A new DNA marker for sex identification in purple asparagus

Garden asparagus (Asparagus officinalis L.) is an economically-important perennial crop. This plant is dioecious, as there are both male and female individuals; male individuals are preferred over females for agricultural production. To reduce the time required for garden asparagus breeding, various male-specific DNA markers are utilized. Male-specific DNA markers, such as Asp1-T7sp and MSSTS710, are currently available for sex identification in many asparagus cultivars. In the current study, we found that these markers are not suitable for sex identification in the purple asparagus cultivar ‘Pacific Purple’, as male-specific amplification of this marker was detected in some male individuals of this cultivar but not in other males. The Asp1-T7sp marker is suitable for use in sex identification in various Asparagus species related to A. officinalis, indicating that the region around this marker is conserved among these species. Thus, we isolated a DNA fragment around this marker by inverse PCR and produced a new DNA marker, MspHd, based on this sequence. However, like Asp1-T7sp and MSSTS710, MspHd was not suitable for sex identification in the cultivar ‘Pacific Purple’. Since all ‘Pacific Purple’ males have morphologically similar male flowers with functional stamens, we produced a new male-specific marker based on the sex determination gene, MSE1/AspMYB35/AspTDF1, which is responsible for stamen development. This marker, named AspMSD, is suitable for sex identification in ‘Pacific Purple’. In addition, this marker can be utilized for sex identification in various asparagus cultivars and some related Asparagus species.     

Sex distinction of asparagus by loop-mediated isothermal amplification and observation of seedling phenotypes

Asparagus (Asparagus officinalis L.) is a dioecious plant. In general, male and female plants are used for open-field culture and intensive cultivation, respectively. Farmers distinguish between the sexes by observing the form of the flower organs. However, because flowering begins 2?C3 years after planting, the sexes cannot be differentiated at transplantation by using this method, and planting of an all-male population is not possible. In this study, the usefulness of loop-mediated isothermal amplification (LAMP), a simple method of gene amplification, for sex distinction at the DNA level was determined. In addition, the phenotypic differences in seeds and seedlings of male and female plants were investigated for application as a method of early sex distinction. By using the LAMP method, the sex could be correctly identified in 100% of the seedlings, suggesting that this method is effective for sex distinction at the gene level. Principal component analysis was conducted with 11 selected parameters after investigating the seeds and seedlings of both male and female plants. The results revealed that male plants tend to have many stalks or cladophylls and female plants tend to have large plant forms, suggesting that the sexes can be distinguished by the external appearance of the seedlings before planting. LAMP and observation of the seedling phenotypes could be useful methods of sex distinction for increasing the efficiency of asparagus breeding.     

Conversion of a male-specific RAPD marker into an STS marker in Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is an economically important plant. This species is dioecious, and male plants are considered to be more desirable than females due to their higher yields. To reduce the time required for asparagus breeding, molecular marker techniques have been employed to identify sex-linked DNA markers. In the present study, we converted the male-specific random amplified polymorphic DNA marker T35R54-1600 into a sequence tagged site marker. We cloned a male-specific DNA fragment amplified with the T35R54 primer and determined the sequence of the fragment. The size of T35R54-1600 is 1,586 bp, and this fragment is not homologous to known sex-linked BAC sequences, indicating that this fragment is a new sex-linked region. Within this fragment, we designed the primer pair ‘MSSTS710’ to amplify a 710 bp region. This marker could be used to identify the sex of eight cultivars of A. officinalis: ‘Mary Washington 500W’, ‘UC157’, ‘Harumachi Green’, ‘Super Welcome’, ‘F4’, ‘Pacific 2000’, ‘F2’ and ‘Backlim’. We also analyzed the applicability of this marker to two dioecious Asparagus species, A. schoberioides and A. kiusianus, which are cross-compatible with A. officinalis. Although male-specific DNA fragments of two dioecious Asparagus species, A. schoberioides and A. kiusianus, were generated using the existing male-specific marker Asp1T7sp, no amplicon was obtained using the MSSTS710 marker. Since MSSTS710 can be employed for sex identification only in A. officinalis and not in closely related Asparagus species, the DNA region around the MSSTS710 marker must be variable among Asparagus species.     

芦笋性别的化学鉴定研究

在成熟芦笋叶片与未知性别的芦笋叶片提取液中加入Ⅰ号裴林试剂和Ⅱ号裴林试剂,静置60分钟后,成熟芦笋雌株上清液呈翠绿色,雄株为黄绿色;而未知性别芦笋上清液颜色无区别,但沉淀量不同。     

石刁柏性别表现与同工酶的关系

采用聚丙烯酰胺凝胶电泳对石刁柏（ＡｓｐａｒａｇｕｓｏｆｆｉｃｉｎａｌｉｓＬ．）雌雄植株不同器官及不同组织的过氧化物酶同工酶谱进行了研究，结果表明，除根部外，尽管鳞片、茎尖、拟叶等器官酶谱有明显差异，但雌雄株间差异有相同的趋势。雄株均比相应的雌株少一条酶带，雌雄植株组织培养获得的愈伤组织和茎尖过氧化物酶同工酶酶谱差异也有类似的规律。说明石刁柏性别差异与过氧化物酶同工酶的数目有关，过氧化物酶同工酶谱的差异可以作为性别鉴定的指标。     

DNA分子标记早期快速鉴别芦笋雌雄株

在相同的生长条件下,芦笋雄株产量比雌株高出25%以上.但芦笋从播种到开花大约需要2年时间,早期难以直接区分芦笋的性别,通过芦笋雌雄株早期快速鉴定,有利于芦笋杂交制种的亲本选择,缩短育种年限,随着DNA分子标记技术的发展,利用性别连锁标记在苗期进行芦笋性别鉴定成为可能.本研究构建了芦笋两性株S1群体共108株以及雌雄株DNA池,利用与芦笋性别决定基因紧密连锁的DNA分子标记Aspl-T7对雄性DNA池和雌性DNA池进行多态性分析,结果表明STS标记Aspl-T7在雄性DNA池与雌性DNA池间具有多态性,可以用来对芦笋两性株S1群体进行性别辅助选择.利用Aspl-T7检测芦笋两性株S1群体,检测结果为雌性(mm)30株,雄性(MM、Mm)78株,卡平方测验结果验证了芦笋性别是由一对等位基因控制,表现出3∶1的分离比例(χc2=0.3086＜χ20.05=3.84).田间性别调查验证和分析结果显示,Aspl-T7可以早期快速有效的鉴别芦笋雌株和雄株.     

Development of a rapid method for identifying asparagus super-males using, N-(4-chloro-2-trifluoromethylphenyl)-N'- propoxyacetamidine to induce flowering

In order to shorten the term for the identification of asparagus super-maleplants, a method usingN-(4-chloro-2-trifluoromethylphenyl)-N'-pro-poxyacetamidine (AM12) toinduce flowering was developed. This method is conducted as follows: seedsfrom andromonoecious flowers are treated with AM12 to induce flowersto form on the seedlings, the new male flowers are crossed with normalfemale flowers, and the progeny seeds are again treated with AM12 toidentify super-male plants from the sex ratio of the seedlings.Asparagus officinalis cv. `UC157' seeds were treated with AM12.The seedlings were induced to flower at a high frequency in 25 days. Thesex ratio of the plants was 1:1 and the male flowers had the pollengermination ability. One male flower induced by AM12 was crossed withfemale plant. This cross produced many progeny seeds. The seeds weretreated again with AM12, and induced to flower. Two super-male plantswere found among the progeny of andromonoecious flowers of the all-malecultivar `Gijnlim' within six months by this method. This method thusshortens the time for identifying super-males. Since the female flowers werefertile as well, AM12 treatment would also be effective for cross breeding.     

Random amplified polymorphic DNA variation among German and Dutch asparagus cultivars

DNA polymorphism among nine cultivars of Asparagus officinalis L. was measured using random amplified polymorphic DNA (RAPD). Of 69 reproducible amplification products from 12 arbitrary decamer primers, 49 RAPD markers were polymorphic and could be used to distinguish six German and three Dutch asparagus cultivars. Even with very small sample sizes, genetic similarity measurements based on the RAPD data allowed accurate grouping of the nine cultivars into distinct clusters, with the exception of two individuals which clustered to closely related varieties. Two German cultivars showed high genetic similarity and were distinct from the remaining German varieties. The German and Dutch cultivars were clearly separated by a relatively large genetic distance.     

Sex identification in Encephalartos natalensis (Dyer and Verdoorn) using RAPD markers

All cycads are strictly dioecious with a long juvenile stage. Currently, there is no method available to determine the sexuality of seedlings prior to the onset of cone formation. This study aimed to develop a sex specific Random Amplified Polymorphic DNA (RAPD) marker for Encephalartos natalensis. Initially, 140 primers were used to amplify the bulk DNA of five individuals each of known male and female sexuality. While a high degree of polymorphism was observed in the amplification profiles of male and female plants, only primer OPD-20 generated a specific band (∼850 bp) in female DNA. To validate this observation, this primer was re-tested with 69 individuals of E. natalensis. The 850 bp DNA band was present in all 38 female individuals tested and it was consistently absent in all 31 male plants tested. The result offers a rapid and simple test to determine sexuality of E. natalensis seedlings at early stages of development, before the onset of reproductive maturity thereby saving time and economic resources when cultivating these specimens.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

小麦基因组的一种简易提取方法

摘要：【研究目的】在综合分析多种提取小麦DNA方法的基础上,改良发展了一种快速简便高效的小麦基因组DNA提取方法。【方法】以春小麦Thatcher和以Thatcher为背景的近等基因系TcLr19,TcLr20, TcLr28为材料,改进后的方法提取的基因组DNA用抗叶锈基因Lr20的STS标记、Lr19和Lr28的SCAR标记、Lr28的SSR 标记进行PCR扩增,【结果】各分子标记都扩增出条带清晰正确的目的片段【结论】这表明该方法能够获得适用于STS、SCAR、SSR标记PCR扩增的高质量的DNA,而且该DNA简易提取方法加快了DNA提取速度、降低了污染源和成本,为大批量提取DNA提供了技术支撑。     

Generation and validation of unique male sex-specific sequence tagged sites (STS) marker from diverse genotypes of dioecious Jojoba-Simmondsia chinensis (Link) Schneider

DNA fingerprinting studies have been carried out with the physiologically mature male and female plants of Jojoba using 80 ISSR primers with a view to generate sex-linked markers. After bulk segregant analysis, two unique ISSR markers, viz. ISSR848₁₅₀₀ and VIS11₁₃₁₇ have been developed which can be used for determining the sex at the seedling stage. Of the eighty primers tested on the pooled male DNA and pooled female DNA samples, six ISSR primers were found to be associated with sex expression. Of the six, only two primers ISSR848 and VIS11 generated unique male sex specific bands of ~1,500 and ~1,300 bp which were consecutively present in all the male genotypes and absent in all the respective female genotypes. The remaining four primers when tried on individuals of different genotypes were confined to their sex specificity in only two female genotypes and absent in their male counterparts. One of the male-sex specific markers, VIS11₁₃₁₇ has also been cloned and sequenced which showed homology with a sex linked gene, DD44 from dioecious Silene species. Furthermore, VIS11₁₃₁₇ was converted into a male sex-specific sequence tagged sites (STS) marker of 584 bp. The male specific STS marker thus developed has been verified and validated on 100 populations of male and female individuals from ten different genotypes of Jojoba to endorse the diagnostic reliability of the STS marker. This can gainfully be employed for screening of sex at seedling stage which would be quite helpful for uprooting the undesired plants, thereby, saving resources like labor, water, fertilizers and space for highly desirable female plants.     

一种快速提取小麦基因组DNA的改良CTAB方法

旨在寻求一种微量、快速提取小麦DNA的方法。以小麦幼叶为材料,用改良CTAB法、改良SDS法、沸水浴法提取小麦叶片总DNA,通过琼脂糖凝胶电泳法、紫外吸收法和PCR检测DNA完整性和纯度。改良CTAB法提取小麦基因组DNA质量和纯度高、无降解,每人每天可以轻松提取200个DNA样品,对转基因植株的PCR检测显示扩增目标条带清晰一致,无假阳性,试验结果理想。改良SDS法所提DNA纯度和浓度虽略不如改良CTAB法,但仍然符合实验要求。沸水浴法提取量少且杂质多,PCR无有效扩增,结果不理想。改良CTAB法提供了一种简便、快速微量小麦DNA提取方法,适用于PCR和其他分子生物学研究。     

工业大麻雄性相关RAPD和SCAR标记的筛选与鉴定

利用42条RAPD（Random Amplified Polymorphic DNA）随机扩增引物分析工业大麻品种“火麻一号”组成的雄性或雌性DNA池（DNA pools）,结果显示,引物OPV-08扩增得到一条大小为869bp与工业大麻雄性相关的特异条带。根据测序结果,合成了两条SCAR（Sequence Characterized Amplified Region）标记引物,该SCAR标记不仅可以对工业大麻雌雄异株材料花期已知性别的雌雄植株进行准确鉴定,还可以对幼苗期未知性别的大麻雌雄植株进行鉴定;也可对雌雄同株材料可能出现的雄化进行早期鉴定。这不仅为工业大麻早期性别鉴定提供基础,且为减少雌雄同株材料的雄化提供支撑。     

Validation of male sex‐specific UBC‐8071200 ISSR marker and its conversion into sequence tagged sites marker in Jojoba: a high precision oil yielding dioecious shrub

There is a recent surge in the marker‐assisted selection of desired sex type among economically important dioecious crops. Simmondsia chinensis (Jojoba), a dioecious crop is of immense agricultural importance where only the female plants are preferred for commerce. DNA fingerprinting technology, ISSR analysis along with bulk segregant analysis (BSA), has been carried out on a diverse set of 17‐ to18‐year‐old mature male and female plants of Jojoba to validate a male sex‐specific ISSR marker, UBC‐807₁₂₀₀ on larger population that comprises 330 female and 255 male plants of Jojoba. This male sex‐specific DNA fragment of ~1200 bp was cloned and sequenced. The sequence was found to be 1120 bp in length and based on the sequence information, a pair of sequence tagged sites primers was developed that amplified a single ~800 bp band, consistently only in all the male populations while no amplification was seen in their female counterparts. The marker was named as Jojoba Male Sex Marker which was further validated on 330 female plants from 22 genotypes and 255 male plants from 17 genotypes.     

Development of male-specific markers and identification of sex reversal mutants in papaya

Papaya is a productive and nutritious fruit grown in tropical and sub-tropical regions worldwide. It is polygamous with three sex types: female, male and hermaphrodite. Sex determination in papaya is controlled by an XY sex chromosome system with two slightly different Y chromosomes, Y for males and Y^h for hermaphrodites. Comparative analysis of the hermaphrodite-specific region of Y^h chromosome (HSY) and male-specific region of Y chromosome (MSY) revealed 99.6% sequence identity, which explains why DNA markers that amplify for both males and hermaphrodites have easily been developed, but not for the male trait specifically. We examined the 0.4% sequence differences, and found 1887 indels and 21,088 SNPs between MSY and HSY. The vast majority of indels are single nucleotide or few base pairs. A large male-specific retrotransposon insertion of 8396 bp was used to develop two papaya male-specific markers, PMSM1 and PMSM2 that amplify 585 and 548 bp fragments, respectively. These two markers were tested in 11 gynodioecious and four dioecious varieties along with autosomal DNA marker 71E and male/hermaphrodite marker W11, and the results showed clear separation of male from hermaphrodite and female. PMSM1 and PMSM2 were also used to test the sex type of six sex male-to-hermaphrodite reversal mutants which are crucial materials for validating candidate genes for sex determination in papaya. Our result showed all six mutants were positive for the male-specific markers. These male-specific markers can be used to distinguish gynodioecious and dioecious cultivars in papaya seed market, and facilitate genetic and genomic research for papaya improvement.     

碱解法快速提取菠菜基因组DNA方法的优化

为了优化碱解法提取菠菜基因组DNA的方法,并为大批量育种材料的分子标记筛选奠定基础,本试验以菠菜幼嫩叶片为试材,以PCR扩增效果为主要依据,研究NaOH浓度、水浴温度与方法、吐温20浓度对菠菜DNA提取质量的影响。结果表明：以0.4 mol/L NaOH为提取液研磨后沸水浴1 min提取的菠菜DNA的PCR扩增效果明显好于常温处理。样品不经研磨、可直接用PCR扩增仪99℃、4 min代替沸水浴加温并省去离心步骤,此时以0.4 mol/L NaOH+0.5%吐温20为提取液时PCR扩增效果最佳。提取DNA的A₂₆₀/A₂₃₀比值较高时PCR扩增效果较好,因此A₂₆₀/A₂₃₀比值是评价提取DNA质量的最优指标。本试验优化了碱解法简便快速提取菠菜DNA的技术规程,达到了理想的PCR扩增效果。     

一种适用于PCR扩增的甜菜干种子DNA快速提取方法的研究

为了寻求一种快速提取甜菜DNA的方法,利用改良CTAB法对甜菜干种子DNA进行了提取,经琼脂糖电泳及SRAP和SSR两种随机引物扩增检测,结果表明该法提取的DNA完整性好,SSR及SRAP扩增条带清晰,符合甜菜SSR-PCR及SRAP-PCR对DNA质量的要求。本研究建立了快速提取甜菜DNA的体系,为分子标记在将来甜菜品种指纹图谱构建及辅助育种等方面的应用奠定了坚实的基础。     

Extraction of Cotton Fiber DNA and Its Application in Traceability

[Objective] To address the questions of lacking monitoring system for origins of varieties, this study explored a DNA extraction method suitable for cotton fiber, aiming to establish an identification system that uses cotton fiber DNA as a medium to trace the authenticity and cultivar of cotton varieties. [Method] By improving and optimizing the extraction method, the DNAs of cotton fibers at different days post anthesis and lint from different years were extracted and used as templates for routine polymerase chain reaction (PCR) amplification. 13 pairs of simple sequence repeat(SSR) primers were selected for SSR-PCR amplification using the DNA of Lu 1127, Shikang 126 and Ruiza 816. [Result] The DNA extraction method can extract cotton fiber DNAs of different developmental stages and different years. With the development of fiber, although the extracted DNA content is reduced, it can still meet the needs of downstream experiments. The extracted cotton fiber DNAs can be subjected to conventional PCR amplification, the PCR amplified bands were clear; 13 pairs of cotton SSR primers were used for SSR-PCR amplification of cotton fiber DNAs of Lu 1127, Shikang 126 and Ruiza 816, and a clear polymorphic band could be amplified and easily distinguished. [Conclusion] This extraction method is suitable for extracting genomic DNA of cotton fiber, and is satisfied with conventional PCR amplification with SSR markers. It is confirmed that the cotton fiber DNA molecular markers are feasible for tracing the source of cotton varieties, and it is expected to provide technical support for ensuring the safety of cotton industry.     

一种适于PCR扩增的蓖麻基因组DNA提取方法

报道了一种从蓖麻叶片中提取DNA的改良SDS方法,该方法提取的DNA经琼脂糖凝胶电泳检测,获得的DNA条带较亮且无明显拖尾现象。利用ISSR引物对提取的蓖麻基因组DNA进行PCR检测,能够获得清晰稳定的条带。说明该方法提取的蓖麻基因组DNA能够满足PCR反应的需要。     

以PCR为目的的大豆叶片DNA提取方法的比较研究

模板DNA的质量直接影响PCR扩增的结果,而不同提取方法及其缓冲液的成份与浓度对提取DNA的质量有重要影响。本文以5个栽培大豆品种的叶片为材料,比较分析了SDS与CTAB两种提取方法以及不同浓度CTAB提取缓冲液对所提取的DNA质量的影响,并通过PCR进行检验。实验结果表明：用1％(W/V)、2％(W/V)浓度的CTAB提取缓冲液和1．25％(WV)SDS提取缓冲液所提取的大豆叶片DNA的质量较好,均能满足PCR扩增模板的需求,其中以1．25％(W／V)SDS提取得到的大豆叶片DNA质量最好,以其为模板扩增的效果最佳,而4％浓度的CTAB不适宜提取大豆叶片DNA。