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.     

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.     

Production of haploids of potato (Solanum tuberosum subsp. tuberosum) and their identification with electrophoretic analysis

Summary The frequency of haploid production following the interspecific pollination of eight tetraploid potato cultivars (Solanum tuberosum subsp. tuberosum) with Solanum phureja clone 1.22 was investigated. A total of 185 haploids were produced with an overall haploid frequency of 3.9 haploids/100 fruits. The haploid frequency was affected by the genotype of the maternal parent. Atlantic, ND860-2, Superior, Saginaw Gold, Spartan Pearl, Nooksack and Onaway had frequencies of 6.2, 5.1, 4.7, 3.9, 2.3 and 0.7 haploids/100 fruits, respectively. There were 60 and 57 haploids produced from Atlantic and Saginaw Gold, respectively, and no haploids were extracted from fruits of Lemhi Russet. Isozyme analysis and visual examination were performed independently to compare the efficiency of discriminating hybrids from haploids. Approximately 80% of total hybrids could be identified by electrophoretic analysis, while 77% were distinguished through visual examination. Pgm-2 ¹, which is unique in the clone 1.22 and absent from all seed parents, was found to be the most useful locus in hybrid identification and 50% of total hybrids could be distinguished by this allele. With similar rationale, Mdh-1 ¹ allozyme, which was absent in six of the eight parents, identified 37% of total hybrids. A combination of both visual and electrophoretic methods made hybrid identification even more efficient, with an average identification efficiency of 91%. A scheme was proposed to develop a new haploid inducer which would be homozygous for both Pgm-2 ¹ and embryo spot.     

A Comparison of Genetic Segregation in Traditional and Microspore-Derived Populations of Brassica juncea L. Czern and Coss.

An isolated microspore culture procedure was used to produce doubled haploid lines of Brassica juncea from F¹ plants of reciprocal crosses between the cultivar‘RLM514’and a canola quality breeding line. The inheritance of two qualitative markers, seed color and leaf hairiness, was compared using traditional and microspore-derived populations from these crosses. Chi-square tests indicated that each trait is controlled by different sets of duplicate pairs of genes. Brown seeds or hairy leaves can result from the presence of either of two dominant alleles, whereas yellow seed or glabrous leaves are produced when alleles at both loci are recessive. The segregation of genes controlling seed color and leaf hairs in doubled haploid progeny did not differ significantly from that expected under random assortment, indicating that doubled haploids can be used in this species for genetic studies, and probably cultivar development as well.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

Genetic,quantitative and microscopic evidence for fusion of haploid nuclei and growth of somatic calli in cultured <Emphasis Type="Italic">ms10</Emphasis><Superscript><Emphasis Type="Italic">35</Emphasis></Superscript> tomato anthers

In plant breeding, androgenic doubled haploids represent powerful tools to save time and resources for pure line generation. While in many species efficient protocols are known, in tomato (Solanum lycopersicum), the knowledge on the induction of androgenesis is still very scarce, and little is known about the particularities of this highly recalcitrant species. The only known method capable of yielding haploid/doubled haploid tomato plants is anther culture. However, this method has important limitations, including low efficiency of haploid induction and a low proportion of spontaneously doubled haploids. To understand these limitations better, we have analyzed the process of callus formation in anthers of tomato lines carrying the ms10 ³⁵ gene for male-sterility, using light and electron microscopy, flow cytometry and genetic analysis with morphological and molecular markers. Our results demonstrate that haploid, doubled haploid and diploid calli occur in tomato anthers, although at different frequencies. Diploid calli derived either from somatic cells or from the fusion of two genetically different haploid nuclei account for more than 90% of the total of calli produced. Somatic calli are derived from the stubs of connective tissue present in the interlocular septa of anthers. This growth is markedly increased in the ms10 ³⁵ mutants, which explains their higher callogenic rates than standard tomato lines. Together, our results reveal serious drawbacks that explain the low efficiency of anther-derived, doubled haploid production in tomato, and stress the need for alternatives towards doubled haploidy.     

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.     

Recessive genes controlling resistance to Helminthosporium leaf blight in synthetic hexaploid wheat

A study was conducted under controlled environment conditions in a phytotron to determine the nature of the inheritance of resistance Helminthosporium leaf blight (HLB) in a synthetic hexaploid wheat line, ‘Chirya‐3’, against the isolate KL‐8 of Bipolaris sorokiniana from the major wheat growing region of India. Crosses were made between two susceptible lines ‘WH 147’ and ‘Chinese Spring’. Analyses of F₁ and F₂ populations of these two crosses (‘WH 147’בChirya‐3’ and ‘Chinese Spring’בChirya‐3’) showed that resistance against the isolate in ‘Chirya‐3’ was governed by two recessive genes functioning in a complementary interaction giving an F₂ segregation pattern of 1 : 15 (resistant : susceptible). The segregation pattern of the resistant F₂ progenies in F₃ families from both crosses confirmed that two homozygous recessive genes were responsible for resistance to the isolate of Bipolaris sorokiniana in the synthetic line ‘Chirya‐3’. It is proposed that the genes be designated as hlbr1 and hlbr2.     

AFLP analysis indicates no introgression of maize DNA in wheat x maize crosses

Amplified fragment length polymorphisms (AFLPs) were used to follow the possible introgression of maize DNA into haploids of wheat as a side‐effect of exploiting wheat x maize hybridization for haploid production. AFLPs were generated with 64 MseI/ EcoRI and 64 MseI/ PstI primer combinations, and the AFLP profiles of haploids were tested against those of maize and of the regular wheat varieties involved in the crosses. On average, 45.1 and 110.7 fragments were produced per assay with the MseI/EcoRI and MseI/PstI combinations, respectively. Different numbers of fragments were produced for wheat and maize: an average of 81 in the haploid, 80 in the wheat parent, and only 67.1 in maize. No evidence was found for introgression of maize into the wheat genome. Three unique AFLP fragments were detected in haploids, which were not present in the parental wheat genotypes. These ‘novel’ AFLP bands in the haploids could be caused by nucleo‐cytoplasmic interaction in the hybrid zygote. Such instability in the wheat genome is defined as temporal, as it was not detected in further generations when colchicine‐doubled progeny of the haploids was tested for the presence of polymorphic fragments.     

Genetics of multiple disease resistance in a doubled-haploid population of barley

The barley accession Q21861 possesses resistance to the stem-rust (Puccinia graminis f.sp. tritici), leaf-rust (P. hordei), and powdery-mildew (Blumeria graminis f.sp. hordei) pathogens. An anther-culture-derived doubled-haploid population was produced from F₁ plants from a cross of this accession and the susceptible breeding line SM89010 as a means of rapidly and efficiently determining the genetics of multiple disease resistance. The doubled-haploid population segregated 1:1 (resistant:susceptible) for resistance to the stem rust pathotype QCC indicating the involvement of a single resistance gene, rpg4. Two-gene (3:1) and one-gene (1:1) segregation ratios were observed for resistance to the stem-rust pathotype MCC at low (23–25°c) and high (27–29°C) temperature, respectively. These different segregation patterns were due to a pathotype × temperature interaction exhibited by rpg4 and Rpg1. another stem-rust-resistance gene present in Q21861. One-gene and two-gene segregation ratios were observed in reaction to the leaf rust and powdery mildew pathogens. These data demonstrate the utility of doubled haploid populations for determining the genetics of multiple disease resistance in barley.     

Identification and inheritance of a partially dominant gene for yellow seed colour in Brassica napus

A yellow‐seeded doubled haploid (DH) line no. 2127‐17, derived from a resynthesized Brassica napus L., was crossed with two black‐seeded Brassica cultivars ‘Quantum’ and ‘Sprint’ of spring type. The inheritance of seed colour was investigated in the F₂, and BC1 populations of the two crosses and also in the DH population derived from the F1 of the cross ‘Quantum’× no. 2127‐17. Seed colour analysis was performed with the colorimeter CR‐300 (Minolta, Japan) together with a visual classification system. The immediate F1 seeds of the reciprocals in the two crosses had the same colour as the self‐pollinated seeds of the respective black‐ and yellow‐seeded female parents, indicating the maternal control of seed colour. The F1 plants produced yellow‐brown seeds that were darker in colour than the seeds of no. 2127‐17, indicating the partial dominance of yellow seed over black. In the segregating BC1 progenies of the two crosses, the frequencies of the black‐ and yellow‐seeded plants fit well with a 1 : 1 ratio. In the cross with ‘Quantum’, the frequencies of yellow‐seeded and black‐seeded plants fit with a 13 : 3 ratio in the F₂ progeny, and with a 3 : 1 ratio in the DH progeny. However, a 49 : 15 segregation ratio was observed for the yellow‐seeded and black‐seeded plants in the F₂ progeny of the cross with ‘Sprint’. It was postulated from these results that seed colour was controlled by three pairs of genes. A dominant yellow‐seeded gene (Y) was identified in no. 2127‐17 that had epistatic effects on the two independent dominant black‐seeded genes (B and C), thereby inhibiting the biosynthesis of seed coat pigments.     

Segregation for isozymes and HMW glutenins in a doubled-haploid cross of winter wheat carrying 1BL.1RS and 5DL.5RS translocations from rye

The doubled haploid (DH) wheat line ‘dh 5841’ carrying two translocations from rye, 5DL.5RS and 1BL.1RS, has been crossed to the subline of wheat cultivar ‘Amadeus 7143’ with a 1BL.1RS translocation. The resulting F₁ hybrid IJ 98 with a heterozygous 5DL.5DS‐5DL.5RS chromosome pair has been used to produce doubled haploids. A total of 57 DH lines were obtained from plantlets regenerated in anther culture after successful colchicine treatment and seed set. These lines were identified regarding the constitution of chromosome 5D (5DL.5DS or 5DL.5RS) by means of isoenzyme marker analysis. Thirty DH lines possessed the 5DL.5DS chromosome, while the remaining 27 lines carried the 5DL.5RS translocation. For some of these lines, the 5DL.5RS chromosome was cytologically confirmed by C‐banding. Furthermore, the DH lines were evaluated for their high molecular weight glutenin subunit composition. All possible combinations for the four independent loci —Skdh, Glu‐Al, Glu‐B1 and Glu‐D1— were detected in only 57 DH lines and no segregation distortion was observed.     

Genetic analysis of total glucosinolate in crosses involving a high glucosinolate Indian variety and a low glucosinolate line of Brassica juncea

Analysis of the glucosinolate content and composition by high‐pressure liquid chromatography indicated that varieties of Brassica juncea bred and grown in India have a high glucosinolate content characterized by the presence of 2‐propenyl (allyl) and 3‐butenyl as the major and 4‐pentenyl as the minor fractions. In contrast, the B. juncea germplasm from other countries is characterized by the presence of 2‐propenyl as the major glucosinolate fraction, trace amounts of 3‐butenyl and a total lack of the 4‐pentenyl types. In order to transfer the low glucosinolate trait to Indian B. juncea, the inheritance of total glucosinolates was investigated using doubled haploid (DH) populations derived from F1 (DH₁) and BC₁ (BC₁DH) of a cross between ‘Varuna’ (the most widely cultivated high glucosinolate variety of India) and ‘Heera’ (a non‐allyl type low glucosinolate line). A total of 752 DH₁ and 1263 BC₁DH gave rise to seven and 11 low glucosinolate (containing less than 18 μmol/g seed) individuals, respectively. On the basis of the frequency of the low glucosinolate individuals, the total glucosinolate was found to be under the control of seven genes. There was presence of both allyl and non‐allyl types in DH₁ and BC₁DH low‐glucosinolate individuals and absence of 3‐butenyl glucosinolate in some of the BC1DH low glucosinolate individuals, indicating segregation for these fractions in the population. The size of the segregating DH population proved to be crucial for precise determination of the number of genes controlling the trait. Because of the large number of genes involved, incorporation of low glucosinolate trait in Indian B. juncea should be approached through doubled haploid (DH) breeding.     

Production of salt tolerant rice breeding line via doubled haploid

Summary A haploid breeding program was initiated to develop doubled haploid salt tolerant rice breeding line via anther culture. Two sensitive breeding lines BR4608-R₁-R₂ and BR4909-R₁-R₂ were crossed with a salt tolerant line IR13146-13-3-3 to transfer its salt tolerant character to the doubled haploids.Anther from confirmed F₁s of the two crosses were cultured in defined medium for callus induction and eventual plant regeneration. Fifteen doubled haploid (DH) lines were obtained from two crosses. Test for salt tolerance were done in vitro. Five out of 15 lines were found tolerant at the level of 8–10 decisiemens/m (ds/m) while the rests were sensitive to that level of salinity.Field experiment was conducted to evaluate the doubled haploids under saline and non saline soil. Five salt tolerant lines produced comparable yield with the resistant control (BR 23) under saline condition, whereas these lines yielded even higher in non saline soil under irrigated condition when evaluated with other 10 sensitive DH linesAbbreviations LSD Least Significant Difference - NAA Napthalene Acetic Acid     

不同除草剂加倍玉米单倍体的效率

通过比较3种除草剂加倍玉米单倍体的效率,提出了利用除草剂加倍玉米单倍体的新方法。以先玉335、中农大4号和8607×8609三个基因型诱导的单倍体籽粒为材料,利用20、40、80和160 μmol L^-1浓度的甲基胺草磷、炔苯酰草胺和氟乐灵作为加倍药剂,在单倍体植株生长到三叶期和五叶期时,用滴心法处理幼苗,选择有花粉的单株自交,收获后调查果穗加倍率;采用细胞学方法观察单倍体的染色体数目和花粉的活性。结果表明,20~160 μmol L^-1的3种除草剂对玉米单倍体加倍均有效果,加倍率在3.42%~26.32%之间。甲基胺草磷、炔苯酰草胺和氟乐灵的加倍率分别为4.29%~26.32%、3.85%~20.81%和3.42%~17.61%;其中80 μmol L^-1甲基胺草磷的加倍效果最佳,使用80 μmol L^-1甲基胺草磷处理3个杂交种的单倍体,平均加倍率分别为25.02%、20.13%和14.99%。方差分析表明,3个基因型间的单倍体加倍率均呈极显著差异,可见使用甲基胺草磷、炔苯酰草胺、氟乐灵可以提高玉米单倍体的加倍频率,但不同基因型单倍体对除草剂的敏感性存在差异。     

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.     

Haploid differentiation in maize kernels based on fluorescence imaging

A new fluorescence‐based method for inbred haploid differentiation in maize kernels was developed by utilizing the R1‐nj colour marker in combination with fluorescence microspectroscopy and imaging. Seven inbred lines with varying R1‐nj expression were used in this study. The fluorescence response of the diploid kernels at the embryonic dye spot was shown to simultaneously exhibit lower intensity and occur at a higher wavelength than the fluorescence of the dye‐lacking haploid embryos. Intensity and area thresholds were applied to fluorescence images to sort the haploids from mixed sample populations, and sorting efficiencies of greater than 80% were achieved in all seven inbred lines (with values greater than 90% for five lines). The potential for high‐throughput sorting when fluorescence imaging is combined with existing technologies for seed handling as well as high sorting efficiency may make fluorescence a viable and promising alternative to current sorting methods for some inbred lines.     

Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize

Doubled haploid plants are useful in genetic studies and plant breeding, but a consistent and satisfactory frequency of production has been difficult to achieve in durum wheat. Triticum turgidum L., using the maize pollen method. The objective of this study was to develop an objective method of producing doubled haploids in durum wheat. Plant growing and handling conditions, aspects of hormone treatments, wheat genotype and pollen source were considered. The number of caryopses, embryos, haploids, doubled plants and doubled plants that set seed were measured. Although growth conditions, pollen source, method of handling plants and wheat genotype are important considerations, the type of hormone was found to be most significant in the production of doubled haploid plants. When 50mg/l dicamba was substituted for 100 mg/l 2,4‐D the number of doubled haploids per spike increased from 0.2 for the best 2,4‐D treatment to 1.3 for the dicamba treatment. This increased frequency was largely attributed to an increase in the number of caryopses generated for each spike emasculated and from an increased frequency of germination of embryos to haploid plantlets. The best production of caryopses was 0.41 caryopses per florest with 2,4‐D. The best production of haploids per 100 florets was 12 with dicamba and 1.65 with 2,4‐D. The frequency of one doubled haploid per emasculated spike through the use of dicamba is a practical level for generating populations for genetic studies.     

Inheritance of anthracnose resistance in the common bean cultivar Widusa

Snap bean (Phaseolus vulgaris L.) cultivar, Widusa, was crossed to Michigan Dark Red Kidney (MDRK), Michelite, BAT 93, Mexico 222, Cornell 49–242, and TO cultivars to study the inheritance of resistance to anthracnose in Widusa. The segregation patterns observed in six F₂ populations supported an expected 3R:1S ratio suggesting that Widusa carries a single dominant gene conditioning resistance to races 7, 65, 73, and 453 of Colletotrichum lindemuthianum, the causal organism of bean anthracnose. Allelism tests conducted with F₂ populations derived from crosses between Widusa and Cornell 49–242 (Co-2), Mexico 222 (Co-3), TO (Co-4), TU (Co-5), AB 136 (Co-6), BAT 93 (Co-9), and Ouro Negro (Co-10), inoculated with races 7, 9, 65 and 73, showed a segregation ratio of 15R:1S. These results suggest that the anthracnose resistance gene in Widusa is independent from the Co-2, Co-3, Co-4,Co-5, Co-6, Co-9, and Co-10 genes. A lack of segregation was observed among 200 F₂ individuals from the cross Widusa/MDRK, and among 138 F₂ individuals from the cross Widusa/Kaboon inoculated with race 65, suggesting that Widusa carries an allele at the Co-1 locus. We propose that the anthracnose resistance allele in Widusa be named Co-1 ⁵ as Widusa exhibits a unique reaction to race 89 compared to other alleles at the Co-1 locus. RAPD marker A18₁₅₀₀ co-segregated in repulsion-phase linkage with the Co-1 ⁵ gene at a distance of 1.2 cM and will provide bean breeders with a ready tool to enhance the use of the Co-1 ⁵ gene in future bean cultivars.     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.