Method for breeding self-compatible potatoes

Abstract

Disclosed is a method for breeding self-compatible potatoes, including the following steps: (1) selecting a self-compatible potato variety material and referring to it as PG6359, and cloning the S-RNase gene of PG6359 through the transcriptome sequencing method; and (2) obtaining two full-length sequences of the S-RNase gene from the cloned S-RNase gene in step (1) and referring to them as S.sub.s11 and S.sub.s12 respectively, and after carrying out an artificial self-pollination for the variety material PG6359, selecting the variety material having the genotype of S.sub.s11S.sub.s11 from the offspring as the female parent, and selecting a self-incompatible material as the male parent, and then obtaining a self-compatible F.sub.1 generation by hybridization. The invention overcomes the self-incompatibility of diploid potatoes, and does not require the introduction of any wild potato gene fragments, thereby avoiding linkage drag, and providing a basis for the rapid creation of a diploid potato inbred line.

Claims

1. A method for breeding self-compatible potatoes, comprising the following steps: (1) selecting a self-compatible potato variety material and referring to it as a variety material PG6359, and cloning the S-RNase gene of the variety material PG6359 through the transcriptome sequencing method; wherein the transcriptome sequencing method comprises: firstly extracting RNA by utilizing the variety material PG6359, and performing the transcriptome sequencing by the sequencing platform to obtain 2 Gb of sequencing data; de novo assembling the transcriptome data by Trinity software, and calculating the expression of each transcript by RNASeq by Expectation Maximization (RSEM) software; then performing the sequence alignment search tool by utilizing the known S-RNase protein sequence in the potato reference genome, and selecting the sequence with an alignment reliability E value less than 1E-5 and an expression level Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) value greater than 200 as a candidate sequence of the S-RNase gene; designing amplification primers to amplify the full length of the S-RNase gene of variety material PG6359, and determining its expression by qPCR; and (2) obtaining two full-length sequences of the S-RNase gene from the cloned S-RNase gene in step (1) and referring to them as S.sub.s11 and S.sub.s12 respectively, wherein the gene sequence of S.sub.s11 is shown in SEQ ID NO:1, and the gene sequence of S.sub.s12 is shown in SEQ ID NO:2; and after carrying out an artificial self-pollination for the variety material PG6359, selecting the variety material having the genotype of S.sub.s11 S.sub.s11 from the offspring as the female parent and referring to it as material A, and selecting a self-incompatible material as the male parent and referring to it as material B, then obtaining a self-compatible F.sub.1 generation by hybridization; performing genotype detection for the F.sub.1 generation to confirm that the F.sub.1 generation contains the S.sub.s11 gene, and detecting that individuals of the F.sub.1 generation are self-compatible after self-pollination of the F.sub.1 generation.

2. The method for breeding self-compatible potatoes according to claim 1, wherein the individual of the F.sub.1 generation is used as a female parent in step (2), and the self-incompatible material B is used as a male parent to perform backcross; then performing genotype detection for the resulting BC.sub.1 generation material to obtain the individual containing S.sub.s11 gene as the female parent, and continuing to backcross with the self-incompatible material B; after multiple generations of backcrossing, then performing another generation of self-crossing.

3. The method for breeding self-compatible potatoes according to claim 1, wherein the sequence of the upstream primer of the amplification primers is shown in SEQ ID NO:3, and the sequence of the downstream primer of the amplification primers is shown in SEQ ID NO:4.

4. A method for generating self-compatible potatoes, comprising performing hybridization by using a potato plant comprising the polynucleotide shown in SEQ ID NO:1 as the first parent, so that the offspring comprises the polynucleotide, wherein the S-RNase genotype of the female parent is S.sub.s11S.sub.s11.

5. A method for generating self-compatible potatoes, comprising performing hybridization by using the potato plant, or a potato plant produced by a plant part, a tuber, a tuber part, a seed or a plant cell thereof comprising SEQ ID NO:1 as the first parent, so that the offspring comprises the polynucleotide, wherein the S-RNase genotype of the female parent is S.sub.s11S.sub.s11.

6. The method for generating self-compatible potatoes according to claim 5, wherein the second parent is a self-incompatible material.

7. The method according to claim 6, further performing backcross by using the offspring comprising the polynucleotide the first parent, and the self-incompatible material as the second parent, so as to obtain a self-compatible material having the genetic background of the second parent.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

(1) Explanation:

(2) The highly polymorphic S-site described herein is the S-RNase protein, which has multiple morphologies, for example, S.sub.s11 and S.sub.s12 are two different morphologies with different amino acid sequences. The S-RNase described herein may represent both the S-RNase protein and the gene determining the expression of the S-RNase protein, and the specific reference may be inferred from the contextual understanding. Similarly, S.sub.s11 and S.sub.s12 may either respectively represent a variant form of the S-RNase protein, or respectively represent the gene determining the variant form, and the specific reference may be inferred from the contextual understanding.

(3) The expression level of the S-RNase gene described herein refers to the content of mRNA transcribed by S-RNase gene.

(4) Example 1: A method for breeding self-compatible potatoes disclosed in the present invention includes the following steps:

(5) (1) performing artificial self-pollination for more than 200 diploid potatoes at flowering stage, selecting a self-compatible potato variety material and referring to it as PG6359, and cloning the S-RNase gene of PG6359 through the transcriptome sequencing method;

(6) (2) obtaining two full-length sequences of the S-RNase gene from the cloned S-RNase gene in step (1) and referring to them as S.sub.s11 and S.sub.s12 respectively, wherein the gene sequence of S.sub.s11 is represented by SEQ ID NO:1, and the gene sequence of S.sub.s12 is represented by SEQ ID NO:2; and after carrying out an artificial self-pollination for the variety material PG6359, selecting the variety material having the genotype of S.sub.s11S.sub.s11 from the offspring as the female parent and referring to it as material A, and selecting a self-incompatible material as the male parent and referring to it as material B, then obtaining a self-compatible F.sub.1 generation by hybridization; performing genotype detection for the F.sub.1 generation to confirm that the F.sub.1 generation contains the S.sub.ss1 gene, and determining that all the F.sub.1 individuals are self-compatible after self-pollination of the F.sub.1 generation.

(7) Two full-length S-RNase sequences of PG6359 are obtained. Based on RSEM calculations, the expression level of S.sub.s11 is 59.42, and the expression level of S.sub.s12 is 5124.98; there is a differential of 100 times. With verification by qPCR, the expression level of S.sub.s12 is 400 times as much as that of S.sub.s11. Since S-RNase gene has nuclease activity, and it can degrade ribosomal RNA in pollen of the same S genotype, thereby inhibiting the extension of the pollen tube. The expression level of S.sub.s11 found in the present invention is relatively low, and it may not be able to exert the effect of inhibiting the extension of the pollen tube. In order to verify this hypothesis, we carry out artificial self-pollination for PG6359, and obtain a large number of selfing offspring. After detection of the S genotypes for 201 offspring, it is found that the genotypes of 105 single plants are S.sub.s11S.sub.s12, and the genotypes of 96 single plants are S.sub.s11S.sub.s11, but no S.sub.s12S.sub.s12 genotype is found. This indicates that due to normal expression of the S.sub.s12 gene in the stigma, the pollen tube containing S.sub.s12 is inhibited from extending, and the low-expressing S.sub.s11 gene cannot reject the pollen containing the S.sub.s11 genotype, thereby resulting in self-compatibility. Since all the offspring of PG6359 contain the lower expression S.sub.s11 gene, they should theoretically be self-compatible. After performing self-pollination for the offspring, it is found that except for several materials without blooming or having poor pollen vitality, the other materials are self-compatible.

(8) The operation methods without specific illustration in this Example all belong to the prior art, so they are not explained too much here.

(9) Example 2: A method for breeding self-compatible potatoes disclosed in the present invention includes the following steps:

(10) (1) selecting a self-compatible potato variety material and referring to it as PG6359, and cloning the S-RNase gene of PG6359 through the transcriptome sequencing method;

(11) (2) obtaining two full-length sequences of the S-RNase gene from the cloned S-RNase gene in step (1) and referring to them as S.sub.s11 and S.sub.s12 respectively, wherein the gene sequence of S.sub.s11 is represented by SEQ ID NO:1, and the gene sequence of S.sub.s12 is represented by SEQ ID NO:2; and after carrying out an artificial self-pollination for the variety material PG6359, selecting the variety material having the genotype of S.sub.s11S.sub.s11 from the offspring as the female parent and referring to it as material A, and selecting a self-incompatible material as the male parent and referring to it as material B, then obtaining a self-compatible F.sub.1 generation by hybridization; performing genotype detection for the F.sub.1 generation to confirm that the F.sub.1 generation contains the S.sub.ss1 gene, and detecting that the F.sub.1 individuals are self-compatible after self-pollination of the F.sub.1 generation.

(12) Particularly, the transcriptome sequencing method in step (1) comprises: firstly extracting RNA by utilizing the style of PG6359, and performing the transcriptome sequencing by Illumina HiSeq® X Ten platform to obtain 2 Gb of sequencing data; de novo assembling the transcriptome data by Trinity software, and calculating the expression of each transcript by RSEM software; then performing BLAST® by utilizing the known S-RNase protein sequence in the potato reference genome, and obtaining a candidate sequence of the S-RNase allele in the transcriptome data; finally based on the alignment results, designing amplification primers to amplify the full length of the S-RNase gene of PG6359, and determining its expression by qPCR.

(13) Further in step (2), the F.sub.1 single plant is used as a female parent, and the self-incompatible material B is used as a male parent to perform backcrossing; then performing genotype detection for the resulting BC.sub.1 generation material to obtain the individual containing S.sub.s11 gene as the female parent, and continuing to backcross with the self-incompatible material B; after multiple generations of backcrossing, then performing another generation of self-crossing, a new self-compatible material with a genetic background of the material B may be obtained.

(14) Two full-length S-RNase sequences of PG6359 are obtained in the invention. Based on RSEM calculations, the expression level of S.sub.s11 is 58.42, and the expression level of S.sub.s12 is 5814.98; there is a differential of 100 times. With verification by qPCR, the expression level of S.sub.s12 is 400 times as much as that of S.sub.s11. It should be explained here that the expression data of S.sub.s11 and S.sub.s12 are obtained from multiple times of parallel experiments. With RSEM calculation and qPCR verification, it can be accurately determined that the expression level of S.sub.s11 is indeed low, and the low-expressing S.sub.s11 gene cannot reject the pollen containing the S.sub.s11 genotype, thereby resulting in self-compatibility.

(15) Further, the sequence of the upstream primer of the amplification primers is represented by SEQ ID NO:3, and the sequence of the downstream primer of the amplification primers is represented by SEQ ID NO:4. The selected amplification primers have strong specificity and can amplify the full-length sequence of S-RNase gene very well and completely.

(16) The operation methods without specific illustration in this Example all belong to the prior art, so they are not explained too much here.

(17) The above descriptions are merely preferred Examples of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent substitution, or improvement made within the spirit and principle of the present invention shall be encompassed in the protection scope of the present invention.