍体籽粒,经自然加倍获得一批纯合双单倍体(DH)系,用测验系京24组配杂交组合分析DH系的配合力表现。结果表明,来源于同一基础材料的不同DH系之间,配合力差异较大,部分DH系的配合力比亲本增加,说明采用单倍体育种可以选育到高配合力的优良玉米自交系,组配出优良品种,从而加快育种进程,应用前景广阔。