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.     

Mapping of maternal QTLs for in vivo haploid induction rate in maize (Zea mays L.)

Haploid technology can significantly shorten the time required for inbred line improvement, accelerate the breeding process, and reduce breeding costs. The production of haploids is not only dependent on the genetics of the paternal haploid inducer, but it is also affected by the genetic background of the maternal donor during the process of haploid production. To address the maternal genetic contribution to haploid production, we pollinated a mapping population consisting of 186 F_2:3 family lines derived from a cross between Zheng58 and Chang7-2 with the inducer line CAU5 and selected haploid kernels using R1-nj kernel markers. Two quantitative trait loci (QTLs), qmhir1 and qmhir2, which contribute to the maternal genetics of haploid induction, were detected on chromosomes 1 and 3, respectively. The qmhir1 locus is located between the flanking marker loci umc1292 and bnlg1014, and it explained 14.70 % of the phenotypic variation. The qmhir2 locus is located between marker loci umc1844 and umc2277 and it explained 8.42 % of the phenotypic variation. The genetic effect of both QTLs is partial dominance.     

Chromosome doubling of haploid maize seedlings using nitrous oxide gas at the flower primordial stage

In maize, inbred lines are used for the production of hybrid varieties. Corn breeders and researchers have considered using haploids to develop inbred lines; however, this procedure has not been practically applied because of the inefficiency of chromosome doubling of maize haploid seedlings. In this report, a procedure has been developed to overcome this difficulty. Maize haploid seedlings obtained from eight different genotypes were treated with nitrous oxide gas (2 days at 600 kPa). Treatment at the six‐leaf stage (flower primordia formation stage) significantly increased the occurrence of fertile sectors on both tassels and ears so that approximately half (44%) of the treated haploids produced kernels after self‐pollination. In the control, only 11% of haploids produced selfed kernels owing to spontaneous chromosome doubling. A strong genotypic effect on the occurrence of fertile sectors after the treatment was observed. This procedure can be used for inbred line development in maize breeding programmes.     

Weighing in on a method to discriminate maize haploid from hybrid seed

The doubled haploid breeding method can produce maize inbred lines faster than traditional methods, but there are challenges associated with it. Sorting haploid from hybrid seed based on visual colour markers is time consuming and can be difficult due to colour inhibitors that obscure pigmentation needed to distinguish between haploid, hybrid and outcrossed seed. In this study, weight was evaluated as a method to sort haploid from hybrid seed. A first experiment utilized two families for analysis in a preliminary study. Eleven haploid and hybrid kernels from both families were weighed for a total of 44 experimental units. A second experiment was carried out using six families, using the same format as the previous, for 132 experimental units. Hybrid seed weighed significantly more than haploid seed in both experiments. However, the interaction between line and kernel type was significant in the second experiment. In conclusion, efficacy of sorting haploid from hybrid kernels based on weight depends on the genotypes involved.     

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.     

Properties of maternal haploid maize plants and potential application to maize breeding

Summary Presented are the results of a two-year study of haploid maize plants in the field. The haploids were produced with the aid of inducer line ZMS. In total, 604 and 1030 haploids were obtained and studied in the first and second years, respectively. Tassels of haploid plants were found to be almost completley sterile. Fertility of ears was studied by pollinating them with the pollen from diploid inbred lines, the cross resulting in almost all of the haploid ears carrying kernels. On average 27.4 kernels per ear of haploid plant were obtained in the first year of study and 26.3 in the second. These gave rise to normal diploid plants. This property allows genotypes selected at the level of haploid plants to be involved in breeding process. Unusual plants were found among haploids, phenotypically resembling homozygous lines. It was assumed that the plants had resulted from spontaneous chromosome doubling in haploids. The results of comparative studies of progenies of unusual plants and inbred lines derived from the same synthetic population are presented.     

Molecular identification of Pm12-carrying introgression lines in wheat using genomic and EST-SSR markers

The traditional process of obtaining maize hybrids involves the generation of inbred lines through successive generations of selfing and subsequent testcrosses in order to identify the best combining ability by allelic complementation. A fast alternative to obtain inbred lines is to induce the formation of haploids followed by chromosome doubling. However, even with the aid of haploid-inducing genetic sources, this strategy has not been widely used in maize breeding programs, partly due to difficulties inherent to haploid generation and identification. In order to evaluate the possibility of using dihaploids to generate homozygous maize tropical lines, we used the androgenetic haploid inducer line W23 as a female parent in crosses with the tropical single-cross hybrid BRS1010. Within the progeny of these crosses, 462 seeds were phenotypically selected as putative haploids by the purple-colored endosperm and colorless embryo conditioned by the R1-nj gene. Among these, only four individuals were confirmed as being haploids using SSR markers, chromosome counting and flow cytometry, showing that the phenotypic marker was not efficient in detecting haploids in the tropical maize genotype used. All four haploids as well as some diploid plants presented reduced size, corroborating the difficulties for haploid identification by phenotypic evaluation. Genetic diversity analysis revealed by SSR markers divided the haploids in two groups represented by flint and dent maize inbred lines, which could be helpful in identifying complementary dihaploid lines. The present article demonstrates that a combination of haploid production and SSR fingerprinting is a feasible strategy for maize hybrid development in tropical germplasm.     

Particle-Bombardment-Mediated Co-Transformation of Maize with a Lysine Rich Protein Gene (sb401) from potato

Summary Little work has been reported on genetic transformation with maize inbred lines, especially elite inbred lines used in breeding. In this work, 7 self-pollinated inbred lines and 4 hybrid lines have been screened. The results revealed that calli derived from immature embryos from two inbred lines X333 and X301 were compact, hyperhydric and unsuitable for transformation, but the calli induced from other inbred lines and all the hybrid lines were friable and yellow and could be used for genetic transformation. The sb401 gene isolated from potato (Solanum berthaultii) encodes a protein with a high lysine content. Maize calli from 5 self-pollinated inbred lines and 4 hybrid lines were transformed using particle-bombardment with different plasmids to simultaneously introduce the sb401 lysine rich gene and the selectable gene hpt respectively. Two hundred and sixty-eight regenerated plants were obtained from these genotypes. Co-insertion was confirmed in 29 regenerated plants by PCR and Southern blot analysis. Transgene segregation of the R1 plants was observed and one marker-free transgenic maize line was recovered. Analysis of the crude protein content in mature seeds of R1 transgenic plants also showed an increase from 36.8% to 48.2%. This study thus provides a workable system for generating transgenic maize free from selectable marker genes and generates valuable resources for obtaining marker free transgenic maize with a high-lysine protein content.     

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.     

Regular segregation of four recessive marker genes among maternal haploids in maize

The objective of the study was to investigate whether a population of maternal haploid plants represents a random gametic array. Four inbred lines of maize (A619, MK01, 092 and 19‐3‐3) were used in the present study. These were crossed with line TO carrying four recessive genes: brown midrib (bm2), liguleless (Ig1), white endosperm (g1) and golden plant (g1). Maternal haploids were produced from the F ¹ hybrids, using a haploid‐inducing pollinator line. Segregation of haploids for the marker genes was estimated under field conditions. The observed segregation data agreed with the expected 1:1 ratio. It is concluded that no segregation distortion occurred during the production of maternal haploids.     

Effectiveness of quantitative resistance conferred by the genetic background of pepper in the control of root‐knot nematodes and influence onto durability of Me1‐ and Me3‐resistant genes in greenhouse conditions

In pepper (Capsicum annuum), the major genes (R‐genes) Me1 and Me3 confer resistance against root‐knot nematodes (Meloidogyne spp.). The combination of R‐genes and quantitative resistance factors in the same genotype is considered a good breeding strategy for increasing the durability of R‐genes. To ascertain this hypothesis, five pepper inbred lines, differing in their quantitative resistance level, were combined with Me1 or Me3 genes in F₁ hybrids. The resistance of inbred lines and F₁ hybrids was evaluated in a greenhouse with soil naturally infected by M. incognita in two successive growing years. In both years, lines carrying Me3 were less infected by the nematode when combined with quantitative resistance. An increase in nematode infection was observed in the second growing year in lines carrying Me1 or Me3, independently of quantitative resistance. The infection level recorded in inbred lines without R‐genes was similar in both years. The effectiveness of quantitative resistance controlling M. incognita is confirmed in greenhouse conditions, although the durability of Me1 and Me3 when combined with quantitative resistance factors was not seen to increase.     

Comparative Field Performance of Tissue Culture-Derived Lines and Breeder Lines of HY320 Spring Wheat

This study examines the performance of somaclonal spring wheat (Triticum aestivum L.) lines developed through culture of somatic embryos. Twenty-nine breeder lines of the spring wheat cv. ‘HY320’ were compared to 51 somaclonal lines of the same cultivar. Somaclonal lines were derived from 27 individual embryos (with up to four lines in each “family”). Somaclonal and breeder lines were evaluated in five field experiments in western Canada. Somaclonal lines were more variable than breeder lines for most agronomic, yield component and quality characters, suggesting that variability among somaclonal lines resulted partly from the tissue culture process. Somaclonal lines yielded, on average, 11 % less than breeder lines. Somaclonal lines had 3.8 % fewer spikelets per spike, 6.5 % fewer kernels per spike and kernels which were 2.7 % lighter. Somaclonal lines had greater test weight, protein concentration, and sedimentation values, and harder kernels.     

利用高油分的花粉直感效应鉴别玉米单倍体

通过对农大高诱1号衍生材料的诱导率和含油量的分析,筛选出了一个诱导率与含油量均较高的高诱株系.该系油分具有显著的花粉直感效应,父本效应值为0.38,以其为父本与普通玉米杂交,杂交当代籽粒含油量显著高于自交籽粒和单倍体籽粒,花粉直感的增油率在30%以上.利用这一区别所发展的单倍体油分筛选法准确率超过90%,明显高于标记     

A new approach to Fourier transform Raman: Identification of haploids in maize (Zea mays)

The objective of this work was to adapt the FT Raman spectroscopy analysis in the differentiation of haploid and diploid kernels in maize, developing a new efficient, agile, precise, and nondestructive methodology. The main difference observed in FT Raman readings was a peak in the region between 1600 and 1700 cm⁻¹ in possibly haploid kernels. It was possible to correlate the characteristics of the kernels with the presence of the R1-nj gene and the readings obtained in the Raman spectrometry technique. Most of the kernels previously classified as haploid showed positive values for principal component analysis (PCAs), indicating a correlation in the identification of haploids by the techniques adopted. The identification of haploids by R-Navajo was superior to FT Raman. However, FT Raman spectroscopy is an agile analysis technique that enables the development of non-invasive and non-destructive analytical methods in maize kernels, in addition to providing relevant information about the chemical structures present in the composition of the samples.     

Development of yellow-seeded Brassica napus of double low quality

Two yellow‐seeded white‐petalled Brassica napus F₇ inbred lines, developed from interspecific crosses, containing 26–28% emcic acid and more than 40 μmol glucosinolates (GLS)/g seed were crossed with two black/dark brown seeded B. napus varieties of double low quality and 287 doubled haploid (DH) lines were produced. The segregation in the DH lines indicated that three to four gene loci are involved in the determination of seed colour, and yellow seeds are formed when all alleles in all loci are in the homozygous recessive state. A dominant gene governed white petal colour and is linked with an erucic acid allele that, in the homozygous condition, produces 26–28% erucic acid. Four gene loci are involved in the control of total GLS content where low GLS was due to the presence of recessive alleles in the homozygous condition in all loci. From the DH breeding population a yellow‐seeded, yellow‐petalled, zero erucic acid line was obtained. This line was further crossed with conventional B. napus varieties of double low quality and, following pedigree selection, a yellow seeded B. napus of double low quality was obtained. The yellow seeds had higher oil plus protein content and lower fibre content than black seeds. A reduction of the concentration of chromogenic substances was found in the transparent seed coat of the yellow‐seeded B. napus.     

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。     

玉米籽粒蛋白质含量的遗传效应及其与产量的关系

用来自5个不同基础群体的14个自选系作母本,不同优势群的5个测验系作父本,采用NC II交配设计配成70个杂交组合,进行了2年3点的田间试验。利用近红外光谱,对亲本及其杂交种的籽粒蛋白质含量进行了分析,同时分析了籽粒产量与蛋白质含量的相关性。结果表明,父、母本及其70个杂交组合间的基因型方差均达到极显著水平;8822和M是蛋白质含量较高的两个基础群体,以之作母本对提高杂交种籽粒的蛋白质含量起主导作用。控制籽粒蛋白质含量的基因以加性效应为主,加性方差占基因型方差的94.29%,但广义遗传力和狭义遗传力相对较低,分别为35.83%和33.94%,说明环境因素对籽粒蛋白含量影响明显。籽粒产量与蛋白质含量相关不显著(r = 0.053),因此,扩大变异选择范围,实现优良基因聚合,产量和蛋白质含量性状可以同步得到改良。     

Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). I. Comparison of sexual and doubled-haploid populations

Summary The genetic consequences of anther culture and chromosome-doubling techniques on burley tobacco (Nicotiana tabacum L.) were examined in this study. Three diploid populations, obtained from a burley tobacco inbred by conventional and anther-culture techniques, were compared. The first population consisted of 50 conventionally-selfed lines; the second population was made up of 35 doubled-haploid lines obtained from individual haploid plants by in vitro techniques (IVDH); and the third population consisted of 20 doubled-haploid lines whose chromosome complements had been doubled with colchicine (CDH). Comparisons of doubled-haploid lines with sexually-derived lines revealed significant differences for yield, maturity, leaf length, and alkaloid content. Yield reductions in the doubled-haploid populations averaged 8.5%. Significant differences observed between the IVDH and CDH populations indicate that the reported deleterious effects of colchicine contributed to the vigor reduction of doubled haploids. The anther derived lines in this study exhibited greater variation than did the sexual materials. This variation could provide useful variation for a breeding program. Variation exhibited by the sexual progeny of the highly inbred line, Kentucky 16, suggests that the differences among anther-derived materials are at least partially due to natural phenomena.Contribution from the department of Agronomy, University of Kentucky, Lexington, KY 40546-0091. This research was supported, in part, by a grant from R. J. Reynolds Tobacco Co. This paper (no 85-3-5) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Effect of 3B, 5A and 3A QTL for Fusarium head blight resistance on agronomic and quality performance of Canadian winter wheat

The objectives of this study were to investigate (i) the correlations between Fusarium head blight (FHB) index, deoxynivalenol (DON) accumulation and percentage of Fusarium‐damaged kernels (FDK) with agronomic and quality traits and (ii) the effect associated with the presence of single QTLs for FHB resistance on agronomic and quality traits in winter wheat. The population was derived from the cross between ‘RCATL33' (FHB resistance derived from ‘Sumai 3’ and ‘Frontana’) and ‘RC Strategy’. Parental lines and recombinant inbred lines (RILs) were genotyped with SSR markers associated with the 3B, 5A and 3A QTLs. The population was planted in FHB‐inoculated nurseries and in agronomy trials. Lines in the 3B QTL class had the lowest FHB index, DON content and FDK level and did not have a significantly lower yield, thousand kernel weight or protein content compared with the lines grouped in other QTL classes (including no QTL class). Marker‐assisted selection of the 3B QTL for FHB resistance into high‐yielding FHB‐susceptible winter wheat is the recommended approach for the development of lines with increased FHB resistance without significant yield and quality penalties.     

Rapid introduction of disease resistance from rye into common wheat by anther culture of a 6x triticale × nulli-tetrasomic wheat

Powdery mildew (caused by Erysiphe graminis) and yellow rust (caused by Puccinia striiformis) are the two most serious wheat diseases found in China. Rye chromosomes, carrying genes for resistance to these diseases, were introduced into common wheat in two generations using chromosome engineering and anther culture. The F₁ hybrids from a cross involving a hexaploid triticale (×Triticosecale Wittmack) בChinese Spring’ nulli‐tetrasomic N6DT6A wheat aneuploid line were anther cultured and doubled‐haploid plants were regenerated. Using genomic in situ hybridization, C‐banding and biochemical marker analyses, one of the anther‐cultured lines (ZH‐1)studied in detail, proved to be a doubled‐haploid with one rye chromosome pair added (1R) and a homozygous 6R/6D substitution (2n= 44). The line was tested for expression of disease resistance and found to be highly resistant to powdery mildew and moderately resistant to yellow rust.