Development and Application of a PCR-Based Molecular Marker for the Identification of High Temperature Tolerant Cabbage (Brassica oleracea var. capitata) Genotypes

Global warming accelerates the development of high temperature (HT)- and high humidity (HH)-tolerant varieties. This is further facilitated by the identification of HTHH-tolerant genes and the development of molecular markers based on these genes. To identify genes involved in HTHH tolerance in cabbage (Brassica oleracea var. capitata), we performed RNA-seq analysis of two inbred lines, BN1 (HTHH-tolerant) and BN2 (HTHH-susceptible), and selected trehalose 6- phosphate phosphatase I-2 (BoTPPI-2) as one of the HTHH-tolerant-associated genes. We also developed a segregating F2 population from a cross between BN1 and BN2. RNA-seq results showed that BoTPPI-2 transcript levels were high in the HTHH-tolerant inbred line BN1, but not detectable in the HTHH-susceptible inbred line BN2. The expression pattern of BoTPPI-2 was not related to the expression of heat shock-related genes. Soft rot resistance, used as an indicator of HTHH tolerance, was higher in BN1 than in BN2. F2 individuals similar to BN1 with respect to phenotype appeared to be HTHH-tolerant, whereas BN2-types were susceptible to HTHH. Analysis of the genomic DNA revealed the presence of a long terminal repeat (LTR; ca. 4.6 kb) in the ninth intron of the BoTPPI-2_BN2 allele, thereby suppressing its transcription and exhibiting HTHH phenotype. Except for the LTR insertion, the sequence of BoTPPI-2_BN2 was almost identical to that of BoTPPI-2_BN1. On the basis of the LTR and BoTPPI-2 sequences, we developed a molecular marker to identify HTHH-tolerant genotypes and validated its efficiency using F2 individuals, inbred lines, and cultivars from diverse sources. The marker explained the genetic basis of HTHH tolerance in at least 80%, but not 100%, of the cabbage genotypes. Thus, additional markers associated with HTHH tolerance are needed for perfect selection.

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Growth, yield and shelf-life of isopentenyltransferase (ipt)-gene transformed broccoli

Canadian Journal of Plant Science ◽

10.4141/cjps08156 ◽

2009 ◽

Vol 89 (4) ◽

pp. 701-711 ◽

Cited By ~ 3

Author(s):

L -F Chan ◽

L. -F.O. Chen ◽

H -Y Lu ◽

C -H Lin ◽

H -C Huang ◽

...

Keyword(s):

Shelf Life ◽

Brassica Oleracea ◽

Inbred Line ◽

Growth Parameters ◽

Growth Yield ◽

Single Copy ◽

Inbred Lines ◽

Yield Potential ◽

Transgenic Lines ◽

Brassica Oleracea L

Loss of chlorophyll leading to floret yellowing limits the post-harvest lifespan of broccoli (Brassica oleracea L. var. italica Plenck). Cytokinins are known to delay floral yellowing of plants. A transgene construct pSG766A, which results in the expression of isopentenyltransferase (ipt), the key enzyme for cytokinin synthesis, has been developed in broccoli. Expression of the ipt transgene is triggered by the senescence-associated gene promoter (SAG-13). Three selfed T5 lines of ipt transformed broccoli (lines 101, 102 and 103) have been obtained through selection for single copy insertion, acceptable horticultural traits and transgene ipt activity. These three transgenic inbred lines were evaluated in the field during 2004-2007 to determine their growth, yield and shelf-life after harvest, relative to a non-transgenic inbred line (104) and the parental variety Green King. For most of the vegetative growth parameters measured, year-to-year variability exceeded line-to-line variability. Inbreeding had little impact on the appearance or yield potential of the broccoli lines. Head yields of the transgenic inbred lines 102 and 103 were comparable to the parental variety Green King, but were significantly higher than the non-transgenic inbred line 104, as lines 102 and 103 produced more plants with heavier flower heads. Cytokinin content in the form of isopentenyladenosine was relatively higher in the transgenic lines than in the two non-transgenic controls. When flower heads were stored at 25 ± 2°C, the period required to cause 50% floret yellowing was 7.5 and 8.5 d for the transgenic lines 102 and 103, respectively, compared with 5.6 d for the non-transgenic line 104, and 5.1 d for the parental variety Green King. This study showed that the ipt-transformed inbred lines of broccoli combined acceptable appearance and yields with enhanced shelf-life.Key words: Brassica oleracea L. var. italica Plenck, transgenic broccoli, isopentenyltransferase gene, genetic characterization, shelf-life

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The relationship between the distribution of genetically distinct inbred lines and upper lethal temperature in Lasaea rubra

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315400036584 ◽

1990 ◽

Vol 70 (3) ◽

pp. 557-570 ◽

Cited By ~ 6

Author(s):

H. Walters-Tyler ◽

J. Davenport

Keyword(s):

Inbred Line ◽

Genetic Basis ◽

Thermal Response ◽

Indirect Evidence ◽

High Water ◽

Inbred Lines ◽

Rocky Shores ◽

Lower Shore ◽

Single Temperature ◽

The Relationship

Lasaea rubra is an inbreeding bivalve species, living at most heights on rocky shores. Freshly collected animals from different shore heights showed significantly different upper median lethal temperatures (MLTs), with upper shore animals having higher MLTs than lower shore specimens. Experiments with animals acclimated for at least one month to a single temperature (15°C) demonstrated that these differences in upper MLT were unaffected by thermal acclimation. Electrophoretic investigation showed that the differences in thermal response had a genetic basis. Homogeneous populations of the high-water inbred line (‘Inbred line A’) had a higher MLT than homogeneous populations of ‘Inbred line C’ which was found on the middle and lower shore. No differences were detected between the MLTs of separate populations of Inbred lines A or C. A third inbred line (‘Inbred line B’) was found on the middle shore, but no homogeneous populations were found. However, indirect evidence suggests that Inbred line B has a thermal response intermediate between those of Inbred lines A and C. Study of populations made up of mixtures of inbred lines confirmed the relationship between upper MLTs and genetic composition of the population.

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Allozyme-Specific Modification of a Maize Seed Chitinase by a Protein Secreted by the Fungal Pathogen Stenocarpella maydis

Phytopathology ◽

10.1094/phyto-100-7-0645 ◽

2010 ◽

Vol 100 (7) ◽

pp. 645-654 ◽

Cited By ~ 24

Author(s):

Todd A. Naumann ◽

Donald T. Wicklow

Keyword(s):

Inbred Line ◽

Fungal Pathogen ◽

Genetic Basis ◽

Inbred Lines ◽

Ear Rot ◽

Maize Seed ◽

Stalk Rot ◽

Stenocarpella Maydis ◽

Commercial Field

Stenocarpella maydis causes both dry-ear rot and stalk rot of maize. Maize inbred lines have varying levels of resistance to ear rot caused by S. maydis. The genetic basis of resistance appears to rely on multiple genetic factors, none of which are known. The commonly used stiff-stalk inbred line B73 has been shown to be strongly susceptible to ear rot caused by S. maydis. Here, we report that the ChitA protein alloform from B73, ChitA-F, encoded by a known allele of the chiA gene, is susceptible to modification by a protein (Stm-cmp) secreted by S. maydis. We also identify a new allele of chiA (from inbred line LH82) which encodes ChitA-S, an alloform of ChitA that is resistant to Stm-cmp modification. Chitinase zymogram analysis of seed from a commercial field showed the presence of both ChitA alloforms in healthy ears, and showed that ChitA-F but not ChitA-S was modified in ears rotted by S. maydis. The ChitA-F protein was purified from inbred line B73 and ChitA-S from LH82. ChitA-F was modified more efficiently than ChitA-S by S. maydis protein extracts in vitro. The chiA gene from LH82 was cloned and sequenced. It is a novel allele that encodes six polymorphisms relative to the known allele from B73. This is the first demonstration that the susceptibility to modification of a fungal targeted plant chitinase differs among inbred lines. These findings suggest that the LH82 chiA allele may be a specific genetic determinant that contributes to resistance to ear rot caused by S. maydis whereas the B73 allele may contribute to susceptibility.

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The ffs Allele Conditions Flower Stem Fasciation in Red Beet

HortScience ◽

10.21273/hortsci.33.3.527f ◽

1998 ◽

Vol 33 (3) ◽

pp. 527f-528

Author(s):

I.L. Goldman

Keyword(s):

Genetic Control ◽

Inbred Line ◽

Goodness Of Fit ◽

Female Parent ◽

Inbred Lines ◽

Chi Square ◽

Recessive Character ◽

Variable Expression ◽

Red Beet ◽

Flower Stem

A fasciated flower stem character arose spontaneously during development of the red beet (Beta vulgaris L.) inbred line W411. The fasciated character is manifest by a flattened flower stem with petioles coalesced into a twisted, ribbonlike appearance. No fasciation is present in the vegetative stem or petioles. An inheritance study was conducted to determine the genetic control of flower stem fasciation. The inbred line W411 was used both as a male and female parent in crosses with four red beet inbred lines. Segregating progenies in both the BC1 and F2 generations were developed and scored for the fasciated flower stem character. Variable expression of the fasciated flower stem phenotype was observed in these progenies; however, the presence of flattened flower stems at the stem/hypocotyl junction was unequivocal. Chi-square goodness-of-fit tests in both the BC1 and F2 generations did not deviate significantly from expected ratios for a monogenic recessive character for each genetic background. No reciprocal differences were detected for any cross in this group of four inbred lines, which suggests the lack of maternal effect for the fasciated character. The symbol ffs is proposed to describe the genetic control of the fasciated flower stem phenotype.

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Development of genic KASP SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize

BMC Plant Biology ◽

10.1186/s12870-021-02932-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Zhengjie Chen ◽

Dengguo Tang ◽

Jixing Ni ◽

Peng Li ◽

Le Wang ◽

...

Keyword(s):

Marker Assisted Selection ◽

Inbred Lines ◽

Average Density ◽

Snp Markers ◽

Data Sets ◽

Rna Seq ◽

Specific Pcr ◽

Maize Inbred Lines ◽

Allele Specific ◽

Allele Specific Pcr

Abstract Background Maize is one of the most important field crops in the world. Most of the key agronomic traits, including yield traits and plant architecture traits, are quantitative. Fine mapping of genes/ quantitative trait loci (QTL) influencing a key trait is essential for marker-assisted selection (MAS) in maize breeding. However, the SNP markers with high density and high polymorphism are lacking, especially kompetitive allele specific PCR (KASP) SNP markers that can be used for automatic genotyping. To date, a large volume of sequencing data has been produced by the next generation sequencing technology, which provides a good pool of SNP loci for development of SNP markers. In this study, we carried out a multi-step screening method to identify kompetitive allele specific PCR (KASP) SNP markers based on the RNA-Seq data sets of 368 maize inbred lines. Results A total of 2,948,985 SNPs were identified in the high-throughput RNA-Seq data sets with the average density of 1.4 SNP/kb. Of these, 71,311 KASP SNP markers (the average density of 34 KASP SNP/Mb) were developed based on the strict criteria: unique genomic region, bi-allelic, polymorphism information content (PIC) value ≥0.4, and conserved primer sequences, and were mapped on 16,161 genes. These 16,161 genes were annotated to 52 gene ontology (GO) terms, including most of primary and secondary metabolic pathways. Subsequently, the 50 KASP SNP markers with the PIC values ranging from 0.14 to 0.5 in 368 RNA-Seq data sets and with polymorphism between the maize inbred lines 1212 and B73 in in silico analysis were selected to experimentally validate the accuracy and polymorphism of SNPs, resulted in 46 SNPs (92.00%) showed polymorphism between the maize inbred lines 1212 and B73. Moreover, these 46 polymorphic SNPs were utilized to genotype the other 20 maize inbred lines, with all 46 SNPs showing polymorphism in the 20 maize inbred lines, and the PIC value of each SNP was 0.11 to 0.50 with an average of 0.35. The results suggested that the KASP SNP markers developed in this study were accurate and polymorphic. Conclusions These high-density polymorphic KASP SNP markers will be a valuable resource for map-based cloning of QTL/genes and marker-assisted selection in maize. Furthermore, the method used to develop SNP markers in maize can also be applied in other species.

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Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Agronomy ◽

10.3390/agronomy11050982 ◽

2021 ◽

Vol 11 (5) ◽

pp. 982

Author(s):

Zhiliang Xiao ◽

Congcong Kong ◽

Fengqing Han ◽

Limei Yang ◽

Mu Zhuang ◽

...

Keyword(s):

Molecular Markers ◽

Brassica Oleracea ◽

Rapid Identification ◽

Inbred Lines ◽

Hybrid Lethality ◽

Specific Pcr ◽

Allele Specific ◽

User Friendly ◽

Allele Specific Pcr ◽

Kasp Marker

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

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Inbreeding and thermal adaptation in Drosophila subobscura

Genome ◽

10.1139/gen-2014-0149 ◽

2014 ◽

Vol 57 (9) ◽

pp. 481-488 ◽

Cited By ~ 4

Author(s):

Goran Zivanovic ◽

Conxita Arenas ◽

Francesc Mestres

Keyword(s):

Inbred Line ◽

Thermal Adaptation ◽

Inbred Lines ◽

Light Period ◽

Drosophila Subobscura ◽

Adaptive Potential ◽

Line Number ◽

Evolutionary Forces ◽

Laboratory Conditions ◽

Significant Line

Using a well-adapted Drosophila subobscura population (Avala, Serbia), a drastic experiment of inbreeding was carried out to assess whether the expected level of homozygosity could be reached or if other evolutionary forces affected the process. In general, no significant changes of inversion (or arrangement) frequencies were detected after 12 brother–sister mating generations. Furthermore, no significant differences were obtained between observed and expected (under the inbreeding model) karyotypic frequencies. Thus, these results seemed to indicate that the main evolutionary factor in the experiment was inbreeding. However, in the G12 generation, complete chromosomal fixation was reached only in two out of the eight final inbred lines. In these lines, the chromosomal compositions were difficult to interpret, but they could be likely a consequence of adaptation to particular laboratory conditions (constant 18 °C, food, light period, etc.). Finally, in a second experiment, the inbred lines presented higher fertility at 18 °C than at 13 °C. Also, there was a significant line effect on fertility: inbred line number 6 (A1, J1, U1+2; U1+2+6, E8, and O3+4+7) presented the highest values, which maybe the result of an adaptation to laboratory conditions. Thus, the results obtained in our experiments reflect the adaptive potential of D. subobscura inversions.

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