CN110129475B - SSR nucleic acid sequence related to cotton high coat score and application thereof - Google Patents
SSR nucleic acid sequence related to cotton high coat score and application thereof Download PDFInfo
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Abstract
The invention discloses a SSR nucleic acid sequence related to cotton high coat division and application thereof. The invention provides a chromosome fragment from abnormal cotton, which is a fragment from an SSR molecular marker JAAS6365 to an SSR molecular marker JAAS5604 on an abnormal cotton No.5 chromosome; the nucleotide sequence of the SSR molecular marker JAAS6365 is shown in SEQ ID No. 1; the nucleotide sequence of the SSR molecular marker JAAS5604 is shown in SEQ ID No. 2. The chromosome fragment provided by the invention has important application value in high-lint cotton breeding, and meanwhile, the SSR molecular marker developed by the invention provides a molecular basis for cultivating high-lint cotton varieties which can be applied to production.
Description
Technical Field
The invention relates to the field of cotton breeding, in particular to a SSR nucleic acid sequence related to cotton high coat division and application thereof.
Background
Cotton is an important economic crop and natural cotton fiber is the main raw material of the world's textile industry. There are four cultivars of cotton (Gossypium spp.) including heterotetraploid upland cotton (Gossypium hirsutum L.) and Gossypium barbadense L., diploid grass cotton (g. herbaceum L.) and Gossypium asianum L can produce natural fibers for textile use. Among them, upland cotton dominates world cotton production due to its high yield and wide adaptability. Increasing yield and improving fiber quality have been two important goals for the improvement of upland cotton varieties. With the rapid development of the textile industry, people have higher and higher requirements on fiber quality, but the demand for excellent fiber quality is pursued and clothes marks are reduced, so that how to coordinate the fiber quality and yield in the genetic improvement of upland cotton is an important research topic at present.
The cotton yield constitutive factors are as follows: the Number of bolls of a single plant (NB), the Seed-cotton yield of a single plant (SY), the Lint yield of a single plant (LY), the Number of seeds of a single plant (NS), the Boll weight (W), the clothing index (LI), the Seed Index (SI), and the clothing score (LP), wherein the Boll Number, the Boll weight, and the clothing score are three major factors constituting the cotton yield, and the clothing score is an important factor determining the cotton fiber yield and has a significant negative correlation with the fiber quality, so that it is increasingly difficult to improve the cotton fiber quality and the cotton yield synchronously by the conventional breeding means. The development of molecular markers provides a rapid, accurate and effective selection method for breeders.
Disclosure of Invention
The invention aims to provide a SSR nucleic acid sequence related to cotton high coat division and application thereof.
In a first aspect, the invention claims a DNA fragment.
The DNA fragment claimed by the invention is a fragment which is located on the abnormal cotton No.5 chromosome and at least contains the SSR molecular marker JAAS6365 to the SSR molecular marker JAAS 5604.
Wherein, the nucleotide sequence of the SSR molecular marker JAAS6365 is shown in SEQ ID No. 1; the nucleotide sequence of the SSR molecular marker JAAS5604 is shown in SEQ ID No. 2.
Further, the DNA fragment is a fragment located on abnormal cotton chromosome 5 from the SSR molecular marker JAAS6365 to the SSR molecular marker JAAS 5604.
In a second aspect, the invention claims a vector, expression cassette, recombinant bacterium or transgenic cell line comprising the DNA segment of the first aspect.
The vector may be an artificial chromosome vector, such as a bacterial artificial chromosome or a yeast artificial chromosome, and the like. The recombinant bacteria and the transgenic cell line contain the artificial chromosome vector.
In a third aspect, the invention claims the use of a DNA fragment as described in the first aspect or a vector, expression cassette, recombinant bacterium or transgenic cell line as described in the second aspect to improve coat length in cotton or to breed coat-improved cotton varieties.
In a fourth aspect, the invention claims an SSR molecular marker or set of SSR molecular markers on the DNA fragment described in the first aspect.
The SSR molecule marker claimed in the present invention is any one of (a1) - (a3) below; the complete set of SSR molecular markers consists of the following components (a1) - (a 3);
(a1) SSR molecular marker JAAS 6365: DNA molecule with nucleotide sequence shown in SEQ ID No. 1;
(a2) SSR molecular marker JAAS 5604: DNA molecule with nucleotide sequence shown in SEQ ID No. 2;
(a3) SSR molecular marker JAAS 0803: DNA molecule with nucleotide sequence shown in SEQ ID No. 3.
In a fifth aspect, the invention claims a primer pair or primer set for identifying the SSR molecular marker or SSR molecular marker set described in the fourth aspect.
The primer pair claimed by the invention is any one of (b1) - (b 3); the primer set consists of the following components (b1) - (b 3);
(b1) primer pair 1 for identifying SSR molecular marker JAAS 6365: designed according to SEQ ID No. 1;
(b2) a primer pair 2 for identifying the SSR molecular marker JAAS 5604: designed according to SEQ ID No. 2;
(b3) primer pair 3 for identifying the SSR molecular marker JAAS 0803: designed according to SEQ ID No. 3.
In a specific embodiment of the invention, the primer pair 1 consists of two single-stranded DNAs shown as SEQ ID No.4 and SEQ ID No. 5; the primer pair 2 consists of two single-stranded DNAs shown as SEQ ID No.6 and SEQ ID No. 7; the primer pair 3 consists of two single-stranded DNAs shown as SEQ ID No.8 and SEQ ID No. 9.
In a sixth aspect, the invention claims a kit comprising a primer pair or a set of primer pairs as described in the fifth aspect.
The kit may further contain any one or more of PCR amplification buffer, double distilled water, DNA polymerase, dNTP, and the like.
In a seventh aspect, the invention claims the use of the SSR molecular marker or set of SSR molecular markers of the fourth aspect, or the primer pair or set of primer pairs of the fifth aspect, or the kit of the sixth aspect, in screening the DNA fragments of the first aspect to breed cotton varieties with increased coat-keeping.
In an eighth aspect, the invention claims the use of the SSR molecular marker or the set of SSR molecular markers in the fourth aspect or the primer pair or the set of primer pairs in the fifth aspect or the kit in the sixth aspect for identifying or assisting in identifying cotton coat trait.
In a ninth aspect, the invention claims a method of breeding cotton varieties with improved seed coats, comprising the steps of: replacing the original chromosome segment of the receptor parent with a segment at least containing from the SSR molecular marker JAAS6365 to the SSR molecular marker JAAS5604 on the abnormal cotton No.5 chromosome (such as a segment from the SSR molecular marker JAAS6365 to the SSR molecular marker JAAS5604 on the abnormal cotton No.5 chromosome), and obtaining the cotton variety with improved dressing score.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method combines the technologies of distant hybridization, molecular marker assisted selection and the like, and replaces the segment of the abnormal cotton No.5 chromosome between SSR molecular markers JAAS6365 and JAAS5604 in the genetic background of Gossypium hirsutum Suo 8289, so as to obtain the high-lint cotton line with chromosome segment replacement. Therefore, the chromosome fragment (the fragment of the abnormal cotton No.5 chromosome between SSR molecular markers JAAS6365 and JAAS5604) developed by the method has important application value in the breeding of high-lint cotton in the future.
(2) The molecular marker assists in target fragment selection, and has the characteristics of early identification, rapid identification and high accuracy and stability, so that the 3 SSR molecular markers and the primer pairs thereof provided by the invention are expected to greatly improve the selection efficiency and breeding speed of the cotton breeding coat character, and have important significance for accelerating the breeding process of the cotton high-coat new variety.
(3) The high-coat-length cotton line provided by the invention can be used for fine positioning of high-coat-length genes and related gene cloning and function analysis through SSR molecular markers and primer pairs thereof, not only can clarify the molecular genetic mechanism of the high-coat-length traits of cotton, but also provides materials and molecular basis for cultivating high-coat-length varieties which can be applied to production.
Drawings
FIG. 1 is a graphical representation of the genotype and distribution of introgression fragments for the abnormal cotton chromosome fragment replacement lines of the present invention. Light grey represents the genotype of the recurrent parent gossypium hirsutum su8289, black represents the genotype of abnormal cotton, dark grey represents the heterozygous segment. The columns represent chromosomes, and are Chr.1-Chr.13 from left to right; the lines represent 74 chromosome fragment substitution lines from top to bottom in sequence, and the right side of each chromosome fragment substitution line corresponds to the information of the number, the length, the proportion of abnormal cotton genome and the proportion of recurrent parent genome of introgression fragments.
FIG. 2 is the coverage of 13 chromosomes with abnormal cotton chromosome fragments from introgression lines.
FIG. 3 shows the BC of the present invention4F4Glue pattern. A is SSR molecular marker JAAS 6365; b is SSR molecular marker JAAS 0803; and C is SSR molecular marker JAAS 5604.
FIG. 4 shows BC of SSR molecular marker JAAS5604 of the present invention4F3Glue pattern.
FIG. 5 is an analysis chart of abnormal cotton No.5 chromosome fragment and clothes mark in the present invention.
FIG. 6 is a bar graph of the differential scores between the high-coat single-fragment replacement line CSSL18 and the recurrent parent threonine 8289 in accordance with the present invention.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Upland cotton 86-1(g.hirsutum var.86-1), upland cotton threo 8289(g.hirsutum var.su8289) and cotton anomaly (Gossypium anomalum): they are described in "Caijiao Zhai, Peng Xu, Xia Zhang et al, Development of Gossypium anomallium derived microsatellite markers and the iron use for genome-wide identification of recombination between the G.anomallium and G.hirsutum genomes, therapeutic and Applied Genetics,2015,128(8): 1531-1540", publicly available from the applicant, and available only for use in the experiments of the instant invention, not for others.
Example 1 determination of chromosome fragments associated with high coat-length trait in Cotton and development of related molecular markers
The high-lint cotton is cultivated by the following steps:
hybridizing the upland cotton 86-1 serving as a donor with the abnormal cotton of the male parent to obtain triploid F1(A1D1B1) Treating young axillary bud of triploid seedling with 0.15% colchicine to obtain fertile hexaploid F1(A1A1D1D1B1B1)；
Hybridizing the hexaploid serving as a female parent with a parent Gossypium hirsutum Soviet 8289 to obtain a pentaploid A1A1D1D1B1Planting all received seeds to obtain 97 single plants;
backcrossing the obtained pentaploid with threo 8289 to obtain BC2F1The seeds of (4) are planted completely, 384 single plants are obtained, 50 recombination types are obtained, and 36 recombination types are fertile;
mixing BC2F1Backcrossing all the individuals with Su8289, planting all the plants to obtain 4331 BC3F1Single plant, 40 recombination types;
combining BC3F1The recombinant individuals and the episomal individuals in the population were continuously backcrossed with threo 8289 to obtain 8540 BC4F1Single plant, 56 recombination types;
mixing BC4F156 recombination types in the populationSelfing to obtain the selfed BC4F2Seed, planting BC4F2And 4543 individuals are obtained in total.
The recombination type is detected by adopting an SSR marker, and the SSR marker is from the following sources: integrating the abnormal cotton specific SSR markers with published cotton whole genome SSR physical maps, screening a set of abnormal cotton specific SSR markers (230) uniformly covering the genome, and analyzing the population to obtain the marker genotype of the abnormal cotton introgression segments.
From BC2F1Initially, the selected recombinant individuals are further backcrossed twice, three times, each generation according to BC2F1And (3) selecting 130 key SSR markers uniformly covering the chromosome set from 230 markers covering the genome according to the marker information of the recombination breakpoint, detecting all recombinant individuals by using a PCR (polymerase chain reaction) technology, wherein the size of each generation population is shown in table 1, and finally obtaining a set of homozygous abnormal cotton chromosome fragment substitution lines (introgression lines).
PCR amplification conditions: using upland cotton threo 8289, abnormal cotton and hexaploid F1And performing genotype detection by taking the filial generation as a template. The PCR reaction system is 10 μ L: DNA template 2. mu.L, 10 XBuffer (Mg)2+) Mu. L, dNTP 0.2. mu.L, Taq DNA polymerase 0.1. mu.L, front and rear primers 0.5. mu. L, ddH each2O 3.7μL。
PCR thermal cycling program: the first step is as follows: pre-denaturation at 95 ℃ for 5 min; the second step: denaturation at 94 ℃ for 30 s; the third step: annealing at 55 ℃ for 45 s; the fourth step: extension at 72 ℃ for 1min (30 cycles of second to fourth steps); the fifth step: extending for 10min at 72 ℃; and a sixth step: storing at 10 deg.C.
Detection of PCR amplification product: the first step is as follows: cleaning the glass plate, and airing for later use; the second step is that: aligning and flatly placing two glass plates in a set, clamping two sides by using a clamp, and placing the two glass plates on a flat plate; the third step: drain with glass rod, slowly pour 8% polyacrylamide gel (29:1), insert comb. Standing for 15 min; the fourth step: after the gel is solidified, loosening the clamp, putting the glass plate with the gel into an electrophoresis tank, pouring 1 xTBE electrophoresis buffer solution into the electrophoresis tank, fastening two sides of the clamp, continuously adding the electrophoresis buffer solution, immersing the gel, and pulling out a comb; the fifth step: spotting (loading 1 μ L), and performing electrophoresis for 1.5h under a 180V constant pressure environment.
BC obtained in 20154F1Selfing all recombinant individuals in the population to obtain BC4F2Seeds, 2016 planting BC in summer4F2Population, a total of 4543 individuals were obtained. According to genetic map and BC2F1Counting the marker information of the data recombination breakpoint, selecting 130 pairs of key SSR primers uniformly covering the chromosome group to detect all BC4F2Population, 51 recombinant types were obtained, 45 of which were homozygous genotypes.
Further planting BC in 2016 (12 months)4F3Population, 1533 individuals in total. According to genetic map and BC2F1Counting the marker information of the data recombination breakpoint, selecting 130 pairs of key SSR primers uniformly covering the chromosome group to detect all BC4F3Population, 53 recombinant types were obtained, 47 of which were homozygous genotypes.
Planting BC in 20174F4In the population, 2225 individuals are obtained in total, 230 SSR (including 130 pairs of primers) primers which are developed and screened by the subject group and uniformly cover an abnormal cotton chromosome group are used for detection, and 74 recombination types are obtained in total, wherein 71 recombination types are homozygous genotypes. The size composition of each generation population is shown in table 1, and the number of recombination types in each generation is shown in table 2.
TABLE 1 BC3F1、BC4F1、BC4F2、BC4F3、BC4F4Population size generation for molecular marker assisted selection
Note: the numbers in parentheses in the second, third and fourth columns indicate the number of selected additional lines.
1、BC4F2、BC4F3And BC4F4Detection of generational recombination types
BC obtained in 20154F1Selfing all recombinant individuals in the population to obtain BC4F2Seeds, 2016 planting BC in summer4F2The population, a total of 4543 individuals were obtained, and genetic mapping and BC were used2F1And (3) selecting 130 key SSR primers which uniformly cover the chromosome group from the marker information of the recombination breakpoint, and detecting to obtain 51 recombination types, wherein 45 recombination types are homozygous genotypes, and the detail is shown in Table 2. And BC4F1Compared with the prior art, 8 new recombination types appear, and the marker intervals of the abnormal cotton chromosome segments are Chr.6: NAU2714-JAAS1095, NAU2714-JAAS1095&NAU1272-JAAS2480, JAAS6227-JAAS 2480; chr.9: JAAS1923-JAAS 0613; chr.10: JAAS1256-JAAS 3294; chr.11: JAAS4829-NAU3703, NAU3703-NAU3234 and Chr.13: JAAS4570-JAAS 2038.
Hainan BC of 2016 months and 12 years4F3Population, 1533 individuals in total, according to genetic map and BC2F1And (3) selecting 130 key SSR primers which uniformly cover the chromosome set from the marker information of the recombination breakpoint to detect, and obtaining 53 recombination types, wherein 47 are homozygous substitution lines (table 2). And BC4F2Compared with at BC4F32 new recombination types appear in the generation, and the marker intervals of the abnormal cotton chromosome segments are Chr.5: DC40130-DC40130 and Chr.11: JAAS0280-JAAS 3199.
Planting BC in 20174F4In the population, 2225 individuals are obtained in total, 230 SSR (including 130 pairs of primers) primers which are developed and screened by the subject group and uniformly cover an abnormal cotton chromosome group are used for detection, and 74 recombination types are obtained in total, wherein 71 recombination types are homozygous genotypes. And BC4F3By comparison, at BC4F420 new recombination types are detected, and the marker intervals of the abnormal cotton chromosome segments are Chr.1: JAAS0826-JAAS1148, NAU3615-JAAS1148,NAU3615-JAAS5817, NAU3615-JAAS0392, NAU5100-NAU4045, NAU2083-NAU2083, NAU2083-JAAS2569 and NAU4045-NAU 4045; chr.4: JAAS2022-JAAS 2076; chr.8: NAU1037-JAAS 6420; chr.10: JAAS3294-JAAS3294, JAAS1256-JAAS 1256; chr.11: JAAS4829-JAAS4829, JAAS3088-JAAS5224, and JAAS4259-JAAS 4259. Since all individuals were subjected to marker scanning at the genome wide level, there were combined types of different chromosomal introgression segments in the new recombination types detected.
The summary of the detection of recombination types in six consecutive generations during the construction of the abnormal cotton chromosome fragment replacement line population is shown in Table 2. From BC2F1To BC4F4Each generation has new recombination type, and simultaneously, due to the reasons of poor individual fertility of offspring, low seed vigor and the like, each generation has the phenomenon of losing the recombination type. To compensate for lost recombination types, at BC3F1、BC4F1And BC4F2Backcrossing of the generation recombination individuals and the recurrent parent is carried out, and simultaneously hybridization of the additional line and the recurrent parent is carried out, so that partial lost recombination types can be recovered, and more new recombination types derived from the lost types are generated. The number of recombination types is from the first 50 (BC)2F1) Increased to 74 (BC)4F4) And finally obtaining 71 abnormal cotton chromosome segment substitution lines (genotype homozygosis) capable of being stably inherited.
Specifically, the number of recombination types identified in different populations is shown in table 2.
TABLE 2 BC2F1、BC3F1、BC4F1、BC4F2、BC4F3And BC4F4Number of recombination types identified in generations
| Chromosome | BC2F1 | BC3F1 | BC4F1 | BC4F2 | BC4F3 | BC4F4 |
| Chr.1 | 3 | 4 | 9 | 8 | 8 | 13 |
| Chr.2 | 4 | 5 | 6 | 3(2) | 3(2) | 3 |
| Chr.3 | 1 | 1 | 3 | 1(1) | 1(1) | 1(1) |
| Chr.4 | 1 | 5 | 3 | 2(1) | 2(1) | 3 |
| Chr.5 | 4 | 6 | 6 | 6 | 7 | 7 |
| Chr.6 | 2 | 2 | 3 | 5 | 5 | 6(1) |
| Chr.7 | 1 | 0 | 1 | 1 | 1 | 1 |
| Chr.8 | 1 | 0 | 3 | 1 | 1 | 2 |
| Chr.9 | 3 | 2 | 2 | 3(1) | 3(1) | 3(1) |
| Chr.10 | 1 | 2 | 2 | 1 | 1 | 3 |
| Chr.11 | 11 | 11 | 11 | 13(1) | 14(1) | 17 |
| Chr.12 | 3 | 2 | 3 | 3 | 3 | 3 |
| Chr.13 | 1 | 0 | 4 | 4 | 4 | 4 |
| Total up to | 36 | 40 | 56 | 51(6) | 53(6) | 74*(3) |
Note: the numbers in parentheses indicate the number of heterozygous recombination types.*Due to being at BC4F4The generation detects recombinant types containing more than two chromosome fragments at the same time, so that the total number is larger than the total number of the recombinant types accumulated by taking the chromosome as a unit.
2. Identification of abnormal cotton chromosome fragment substitution lines
At BC4F4Generating, performing whole genome foreground and background identification on all recombinant individuals by using 230 specific SSR primers uniformly covering an abnormal cotton genome, and finally determining 74 stably inherited chromosome fragment substitution lines which are sequentially numbered by CSSL1-CSSL74, wherein the number of the single fragment substitution lines is 43, the number of the two fragment substitution lines is 24, and the number of the three fragment substitution lines is 7; the introgression lines of 1 single-fragment and 2 two-fragment lines also remained heterozygous, and the remaining 71 lines were homozygous chromosome fragment lines. The chromosome segments of the two-segment and three-segment replacement lines were mainly the combinations between the introgressed segments on chr.1, chr.2, chr.5, chr.6, chr.9, chr.10, chr.11, chr.12 and chr.13. The types of chromosome fragment substitution lines and the corresponding types of introgression fragments are shown in Table 3.
TABLE 3 BC4F4Detected abnormal cotton chromosome introgression segment and obtained chromosome segment substitution line
Note:*the introgression fragment is in heterozygous state. Underlined notation indicates backcrossing with the addition line.+The designation is a two-fragment substitution line,#the three-segment substitution lines are labeled, and the single-segment substitution lines are not labeled.
The chromosomal composition of the strain is deduced by the method of Young software (Van Berllo R.GGT 2.0: Versatile software for visualization and analysis of genetic data. journal of knowledge, 2008,99(2):232-236.https:// doi.org/10.1093/joined/esm 109), and Young and Tanksley (Young ND, Tanksley SD.Restriction acquisition length fragment fragments and the concept of genetic applications, 1989,77(1):95-101.https:// doi.org/10.1007/bf00292322), defining the position and size of the donor fragment, etc. Mapping the distribution of the chromosome fragments of the 74 chromosome fragment replacement lines (fig. 1) it can be seen that the abnormal cotton fragments (black) on the diagonal cover most of the genome, with overlap between the fragments of the majority of the chromosome fragment replacement lines. The length of the abnormal cotton segment in the chromosome segment substitution line is between 4.75cM (CSSL10) and 267.45cM (CSSL59), the average length is 68.91cM, and the cumulative length is 4972.79cM, which is equivalent to 2.15 times of the total length of the abnormal cotton genome. Whereas all introgression fragments covered the total length of the abnormal cotton chromosome by 1607.1cM due to the overlap between the introgression fragments, the coverage was about 70% (fig. 2). The chromosome fragment substitution coefficients of the chromosome fragments with Chr.1 and Chr.11 are more, 20 and 27 respectively, and the coverage rate of the two abnormal cotton chromosomes reaches 100 percent. The number of substitution coefficients of chromosome fragments containing both Chr.3 and Chr.7 chromosome fragments is only 1, but the chromosome coverage is not low (59.9% and 61.2%, respectively) due to the longer length of the fragment. The lowest coverage was chr.10, which was only 17.9%. It can also be seen from figure 1 that the dark and dark grey sections are not abundant in the light grey regions outside the diagonal, indicating that the genetic background recovery of gossypium hirsutum threonine 8289 for this set of chromosome segment replacement materials is high, ranging from 88.43% (CSSL59) to 99.79% (CSSL10), with an average recovery of 97.09%.
BC4F4The SSR detection gel map of the strain is shown in FIG. 3.
In fig. 3, a is a detection result of SSR molecular marker JAAS 6365; marker in lane 1, genotype of gossypium hirsutum su8289 in lane 2, genotype of gossypium hirsutum 86-1 in lane 3, genotype of abnormal cotton in lane 4, and hexaploid F in lane 51The genotype of (1), the genotype of CSSL18 in lane 6, the genotype of CSSL50 in lane 7, the genotype of CSSL51 in lane 8, the genotype of CSSL19 in lane 9, the genotype of CSSL52 in lane 10 and the genotype of CSSL53 in lane 11.
In FIG. 3, B is the detection result of SSR molecular marker JAAS 0803; marker in lane 1, genotype of threo 8289 in lane 2, genotype of 86-1 in lane 3, genotype of abnormal cotton in lane 4, and hexaploid F in lane 51 Lane 6 is the genotype of CSSL18, lane 7 is the genotype of CSSL50, lane 8 is the genotype of CSSL51, lane 9 is the genotype of CSSL19, lane 10 is the genotype of CSSL52, lane 11 is the genotype of CSSL 53.
In fig. 3, C is a detection result of SSR molecular marker JAAS 5604; marker in lane 1, genotype of threo 8289 in lane 2, genotype of 86-1 in lane 3, genotype of abnormal cotton in lane 4, and hexaploid F in lane 51 Lane 6 is the genotype of CSSL18, lane 7 is the genotype of CSSL50, lane 8 is the genotype of CSSL51, lane 9 is the genotype of CSSL19, lane 10 is the genotype of CSSL52, lane 11 is the genotype of CSSL 53. Genotypes of CSSL18, CSSL50 and CSSL51 with hexaploid F1The genotypes of (a) and (b) are the same for the following reasons: abnormal cotton B1And landThe sub-group of the gossypium hirsutum At is easy to recombine. In general, the Dt subgroup of Gossypium hirsutum also presents an SSR site homologous to the At subgroup, where no recombination has occurred. During PCR amplification, the primer and the abnormal cotton B1While the group SSR sites bind, they also bind to SSR sites of the Dt subgroup, and thus the genotypes of CSSL18, CSSL50 and CSSL51 are associated with hexaploid F1The same is true. To verify this conclusion, BC at CSSL184F3In the population, 20 individuals subjected to selfing are selected for genotype detection, and the genotype is not separated as a result, which shows that the three chromosome fragment replacement lines are stably inherited and are homozygous chromosome fragment replacement lines (figure 4).
FIG. 4 shows BC of SSR molecular marker JAAS5604 in CSSL184F3And (5) generation detection results. Marker in lane 1, genotype of threo 8289 in lane 2, genotype of 86-1 in lane 3, genotype of abnormal cotton in lane 4, and hexaploid F in lane 51The genotype of (4), lanes 6-25 are BC4F3Genotype of 20 individuals in the population.
SSR marker analysis: the same band pattern as that of Su8289 and 86-1 was designated as "1", the same band pattern as that of abnormal cotton was designated as "2", and F was designated as that of abnormal cotton at the time of genotype data collection1The same band pattern is denoted as "3" and the absence or ambiguity of the band pattern is denoted as "-".
BC shown by FIG. 34F4As can be seen from SSR detection gel images of strains, in the detection results of SSR molecular markers JAAS6365 and JAAS0803, the band types of three chromosome fragment substitution lines CSSL50, CSSL51 and CSSL18 are consistent with abnormal cotton band types (2). The SSR molecular marker JAAS5604 has band pattern similar to that of hexaploid F in three chromosome segment substitution lines CSSL50, CSSL51 and CSSL181The belt patterns are identical ("3"), but BC at CSSL50, CSSL51 and CSSL184F3The generations did not segregate (FIG. 4), indicating that the three chromosome fragment replacement lines had stably inherited and were homozygous chromosome fragment replacement lines. Thus, CSSL50, CSSL51, and CSSL18 all contained SSR molecular markers JAAS6365, JAAS5604, and JAAS0803 located on abnormal cotton chromosome 5.
Statistical analysis shows that the introgression fragments from the abnormal cotton chromosome 5 exist in all the high-lint cotton lines.
To verify the genetic contribution of the abnormal cotton fragment introgressed by Gossypium hirsutum Chr.5 to the score, we analyzed the genotype and the score of the chromosome fragment replacement line of chromosome 5. The results are shown in FIG. 5. In FIG. 5, the left part is the genetic background analysis of the different chromosome fragment substitution line of chromosome 5 on chromosome 5. Light gray indicates threo 8289 gene fragment and black indicates abnormal cotton gene fragment. The right hand part was the coat phenotype statistics for the different chromosome fragment replacement lines and the recurrent parent su 8289.*Indicating significant difference.
As can be seen from fig. 5, the chromosome segment substitution lines of chromosome 5 have different substitution segments, wherein each of the 3 chromosome segment substitution lines CSSL50, CSSL51, and CSSL18 contains a specific abnormal cotton SSR molecular marker JAAS6365, JAAS0803, and JAAS5604 (i.e., each of the 3 chromosome segment substitution lines CSSL50, CSSL51, and CSSL18 contains a segment located on the abnormal cotton chromosome 5 from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604), and has a different degree of improvement in the clothing scores compared to recurrent parent su 8289. And the rest 3 chromosome fragment substitution lines do not contain the abnormal cotton chromosome fragments marked by the three SSR molecules, and the difference of the coat score of the abnormal cotton chromosome fragment is not significant compared with the recurrent parent threonine 8289. The genetic contribution of the abnormal cotton chromosome segments of the three SSR molecular markers JAAS6365, JAAS0803 and JAAS5604 to the high-clothes score is verified to a certain extent.
Adopting a single marker analysis method of WinQTLCart 2.5 software, carrying out correlation analysis on all 23 markers of the No.5 chromosome and the clothes character, and displaying the results: markers JAAS6365, JAAS0803 and JAAS5604 had significant correlation with the scores, and the remaining markers had no correlation with the scores (table 4).
TABLE 4 correlation analysis of signatures JAAS6365, JAAS0803 and JAAS5604 with the scores of clothes
| Traits | Chromosome | Marking | P value | R2 |
| Clothes divider | Chr.5 | JAAS6365 | 0.00832195 | 0.111 |
| Clothes divider | Chr.5 | JAAS0803 | 0.00832195 | 0.111 |
| Clothes divider | Chr.5 | JAAS5604 | 0.00832195 | 0.111 |
Line CSSL18 was planted in a different environment, environment 1: lishu plant science base of agricultural science institute of Jiangsu, Nanjing Jiangsu province in 2017; environment 2: lishu plant science base of agricultural science institute of Jiangsu Nanjing Jiangsu province in 2018; environment 3: xinjiang Korla in 2018. Repeating: all test points are arranged in random blocks and are repeated for 3-4 times, and the planting density and the production management are the same as the local field production of the test points.
The scores were counted as shown in Table 5.
TABLE 5 Perkins phenotype statistics of lines CSSL18 and recurrent parent Su8289
Note: "-" indicates no detection.
As can be seen from table 5, a single-segment substitution line CSSL18 of chromosome 5 (during the process of constructing a set of abnormal cotton chromosome segment substitution line population, identified by SSR molecular marker-assisted selection at each generation, CSSL18 only differs from the recurrent parent ja 8289 in that the corresponding segment on chromosome 5 is replaced by the "segment located on the abnormal cotton chromosome 5 from SSR molecular marker JAAS6365 to SSR molecular marker JAAS 5604", and the rest of the genome is completely identical to the recurrent parent ja 8289), has a score of 41.97-46.57 in different environments, is significantly higher than that of the recurrent parent ja 8289(39.87-43.13), and can be used as an important material for high-yield breeding of cotton and research on molecular genetic mechanisms of cotton.
Fig. 6 corresponds to the numerical values of table 5.
Fig. 6 shows that the difference between the scores of CSSL18 and threo 8289 is significant.
From the above experimental results, it can be seen that: the cotton lint can be improved by containing a chromosome segment from the SSR molecular marker JAAS6365 to the SSR molecular marker JAAS5604 on the abnormal cotton No.5 chromosome. The 3 SSR molecular markers (JAAS6365, JAAS0803 and JAAS5604) on the chromosome segment and the developed primer pair can be used for screening a target chromosome segment to culture cotton varieties with improved clothes.
Wherein, the specific information of each SSR molecular marker and the detection primer thereof is as follows:
the nucleotide sequence of the SSR molecular marker JAAS6365 is shown in SEQ ID No. 1.
The nucleotide sequence of SSR molecular marker JAAS5604 is shown in SEQ ID No. 2;
the nucleotide sequence of SSR molecular marker JAAS0803 is shown in SEQ ID No. 3.
The primers (i.e. the primer pair used for detection in a in fig. 3) for detecting the SSR molecular marker JAAS6365 are as follows:
JAAS6365-F：5’-AGCATCCAAAACCCATTTGCT-3’(SEQ ID No.4)；
JAAS6365-R：5’-ACCGCATCCTAAGGAAAGCT-3’(SEQ ID No.5)。
the primers (i.e. primer pair used for detection in C in fig. 3 and detection in fig. 4) for detecting the SSR molecular marker JAAS5604 are as follows:
JAAS5604-F：5’-TGACGTCGTTGATCCACCTC-3’(SEQ ID No.6)；
JAAS5604-R：5’-TCCCATGGGTGTGGTAAAACC-3’(SEQ ID No.7)。
the primers used for detecting the SSR molecular marker JAAS0803 (i.e. the primer pair used for detection B in fig. 3) are as follows:
JAAS0803-F：5’-ACTTTTTGCATTATCTAAGGTTCTGT-3’(SEQ ID No.8)；
JAAS0803-R：5’-ACCGATACTCTTTTTCCCTGCA-3’(SEQ ID No.9)。
while particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
<110> agricultural science and academy of Jiangsu province
<120> SSR nucleic acid sequence related to cotton high coat score and application thereof
<130> GNCLN190978
<160> 9
<170> PatentIn version 3.5
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Claims (4)
1. The application of the primer set or the kit containing the primer set in screening DNA fragments to assist in breeding generation populations with improved clothes and using upland cotton and abnormal cotton as parents;
the DNA fragment has the same sequence with a fragment located on an abnormal cotton No.5 chromosome from an SSR molecular marker JAAS6365 to an SSR molecular marker JAAS 5604;
the DNA fragment comprises the SSR molecular marker JAAS6365, the SSR molecular marker JAAS0803 and the SSR molecular marker JAAS5604 which are positioned on an abnormal cotton No.5 chromosome;
the nucleotide sequence of the SSR molecular marker JAAS6365 is shown in SEQ ID No. 1; the nucleotide sequence of the SSR molecular marker JAAS5604 is shown in SEQ ID No. 2; the nucleotide sequence of the SSR molecular marker JAAS0803 is SEQ ID No. 3;
the primer set consists of the following components (b1) - (b 3);
(b1) primer pair 1 for identifying the SSR molecular marker JAAS 6365: designed according to SEQ ID No. 1;
(b2) a primer pair 2 for identifying the SSR molecular marker JAAS 5604: designed according to SEQ ID No. 2;
(b3) primer pair 3 for identifying the SSR molecular marker JAAS 0803: designed according to SEQ ID No. 3;
the plant clothes score containing the SSR molecular marker JAAS6365, the SSR molecular marker JAAS0803 and the SSR molecular marker JAAS5604 on the abnormal cotton No.5 chromosome is improved.
2. Use according to claim 1, characterized in that: the primer pair 1 consists of two single-stranded DNAs shown as SEQ ID No.4 and SEQ ID No. 5; the primer pair 2 consists of two single-stranded DNAs shown as SEQ ID No.6 and SEQ ID No. 7; the primer pair 3 consists of two single-stranded DNAs shown as SEQ ID No.8 and SEQ ID No. 9.
3. The application of the primer set or the kit containing the primer set in the auxiliary identification of the seed coat traits of the generation population taking upland cotton and abnormal cotton as parents;
the primer set consists of the following components (b1) - (b 3);
(b1) primer pair 1 for identifying SSR molecular marker JAAS 6365: the SSR molecular marker is designed according to the nucleotide sequence SEQ ID No.1 of JAAS 6365;
(b2) primer pair 2 for identifying SSR molecular marker JAAS 5604: the SSR molecular marker is designed according to a nucleotide sequence SEQ ID No.2 of JAAS 5604;
(b3) primer pair 3 for identifying SSR molecular marker JAAS 0803: the SSR molecular marker is designed according to a nucleotide sequence SEQ ID No.3 of JAAS 0803;
and the plant coat length containing the SSR molecular marker JAAS6365, the SSR molecular marker JAAS0803 and the SSR molecular marker JAAS5604 on the abnormal cotton No.5 chromosome is increased.
4. Use according to claim 3, characterized in that: the primer pair 1 consists of two single-stranded DNAs shown as SEQ ID No.4 and SEQ ID No. 5; the primer pair 2 consists of two single-stranded DNAs shown as SEQ ID No.6 and SEQ ID No. 7; the primer pair 3 consists of two single-stranded DNAs shown as SEQ ID No.8 and SEQ ID No. 9.
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| PCT/CN2020/089196 WO2020224636A1 (en) | 2019-05-09 | 2020-05-08 | Gossypium anomalum-sourced ssr sequence associated with high lint percentage and drought tolerance and application thereof |
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